ALBERT

Description: 〈h3〉Abstract〈/h3〉 〈p〉Accumulation of acetate is a limiting factor in recombinant production of (〈em〉R〈/em〉)-3-hydroxybutyrate (3HB) by 〈em〉Escherichia coli〈/em〉 in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) deletion of phosphotransacetylase (〈em〉pta〈/em〉), pyruvate oxidase (〈em〉poxB〈/em〉), and/or the isocitrate lyase regulator (〈em〉iclR〈/em〉), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different 〈em〉E. coli〈/em〉 strain backgrounds (B, BL21, W, BW25113, MG1655, W3110, and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of 〈em〉pta〈/em〉 and 〈em〉pta-poxB〈/em〉 in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different 〈em〉E. coli〈/em〉 strains confirmed BL21 as a low acetate-forming background. Despite low 3HB titers in low-cell-density screening, 3HB-producing BL21 produced five times less acetic acid per mole of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L〈sup〉−1〈/sup〉 h〈sup〉−1〈/sup〉) and the highest 3HB concentration (16.3 g L〈sup〉−1〈/sup〉) achieved by recombinant 〈em〉E. coli〈/em〉. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.〈/p〉

Description: 〈p〉The images of cells under microscope in Figure 2 and Figure S2 were misused from 〈em〉Wang G〈/em〉 et al. 〈em〉Front Cell Infect Microbiol〈/em〉. 2018 Nov 30;8:418. These images were generated in the same set of assays.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Some members of the 〈em〉Bacillus velezensis〈/em〉 (〈em〉Bv〈/em〉) group (e.g., 〈em〉Bv〈/em〉 FZB42T and AS3.43) were previously assigned grouping with 〈em〉B. subtilis〈/em〉 and 〈em〉B〈/em〉. 〈em〉amyloliquefaciens〈/em〉, based on the fact that they shared a 99% DNA–DNA percentage phylogenetic similarity. However, hinging on current assessments of the pan-genomic reassignments, the differing phylogenomic characteristics of 〈em〉Bv〈/em〉 from 〈em〉B〈/em〉. 〈em〉subtilis〈/em〉 and B. 〈em〉amyloliquefaciens〈/em〉 are now better understood. Within this re-grouping/reassignment, the various strains within the 〈em〉Bv〈/em〉 share a close phylogenomic resemblance, and a number of these strains have received a lot of attention in recent years, due to their genomic robustness, and the growing evidence for their possible utilization in the agricultural industry for managing plant diseases. Only a few applications for their use medicinally/pharmaceutically, environmentally, and in the food industry have been reported, and this may be due to the fact that the majority of those strains investigated are those typically occurring in soil. Although the intracellular unique biomolecules of 〈em〉Bv〈/em〉 strains have been revealed via in silico genome modeling and investigated using transcriptomics and proteomics, a further inquisition into the 〈em〉Bv〈/em〉 metabolome using newer technologies such as metabolomics could elucidate additional applications of this economically relevant 〈em〉Bacillus〈/em〉 species, beyond that of primarily the agricultural sector.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Poly-γ-glutamic acid (γ-PGA) is an extracellularly produced biodegradable polymer, which has been widely used as agricultural fertilizer, mineral fortifier, cosmetic moisturizer, and drug carrier. This study firstly discovered that lichenysin, as a biosurfactant, showed the capability to enhance γ-PGA production in 〈em〉Bacillus licheniformis〈/em〉. The exogenous addition of lichenysin improved the γ-PGA yield up to 17.9% and 21.9%, respectively, in the native strain 〈em〉B. licheniformis〈/em〉 WX-02 and the lichenysin-deficient strain 〈em〉B. licheniformis〈/em〉 WX02-Δ〈em〉lchAC.〈/em〉 The capability of intracellular biosynthesis of lichenysin was positively correlated with γ-PGA production. The yield of γ-PGA increased by 25.1% in the lichenysin-enhanced strain 〈em〉B. licheniformis〈/em〉 WX02-Psrflch and decreased by 12.2% in the lichenysin-deficient strain WX02-Δ〈em〉lchAC.〈/em〉 Analysis of key enzyme activities and gene expression in the TCA cycle, precursor glutamate synthesis, and γ-PGA synthesis pathway revealed that the existence of lichenysin led to increased γ-PGA via shifting the carbon flux in the TCA cycle towards glutamate and γ-PGA biosynthetic pathways, minimizing by-product formation, and facilitating the uptake of extracellular substrates and the polymerization of glutamate to γ-PGA. Insight into the mechanisms of enhanced production of γ-PGA by lichenysin would define the essential parameters involved in γ-PGA biosynthesis and provide the basis for large-scale production of γ-PGA.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Extremely acidic soils of natural forests in Nanling National Nature Reserve have been previously investigated and revisited in two successive years to reveal the active ammonia oxidizers. Ammonia-oxidizing archaea (AOA) rather than ammonia-oxidizing bacteria (AOB) were found more functionally important in the extremely acidic soils of the natural forests in Nanling National Nature Reserve. The relative abundances of 〈em〉Nitrosotalea〈/em〉, 〈em〉Nitrososphaera〈/em〉 sister group, and 〈em〉Nitrososphaera〈/em〉 lineages recovered by ammonia monooxygenase subunit A (〈em〉amoA〈/em〉) transcripts were reassessed and compared to AOA communities formerly detected by genomic DNA. 〈em〉Nitrosotalea〈/em〉, previously found the most abundant AOA, were the second-most-active lineage after 〈em〉Nitrososphaera〈/em〉 sister group. Our field study results, therefore, propose the acidophilic AOA, 〈em〉Nitrosotalea〈/em〉, can better reside in extremely acidic soils while they may not contribute to nitrification proportionately according to their abundances or they are less functionally active. In contrast, the functional importance of 〈em〉Nitrososphaera〈/em〉 sister group may be previously underestimated and the functional dominance further extends their ecological distribution as little has been reported. 〈em〉Nitrososphaera gargensis〈/em〉–like AOA, the third abundant lineage, were more active in summer. The analyses of AOA community composition and its correlation with environmental parameters support the previous observations of the potential impact of organic matter on AOA composition. Al〈sup〉3+〈/sup〉, however, did not show a strong adverse correlation with the abundances of functional AOA unlike in the DNA-based study. The new data further emphasize the functional dominance of AOA in extremely acidic soils, and unveil the relative contributions of AOA lineages to nitrification and their community transitions under the environmental influences.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉〈em〉Pseudomonas aeruginosa〈/em〉 are ubiquitous γ-proteobacteria capable of producing the biosurfactant rhamnolipids (RL) and the polymer polyhydroxyalkanoate (PHA). RL are glycolipids with high biotechnological potential, whereas PHA is used for the production of biodegradable plastics. It has been proposed that the β-oxidation pathway provides intermediates for RL biosynthesis, even when using a non-fatty acid carbon source for growth, while an intermediate of de novo fatty acid biosynthesis (FASII) pathway [(〈em〉R〈/em〉)-3-hydroxyacyl-ACP] is used for PHA biosynthesis. The aim of this work is to study the inter-relationship of the RL and PHA biosynthetic pathways in a culture medium with a non-fatty acid carbon source, focusing on the role of FASII and β-oxidation in supplying the substrates for the first step in RL and PHA synthesis, carried out by the RhlA and PhaG enzymes, respectively. The PHA synthases (PhaC1 and PhaC2) are only able to use CoA-linked 3-hydroxy acids and the PhaG enzyme catalyzes the conversion of (〈em〉R〈/em〉)-3-hydroxyacyl-ACP to (〈em〉R〈/em〉)-3-hydroxyacyl-CoA, the substrate of PhaC1 and PhaC2. RhlA in turn catalyzes the synthesis of the RL precursor 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAA) by the dimerization of two 3-hydroxyalkanoic acid molecules (that have been shown to be also (〈em〉R〈/em〉)-3-hydroxyacyl-ACP). In this work, we show that RhlA can produce both RL and PHA precursors (presumably CoA-linked HAA), that the blockage of carbon flux through β-oxidation pathway does not decrease RL titer, and that the enoyl-CoA hydratase RhlY and enoyl-CoA hydratase/isomerase RhlZ produce the main fatty acids precursor of RL using as substrate also a FASII intermediate (presumably (〈em〉S〈/em〉)-3-hydroxyacyl-CoA).〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The aim of this research was to analyze the antioxidant and prebiotic properties of lactobionic acid and to develop a method of producing it from whey using the bacterium 〈em〉Pseudomonas taetrolens〈/em〉. Prebiotic properties were tested with selected bacterial strains that exhibit probiotic properties, while the antioxidant efficacy was tested using cold-pressed rapeseed oil. A particularly evident prebiotic effect was observed with the bacterium 〈em〉Lactobacillus fermentum〈/em〉 with a lactobionic acid concentration of 16 mg/cm〈sup〉3〈/sup〉. The growth curves of microorganisms in a substrate with various levels of lactobionic acid showed similarities between 〈em〉Lactococcus lactis〈/em〉, 〈em〉Lactobacillus acidophilus〈/em〉 DSM 20242, 〈em〉Lactobacillus acidophilus〈/em〉 L-AH1, 〈em〉Lactobacillus acidophilus〈/em〉 NCDO, 〈em〉Lactobacillus delbrueckii〈/em〉 A, 〈em〉Lactobacillus casei〈/em〉, 〈em〉Lactobacillus casei〈/em〉 Shirota, 〈em〉Bifidobacterium bifidum〈/em〉 DSM 20215, and 〈em〉Bifidobacterium bifidum〈/em〉 DSM 20456, where a short logarithmic growth phase could be distinguished, in comparison to the growth of 〈em〉Lactobacillus fermentum〈/em〉 and 〈em〉Lactobacillus acidophilus〈/em〉 CH-5, where the logarithmic growth phase was extended. 〈em〉Bifidobacterium bifidum〈/em〉 DSM 20082 and 〈em〉Bifidobacterium bifidum〈/em〉 DSM 20239 form a separate group. The greater the amount of lactobionic acid added, the higher its activity. The greatest oxidation inhibition efficacy in rapeseed oil was recorded on day 10 of storage at 60 °C with an acid content of 10 mg/cm〈sup〉3〈/sup〉. Expressed as a percentage reduction of peroxide value, this effect was 19.6%. The best result for preparations of lactobionic acid were found at 1 cm〈sup〉3〈/sup〉 (22.03 mg/cm〈sup〉3〈/sup〉), amounting to 7.3% on day 10 of the rapeseed oil thermostat test.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Lasso peptides are ribosomally synthesized and post-translationally modified natural products with a characteristic slipknot-like structure, which confers these peptides remarkable stability and diverse pharmacologically relevant bioactivities. Among all the reported lasso peptides, lassomycin and lariatins are unique lasso peptides that exhibit noticeable anti-tuberculosis (TB) activity. Due to the unique threaded structure and the unusual bactericidal mechanism toward 〈em〉Mycobacterium tuberculosis〈/em〉, these peptides have drawn considerable interest, not only in the field of total synthesis but also in several other fields including biosynthesis, bioengineering, and structure-activity studies. During the past few years, significant progress has been made in understanding the biosynthetic mechanism of these intriguing compounds, which has provided a solid foundation for future work. This review highlights recent achievements in the discovery, structure elucidation, biological activity, and the unique anti-TB mechanism of lasso peptides. Moreover, the discovery of their biosynthetic pathway has laid the foundation for combinatorial biosynthesis of their analogs, which provides new perspectives for the production of novel anti-TB lasso peptides.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Lactose is a natural disaccharide obtained from the milk of most mammals and a waste product of cheese and casein manufacturing. Over the past decades, lactose in whey has increasingly been promoted as an important resource, and an increasing number of significant advances have been made to investigate its healthy and functional properties. Lactose can be biotransformed into many kinds of derivatives, including galacto-oligosaccharides, epilactose, lactulose, lactosucrose, and 〈span〉d〈/span〉-tagatose. Biological efficiency and safety are critical for the enzymatic production of lactose derivatives from lactose. These lactose derivatives show a range of prominent physiological features and effects, such as prebiotic properties, indigestibility, and obesity prevention, which can be utilized in the pharmaceutical, health, and food industries. In this review, we present the properties and physiological effects of lactose derivatives, detailing their biological production by various enzymes and their applications in dairy products, especially directly in the milk industry.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Raspberry ketone is an important ingredient in the flavor and fragrance industries. Due to its low content in fruits and vegetables, the production of natural raspberry ketone using heterologous synthesis in microbial strains is recently attracting increased attention. In this work, a heterologous pathway to produce raspberry ketone from 〈em〉p〈/em〉-coumaric acid, including 4-coumarate: CoA ligase (4CL), benzalacetone synthase (BAS), and raspberry ketone/zingerone synthase (RZS1) from plants, was successfully assembled in 〈em〉Escherichia coli〈/em〉. When the RZS1 gene was introduced into 〈em〉E. coli〈/em〉 and co-expressed with two other genes, the intermediate 4-hydroxybenzylidene acetone in the pathway was almost completely transformed into a raspberry ketone. Substituting TB medium for M9 medium increased raspberry ketone titers by 3–4 times. Furthermore, the heterologous pathway was partitioned into two modules; module one produced 〈em〉p〈/em〉-coumaroyl-CoA from 〈em〉p〈/em〉-coumaric acid by 4CL, and module two produced raspberry ketone from coumaroyl-CoA by the action of BAS and RZS1. Optimizing the balanced expression of the two modules, it was shown that moderate expression of module one and high expression of module two was the best combination to enhance raspberry ketone production. The engineered strain CZ-8 reached 90.97 mg/l of raspberry ketone, which was 12 times higher than previously reported. In addition, the preferred approach of the heterologous pathway was related to the heterologous genes from different sources; for example, 4CL from 〈em〉Arabidopsis thaliana〈/em〉 seemed to be more suitable for raspberry ketone production than that from 〈em〉Petroselinum crispum〈/em〉. This work paves an alternative way for future economic production of natural raspberry ketone.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Spatial separation of metabolic stages in anaerobic digesters can increase the methane content of biogas, as realized in a tube anaerobic baffled reactor. Here, we investigated the performance and microbial community dynamics of a laboratory-scale mesophilic anaerobic baffled reactor with four compartments treating an artificial substrate. Due to the activity of fermentative bacteria, organic acids mostly accumulated in the initial compartments. The methane content of the biogas increased while hydrogen levels decreased along the compartments. Microbial communities were investigated based on bacterial 16S rRNA genes, 〈em〉hydA〈/em〉 genes encoding Fe–Fe-hydrogenases, and 〈em〉mcrA〈/em〉 genes/transcripts encoding the methyl-CoM reductase. The metaproteome was analyzed to identify active metabolic pathways. During the reactor operation, 〈em〉Clostridia〈/em〉 and 〈em〉Bacilli〈/em〉 became most abundant in the first compartment. Later compartments were dominated by 〈em〉Sphingobacteriia〈/em〉, 〈em〉Deltaproteobacteria〈/em〉, 〈em〉Clostridia〈/em〉, 〈em〉Bacteroidia〈/em〉, 〈em〉Synergistia〈/em〉, 〈em〉Anaerolineae〈/em〉, 〈em〉Spirochaetes〈/em〉, vadinHA17, and W5 classes. Methanogenic communities were represented by 〈em〉Methanomicrobiales〈/em〉, 〈em〉Methanobacteriaceae〈/em〉, 〈em〉Methanosaeta〈/em〉, and 〈em〉Methanosarcina〈/em〉 in the last compartments. Analysis of 〈em〉hydA〈/em〉 and 〈em〉mcrA〈/em〉 genes and metaproteome data confirmed the spatial separation of metabolic stages. In the first compartment, proteins of carbohydrate transport and metabolism were most abundant. Proteins assigned to coenzyme metabolism and transport as well as energy conservation dominated in the other compartments. Our study demonstrates how the spatial separation of metabolic stages by reactor design is underpinned by the adaptation of the microbial community to different niches.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉This study proposes the investigation of curcumin encapsulation into 〈em〉Saccharomyces cerevisiae〈/em〉 cells through osmoporation as an efficient way of increasing curcumin stability. The influence of three process parameters (cell, ethanol and curcumin concentrations) on the encapsulation process was evaluated, and the obtained biocapsules were characterised for physical and photochemical stabilisation. Results showed that encapsulation efficiency was favoured by the increase of cells/curcumin ratio and ethanol concentration up to 60%. Differential scanning calorimetry (DSC) curves revealed that yeast encapsulation delayed the curcumin melting point up to 207 °C. Encapsulated curcumin retained over 80% of antioxidant activity after thermal treatment (150 °C) and over 70% after a 50-day exposure to artificial light. Photochemical stability of yeast-encapsulated curcumin was increased by 5.7-fold, and half-life time reached 181 days under illumination conditions. Overall, osmoporation-produced yeast biocapsules confirmed the versatility of osmoporation as an encapsulation technique and successfully improved curcumin stability.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The focus of this study was to investigate the effects of 〈em〉luxS〈/em〉, a key regulatory gene of the autoinducer-2 (AI-2) quorum sensing (QS) system, on the biofilm formation and biocontrol efficacy against 〈em〉Ralstonia solanacearum〈/em〉 by 〈em〉Paenibacillus polymyxa〈/em〉 HY96-2. 〈em〉luxS〈/em〉 mutants were constructed and assayed for biofilm formation of the wild-type (WT) strain and 〈em〉luxS〈/em〉 mutants of 〈em〉P. polymyxa〈/em〉 HY96-2 in vitro and in vivo〈em〉.〈/em〉 The results showed that 〈em〉luxS〈/em〉 positively regulated the biofilm formation of HY96-2. Greenhouse experiments of tomato bacterial wilt found that from the early stage to late stage postinoculation, the biocontrol efficacy of the 〈em〉luxS〈/em〉 deletion strain was the lowest with 50.70 ± 1.39% in the late stage. However, the 〈em〉luxS〈/em〉 overexpression strain had the highest biocontrol efficacy with 75.66 ± 1.94% in the late stage. The complementation of 〈em〉luxS〈/em〉 could restore the biocontrol efficacy of the 〈em〉luxS〈/em〉 deletion strain with 69.84 ± 1.09% in the late stage, which was higher than that of the WT strain with 65.94 ± 2.73%. Therefore, we deduced that 〈em〉luxS〈/em〉 could promote the biofilm formation of 〈em〉P. polymyxa〈/em〉 HY96-2 and further promoted its biocontrol efficacy against 〈em〉R. solanacearum〈/em〉.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Cell physiology parameters are essential aspects of biological processes; however, they are difficult to determine on-line. Dielectric spectroscopy allows the on-line estimation of viable cells and can provide important information about cell physiology during culture. In this study, we investigated the dielectric property variations in 〈em〉Kluyveromyces marxianus〈/em〉 SLP1 and 〈em〉Saccharomyces cerevisiae〈/em〉 ERD yeasts stressed by 5-hydroxymethyl-2-furaldehyde and 2-furaldehyde during aerobic growth. The dielectric properties of cell permittivity, specific membrane capacitance (〈em〉Cm〈/em〉), and intracellular conductivity (〈em〉σ〈/em〉〈sub〉In〈/sub〉) were considerably affected by furan aldehydes in the same way that the cell population, viability, cell size, substrate consumption, organic acid production, and respiratory parameters were. The yeasts stressed with furan aldehydes exhibited three physiological states (〈em〉φ〈/em〉): adaptation, replicating, and nonreplicating states. During the adaptation state, there were small and stable signs of permittivity, 〈em〉Cm〈/em〉, and 〈em〉σ〈/em〉〈sub〉In〈/sub〉; additionally, no cell growth was observed. During the replicating state, cell growth was restored, and the cell viability increased; in addition, the permittivity and 〈em〉σ〈/em〉〈sub〉In〈/sub〉 increased rapidly and reached their maximum values, while the 〈em〉Cm〈/em〉 decreased. In the nonreplicating state, the permittivity and 〈em〉σ〈/em〉〈sub〉In〈/sub〉 were stable, and 〈em〉Cm〈/em〉 decreased to its minimum value. Our results demonstrated that knowing dielectric properties allowed us to obtain information about the physiological state of the cells under control and stressed conditions. Since the permittivity, 〈em〉Cm〈/em〉, and 〈em〉σ〈/em〉〈sub〉In〈/sub〉 are directly associated with the physiological state of the yeast, these results should contribute to a better understanding of the stress response of yeasts and open the possibility to on-line monitor and control the physiological state of the cell in the near future.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Type I polyketide synthases (PKSs) are large multi-domain proteins converting simple acyl-CoA thioesters such as acetyl-CoA and malonyl-CoA to a large diversity of biotechnologically interesting molecules. Such multi-step reaction cascades are of particular interest for applications in engineered microbial cell factories, as the introduction of a single protein with many enzymatic activities does not require balancing of several individual enzymatic activities. However, functional introduction of type I PKSs into heterologous hosts is very challenging as the large polypeptide chains often do not fold properly. In addition, PKS usually require post-translational activation by dedicated 4′-phosphopantetheinyl transferases (PPTases). Here, we introduce an engineered 〈em〉Corynebacterium glutamicum〈/em〉 strain as a novel microbial cell factory for type I PKS-derived products. Suitability of 〈em〉C. glutamicum〈/em〉 for polyketide synthesis could be demonstrated by the functional introduction of the 6-methylsalicylic acid synthase ChlB1 from 〈em〉Streptomyces antibioticus〈/em〉. Challenges related to protein folding could be overcome by translation fusion of ChlB1〈sub〉〈em〉Sa〈/em〉〈/sub〉 to the C-terminus of the maltose-binding protein MalE from 〈em〉Escherichia coli〈/em〉. Surprisingly, ChlB1〈sub〉〈em〉Sa〈/em〉〈/sub〉 was also active in the absence of a heterologous PPTase, which finally led to the discovery that the endogenous PPTase PptA〈sub〉〈em〉Cg〈/em〉〈/sub〉 of 〈em〉C. glutamicum〈/em〉 can also activate ChlB1〈sub〉〈em〉Sa〈/em〉〈/sub〉. The best strain, engineered to provide increased levels of acetyl-CoA and malonyl-CoA, accumulated up to 41 mg/L (0.27 mM) 6-methylsalicylic acid within 48 h of cultivation. Further experiments showed that PptA〈sub〉〈em〉Cg〈/em〉〈/sub〉 of 〈em〉C. glutamicum〈/em〉 can also activate nonribosomal peptide synthetases (NRPSs), rendering 〈em〉C. glutamicum〈/em〉 a promising microbial cell factory for the production of several fine chemicals and medicinal drugs.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Microbial production of fuels and chemicals offers a means by which sustainable product manufacture can be achieved. In this regard, 〈em〉Yarrowia lipolytica〈/em〉 is a unique microorganism suitable for a diverse array of biotechnological applications. As a robust oleaginous yeast, it has been well studied for production of fuels and chemicals derived from fatty acids. However, thanks in part to newfound genetic tools and metabolic understanding, 〈em〉Y. lipolytica〈/em〉 has been explored for high-level production of a variety of non-lipid products. This mini-review will discuss some of the recent research surrounding the ability of 〈em〉Y. lipolytica〈/em〉 to support bio-based chemical production outside the realm of fatty acid metabolism including polyketides, terpenes, carotenoids, pentose phosphate-derived products, polymers, and nanoparticles.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Exploring a cheap and clean renewable energy has become a common destination round the world with the depletion of oil resources and the concerns of increasing energy demands. Lignocellulosic biomass is the most abundant renewable resource in the biosphere, and the total biomass formed by plant photosynthesis reached more than 200 billion tons every year. Cellulase and hemicellulose and lignin degradation enzymes, the efficient biocatalyst, could efficiently convert the lignocellulosic biomass into sugars that could be further processed into biofuels, biochemical, and biomaterial for human requirement. The utilization and conversion of cellulosic biomass has great significance to solve the problems such as environmental pollution and energy crisis. Lignocellulosic materials are widely considered as important sources to produce sugar streams that can be fermented into ethanol and other organic chemicals. Pretreatment is a necessary step to overcome its intrinsic recalcitrant nature prior to the production of important biomaterial that has been investigated for nearly 200 years. Emerging research has focused in order of economical, eco-friendly, and time-effective solutions, for large-scale operational approach. These new mentioned technologies are promising for lignocellulosic biomass degradation in a huge scale biorefinery. This review article has briefly explained the emerging technologies especially the consolidated bioprocessing, chemistry, and physical base pretreatment and their importance in the valorization of lignocellulosic biomass conversion.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Although poly (ADP-ribose) polymerase (PARP) inhibitors, as anti-tumor drugs targeting the DNA damage response (DDR), have been used for the therapy of various tumors, few researches reported their effect on laryngeal squamous cell carcinoma (LSCC). Here, we first discovered that the PARP-1/2 inhibitor Niraparib could simultaneously induce cell growth inhibition and autophagy in LSCC TU212 and TU686 cells. Niraparib decelerated cell cycle of LSCC by arresting G1 phase and preventing the cells from entering S phase. DNA lesions were also observed upon Niraparib treatment as evidenced by the accumulation of γH2AX and abatement of pRB expression. In addition, autophagy generation was confirmed by the observation of autophagosomes, LC3-positive autophagy-like vacuoles, and obvious conversion of LC3-I to LC3-II. Moreover, blocking autophagy enhanced Niraparib-induced growth inhibition and DNA lesions. Further studies suggested that autophagy suppression could obstruct the activation of checkpoint kinase 1 (Chk1) through elevating proteasomal activity and then impair the capacity of homologous recombination (HR), thereby improving the anti-LSCC efficiency of Niraparib. Collectively, these findings suggested that simultaneous targeting of Niraparib and autophagy might be a promising therapeutic schedule for LSCC in clinic.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Non-dairy milk alternatives (or milk analogues) are water extracts of plants and have become increasingly popular for human nutrition. Over the years, the global market for these products has become a multi-billion dollar business and will reach a value of approximately 26 billion USD within the next 5 years. Moreover, many consumers demand plant-based milk alternatives for sustainability, health-related, lifestyle and dietary reasons, resulting in an abundance of products based on nuts, seeds or beans. Unfortunately, plant-based milk alternatives are often nutritionally unbalanced, and their flavour profiles limit their acceptance. With the goal of producing more valuable and tasty products, fermentation can help to the improve sensory profiles, nutritional properties, texture and microbial safety of plant-based milk alternatives so that the amendment with additional ingredients, often perceived as artificial, can be avoided. To date, plant-based milk fermentation mainly uses mono-cultures of microbes, such as lactic acid bacteria, bacilli and yeasts, for this purpose. More recently, new concepts have proposed mixed-culture fermentations with two or more microbial species. These approaches promise synergistic effects to enhance the fermentation process and improve the quality of the final products. Here, we review the plant-based milk market, including nutritional, sensory and manufacturing aspects. In addition, we provide an overview of the state-of-the-art fermentation of plant materials using mono- and mixed-cultures. Due to the rapid progress in this field, we can expect well-balanced and naturally fermented plant-based milk alternatives in the coming years.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Directed evolution is frequently applied to identify genetic variants with improvements in a single or multiple properties. When used to improve multiple properties simultaneously, a common strategy is to apply alternating rounds of selection criteria to enrich for variants with each desirable trait. In particular, counterselection, or selection against undesired traits rather than for desired ones, has been successfully employed in many studies. Although the sequence and stringency of alternating selective pressures for different traits are known to be highly consequential for the outcome of the screen, the effects of these parameters have not been systematically evaluated. We developed a method for producing a statistical modeling framework to elucidate these effects. The model uses single-cell fluorescence intensity distributions to estimate the proportions of phenotypic populations within a library and then predicts the changes in these proportions depending on specified positive selective or counterselective pressures. We validated the approach using recently described systems for metabolite-responsive bacterial transcription factors and yeast G-protein-coupled receptors. Finally, we applied the model to identify biological sources that exert undesirable selective pressure on libraries during sorting. Notably, these pressures produce substantial artifacts that, if unaddressed, can lead to failure of the screen. This method for model generation can be applied to FACS-based directed evolution experiments to create a quantitative framework that identifies subtle population effects. Such models can guide the choice of experimental design parameters to better enrich for true positive genetic variants and improve the chance of successful directed evolution.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Outer membrane vesicles (OMVs) are nanoparticles secreted by Gram-negative bacteria that can be used for diverse biotechnological applications. Interesting applications have been developed, where OMVs are the basis of drug delivery, enzyme carriers, adjuvants, and vaccines. Historically, OMV research has mainly focused on vaccines. Therefore, current OMV production processes have been based on batch processes. The production of OMVs in batch mode is characterized by relatively low yields and high costs. Transition of OMV production processes from batch to continuous processes could increase the volumetric productivity, reduce the production and capital costs, and result in a higher quality product. Here, we study the continuous production of 〈em〉Neisseria meningitidis〈/em〉 OMVs to improve volumetric productivity. Continuous cultivation of 〈em〉N〈/em〉. 〈em〉meningitidis〈/em〉 resulted in a steady state with similar high OMV concentrations as are reached in current batch processes. The steady state was reproducible and could be maintained for at least 600 h. The volumetric productivity of a continuous culture reached 4.0 × 10〈sup〉14〈/sup〉 OMVs per liter culture per day, based on a dilution rate of 1/day. The tested characteristics of the OMVs did not change during the experiments showing feasibility of a continuous production process for the production of OMVs for any application.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Sialylated oligosaccharides are known to have beneficial effects, such as increasing the level of bifidobacteria, reducing the levels of blood endotoxin and blood ammonia, and enhancing the body’s immune system. However, it is unknown whether sialylated lactuloses have modulatory effects on the intestinal microbiota. In this study, 60 healthy mice were randomly divided into six groups, namely, a normal control group, a lactulose group, a Kdn-α2,3-lactulose group, a Kdn-α2,6-lactulose group, a Neu5Ac-α2,3-lactulose group, and a Neu5Ac-α2,6-lactulose group. After 14 days of lactulose administration, the feces of three mice from each group were collected, and the intestinal microbiota were detected by Illumina MiSeq high-throughput sequencing targeting the V3–V4 region of the 16S rDNA gene. At the phylum level, the relative abundance of 〈em〉Firmicutes〈/em〉 was increased in the sialylated lactulose groups, while the abundance of 〈em〉Bacteroidetes〈/em〉 was decreased. At the family level, sialylated lactulose intervention decreased the relative abundance of 〈em〉Bacteroidales S24-7 group〈/em〉 and 〈em〉Helicobacteraceae〈/em〉 and enhanced the abundance of 〈em〉Lactobacillaceae〈/em〉, which reflects the modulatory effect of sialylated lactulose on intestinal microbiota. Diversity analysis indicated that the index of Chao was higher in the sialylated lactulose groups than in the normal control group, and the Shannon and Simpson diversity indices were higher in the Kdnα-2,6-lactulose group and the Neu5Ac-α2,3-lactulose group than in the normal control group. The results of the intestinal microbiota sample composition indicated that there were differences between the sialylated lactulose groups and the normal control group. Thus, sialylated lactulose could be used as a functional food component with potential therapeutic applications in manipulating intestinal microbiota to exert beneficial effects on the host’s health.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉〈em〉Pseudomonas stutzeri〈/em〉 strain XL-2 exhibited significant performance on biofilm formation. Extracellular polymeric substances (EPS) secreted by strain XL-2 were characterized by colorimetry and Fourier transform infrared (FT-IR) spectroscopy. The biofilm growth showed a strong positive correlation (〈em〉r〈/em〉〈sub〉P〈/sub〉=0.96, 〈em〉P〈/em〉〈0.01) to extracellular protein content, but no correlation to exopolysaccharide content. Hydrolyzing the biofilm with proteinase K caused a significant decrease in biofilm growth (〈em〉t〈/em〉=3.7, 〈em〉P〈/em〉〈0.05), whereas the changes in biofilm growth were not significant when the biofilm was hydrolyzed by α-amylase and β-amylase, implying that proteins rather than polysaccharides played the dominant role in biofilm formation. More specifically, confocal laser scanning microscopy (CLSM) revealed that the extracellular proteins were tightly bound to the cells, resulting in the cells with EPS presenting more biofilm promotion protein secondary structures, such as three-turn helices, β-sheet, and α-helices, than cells without EPS. Both bio-assays and quantitative analysis demonstrated that strain XL-2 produced signal molecules of 〈em〉N〈/em〉-acylhomoserine lactones (AHLs) during biofilm formation process. The concentrations of C〈sub〉6〈/sub〉-HLS and C〈sub〉6〈/sub〉-〈em〉oxo〈/em〉-HLS were both significantly positively correlated with protein contents (〈em〉P〈/em〉〈0.05). Dosing exogenous C〈sub〉6〈/sub〉-HLS and C〈sub〉6〈/sub〉-〈em〉oxo〈/em〉-HLS also resulted in the increase in protein content. Therefore, it was speculated that C〈sub〉6〈/sub〉-HLS and C〈sub〉6〈/sub〉-〈em〉oxo〈/em〉-HLS released by strain XL-2 could up-regulate the secretion of proteins in EPS, and thus promote the formation of biofilm.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Oxidative stress is an unavoidable consequence of interactions with various reactive oxygen species (ROS)-inducing agents that would damage cells or even cause cell death. Bacteria have developed defensive systems, including induction of stress-sensing proteins and detoxification enzymes, to handle oxidative stress. Cyclic diguanylate (c-di-GMP) is a ubiquitous intracellular bacterial second messenger that coordinates diverse aspects of bacterial growth and behavior. In this study, we revealed a mechanism by which c-di-GMP regulated bacterial oxidative stress resistance in 〈em〉Pseudomonas putida〈/em〉 KT2440. High c-di-GMP level was found to enhance bacterial resistance towards hydrogen peroxide. Transcription assay showed that expression of two oxidative stress resistance genes, 〈em〉fpr-1〈/em〉 and 〈em〉katE〈/em〉, was promoted under high c-di-GMP level. Deletion of 〈em〉fpr-1〈/em〉 and 〈em〉katE〈/em〉 both decreased bacterial tolerance to hydrogen peroxide and weakened the effect of c-di-GMP on oxidative stress resistance. The promoted expression of 〈em〉fpr-1〈/em〉 under high c-di-GMP level was caused by increased cellular ROS via a transcriptional regulator FinR. We further demonstrated that the influence of high c-di-GMP on cellular ROS depend on the existence of FleQ, a transcriptional regulatory c-di-GMP effector. Besides, the regulation of 〈em〉katE〈/em〉 by c-di-GMP was also FleQ dependent in an indirect way. Our results proved a connection between c-di-GMP and oxidative stress resistance and revealed a mechanism by which c-di-GMP regulated expression of 〈em〉fpr-1〈/em〉 and 〈em〉katE〈/em〉 in 〈em〉P. putida〈/em〉 KT2440.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The maltooligosaccharide-forming amylase from 〈em〉Bacillus stearothermophilus〈/em〉 STB04 (Bst-MFA) randomly cleaves the α-1,4 glycosidic linkages of starch to produce predominantly maltopentaose and maltohexaose. The three-dimensional co-crystal structure of Bst-MFA with acarbose highlighted the stacking interactions between Trp139 and the substrate in subsites − 5 and − 6. Interactions like this are thought to play a critical role in maltopentaose/maltohexaose production. A site-directed mutagenesis approach was used to test this hypothesis. Replacement of Trp139 by alanine, leucine, or tyrosine dramatically increased maltopentaose production and reduced maltohexaose production. Oligosaccharide degradation indicated that these mutants also enhance productive binding of the substrate aglycone, leading to a high maltopentaose yield. Therefore, the aromatic stacking between Trp139 and substrate is suggested to control product specificity and the oligosaccharide cleavage pattern.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Carotenoids are naturally synthesized in some species of bacteria, archaea, and fungi (including yeasts) as well as all photosynthetic organisms. 〈em〉Escherichia coli〈/em〉 has been the most popular bacterial host for the heterologous production of a variety of carotenoids, including even xanthophylls unique to photosynthetic eukaryotes such as lutein, antheraxanthin, and violaxanthin. However, conversion efficiency of these epoxy-xanthophylls (antheraxanthin and violaxanthin) from zeaxanthin remained substantially low. We here examined several factors affecting their productivity in 〈em〉E. coli〈/em〉. Two sorts of plasmids were introduced into the bacterial host, i.e., a plasmid to produce zeaxanthin due to the presence of the 〈em〉Pantoea ananatis crtE〈/em〉, 〈em〉crtB〈/em〉, 〈em〉crtI〈/em〉, 〈em〉crtY〈/em〉, and 〈em〉crtZ〈/em〉 genes in addition to the 〈em〉Haematococcus pluvialis IDI〈/em〉 gene, and one containing each of zeaxanthin epoxidase (〈em〉ZEP〈/em〉) genes originated from nine photosynthetic eukaryotes. It was consequently found that paprika (〈em〉Capsicum annuum〈/em〉) ZEP (CaZEP) showed the highest conversion activity. Next, using the 〈em〉CaZEP〈/em〉 gene, we performed optimization experiments in relation to 〈em〉E. coli〈/em〉 strains as the production hosts, expression vectors, and ribosome-binding site (RBS) sequences. As a result, the highest productivity of violaxanthin (231 μg/g dry weight) was observed, when the pUC18 vector was used with 〈em〉CaZEP〈/em〉 preceded by a RBS sequence of score 5000 in strain JM101(DE3).〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Polyhydroxyalkanoates (PHAs) are considerable biopolymers that have gained an increasing biotechnological interest in different applications, although their industrial production presents several limitations. Filamentous bacterial cells could represent a possible strategy to increase PHA yield, since more abundant PHA inclusions can be stored in elongated than in rod-shaped cells. At first, we determined the optimal batch culture conditions to induce filamentation in 〈em〉Pseudomonas mediterranea〈/em〉 CFBP-5447T, using glutamine, glycerol, glucose, and sodium octanoate, as the sole carbon source, at low- (100 rpm) or high- (250 rpm) shaking speeds. Successively, a fermentative process was set up using glutamine in a co-metabolic strategy with glycerol, and the PHAs production was compared in rod-shaped and filamentous cells. High glutamine concentrations (from 28 to 56 mM) were able to induce alone filamentation, whereas at lower glutamine concentrations (5–10 mM), the shaking speeds became critical to allow or not filamentous phenotype. PHA granule production was higher in filamentous than in rod-shaped cells, when glycerol (46.6 mM) was added to glutamine (5 mM) in co-metabolism, and fermentation was performed at a low-shaking speed. After extraction and precipitation, PHA yield was about two times higher in filamentous than that rod-shaped cells. Our results provide new insights into filament-inducing conditions and indicate a potential use of filamentous 〈em〉P. mediterranea〈/em〉 CFBP-5447T cells to increase PHA yield. These findings could have great advantages in PHAs recovering during downstream processes, since the harvesting of elongated cells is much less time-consuming and energy expensive than required with rod-shaped cells.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Outcome of patients with blood stream infections (BSI) depends on the rapid initiation of adequate antibiotic therapy, which relies on the fast and reliable identification of the underlying pathogen. Blood cultures (BC) using CO〈sub〉2〈/sub〉-sensitive colorimetric indicators and subsequent microbiological culturing are the diagnostic gold standard but turnaround times range between 24 and 48 h. The detection of volatile organic compounds of microbial origin (mVOC) has been described as a feasible method for identifying microbial growth and to differentiate between several microbial species. In this study, we aimed to investigate the ability of mVOC analyses using a gas chromatograph coupled to an ion mobility spectrometer (GC-IMS) for the recognition of bacterial growth and bacterial differentiation in BCs. Therefore, samples of whole blood and diluted bacterial suspension were injected into aerobic and anaerobic BC bottles and incubated for 8 h. Headspace samples from cultures of 〈em〉Escherichia coli〈/em〉 (DSM 25944), 〈em〉Staphylococcus aureus〈/em〉 (DSM 13661), and 〈em〉Pseudomonas aeruginosa〈/em〉 (DSM 1117) were investigated hourly and we determined at which point of time a differentiation between the bacteria was possible. We found specific mVOC signals in the headspace over growing BCs of all three bacterial species. GC-IMS headspace analyses allowed faster recognition of bacterial growth than the colorimetric indicator of the BCs. A differentiation between the three investigated species was possible after 6 h of incubation with a high reliability in the principal component analysis. We concluded that GC-IMS headspace analyses could be a helpful method for the rapid detection and identification of bacteria in BSI.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Although the potential of heterotrophic microalgae served as a sustainable source for lutein, it was still crucial to formulate a suitable medium to offset the cost involved in algal biomass cultivation while improve inherent lutein productivity. The objective of this study was to investigate the feasibilities of waste 〈em〉Monascus〈/em〉 fermentation broth medium (MFBM) toward heterotrophic 〈em〉Chlorella protothecoides〈/em〉-enriched lutein. The results indicated that 〈em〉C. protothecoides〈/em〉 subjected to MFBM batch feeding achieved 7.1 g/L biomass and 7.27 mg/g lutein. The resulting lutein productivity (7.34 mg/L/day) represented 1.54-fold more than that of frequently used Basal medium. Concurrently, the effective metabolism and absorption of carbon, nitrogen, and phosphorus in MFBM by 〈em〉C. subellipsoidea〈/em〉 cultivation make it easily complied with the permissible dischargeable limits for fermentation broth. When response to fed-batch culture mode, the biomass and lutein productivity peaked 20.4 g/L and 9.11 mg/L/day with concentrated MFBM feeding. Transcriptomics data hinted that MFBM feeding manipulated lutein biosynthesis key checkpoints (e.g., lycopene 〈em〉β〈/em〉-cyclase and lycopene 〈em〉ε〈/em〉-cyclase) while accelerated energy pathways (e.g., glycolysis and TCA cycle) to contribute such high lutein productivity in 〈em〉C. protothecoides〈/em〉. These encouraging findings not only provided indications in applying nutrient-rich fermentation broth for affordable microalgae cultivation but also presented possibilities in linking algal high value-added products like lutein with high-efficient biological nutrition removal from industrial fermentation processing.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉〈em〉Ganoderma〈/em〉 have been regarded as a traditional source of natural bioactive compounds for centuries and have recently been exploited for potential components in the cosmetics industry. Besides 〈em〉Ganoderma〈/em〉 polysaccharides and triterpenes, multiple proteins have been found in 〈em〉Ganoderma〈/em〉. With the in-depth study of these proteins, various pharmacological functions of 〈em〉Ganoderma〈/em〉 have become important in the discovery and development of new products. In the review, we summarized and discussed the kinds and characteristics of 〈em〉Ganoderma〈/em〉 proteins, especially on fungal immunomodulatory proteins (FIPs) which can be potentially developed into cosmeceuticals or nutricosmetics and are a suitable target for production using established biotechnological methods. Furthermore, we discuss their pharmacological activities of the proteins with a focus on their pharmacological functions related to cosmetics, such as antioxidant activity, inhibition of melanin, antibacterial activity, and regulation of inflammatory mediators. Numerous other questions also are addressed before the proteins can be widely accepted and used as cosmetic additives.〈/p〉

Description: 〈p〉The first funding No. should be 41671298 instead of 41301298.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Chitinases are hydrolases that catalyze the cleavage of the β-1,4-O-glycosidic linkages in chitin, a polysaccharide abundantly found in nature. Although numerous chitinolytic enzymes have been studied in detail, relatively little is known about chitinases capable of broad specificity. Broad-specificity chitinases are a sort of novel chitinases possessing two or three different catalytic activities among exochitinase, endochitinase, and N-acetylglucosaminidase. In the light of the difference of module composition and catalytic mechanism, the broad-specificity chitinases included two broad categories, broad-specificity chitinases with a single catalytic domain or multi-catalytic domains. This broad-specificity chitinases have great potential in chitin conversion. In this review, we summarize all reported cases of broad-specificity chitinases and provide an overview of the recent findings on their origin, characterization, catalytic mechanism, and potential application. Moreover, in-depth study into these chitinases could contribute to our understanding of other broad-specificity enzymes which may have some benefits on progress of biotechnology.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉At present, anti-virulence drugs are being considered as potential therapeutic alternatives and/or adjuvants to currently failing antibiotics. These drugs do not kill bacteria but inhibit virulence factors essential for establishing infection and pathogenesis through targeting non-essential metabolic pathways reducing the selective pressure to develop resistance. We investigated the effect of naturally isolated plant compounds on the repression of the quorum sensing (QS) system which is linked to virulence/pathogenicity in 〈em〉Pseudomonas aeruginosa〈/em〉. Our results show that 〈em〉trans〈/em〉-cinnamaldehyde (CA) and salicylic acid (SA) significantly inhibit expression of QS regulatory and virulence genes in 〈em〉P. aeruginosa〈/em〉 PAO1 at sub-inhibitory levels without any bactericidal effect. CA effectively downregulated both the 〈em〉las〈/em〉 and 〈em〉rhl〈/em〉 QS systems with 〈em〉lasI〈/em〉 and 〈em〉lasR〈/em〉 levels inhibited by 13- and 7-fold respectively compared to 3- and 2-fold reductions with SA treatment, during the stationary growth phase. The QS inhibitors (QSI) also reduced the production of extracellular virulence factors with CA reducing protease, elastase and pyocyanin by 65%, 22% and 32%, respectively. The QSIs significantly reduced biofilm formation and concomitantly with repressed rhamnolipid gene expression, only trace amount of extracellular rhamnolipids were detected. The QSIs did not completely inhibit virulence factor expression and production but their administration significantly lowered the virulence phenotypes at both the transcriptional and extracellular levels. This study shows the significant inhibitory effect of natural plant-derived compounds on the repression of QS systems in 〈em〉P. aeruginosa〈/em〉.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Iodine is an important trace element involved in thyroid hormone biosynthesis, while diet-induced obesity is reported to disturb the trace element metabolic balance. Herein, we studied the host-specific responses involved in modulating thyroid function and gut microbiota in obese mice after the iodine treatment and analyzed the possible causes for these responses. Obesity in the mice was induced by a high-fat diet, and the obese and normal mice were treated with the same iodine dosage (18 μg/kg/day) continuously for 8 weeks. Iodine treatment in the obese mice showed a weight-reducing effect, increased the thyroid hormone concentrations, altered the transcriptions of genes involved in thyroid hormone biosynthesis, and modulated the gut microbiota with an increased abundance of pathogenic bacteria and decreased the proportion of beneficial bacteria. However, completely different or even opposite response profiles were observed in the normal hosts. Our work indicated that obesity may exacerbate the risk of thyroid disease with a relatively safe dose of iodine, and individual differences should be considered with trace element supplementation.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Due to increasingly limited water resources, diminishing farmland acreage, and potentially negative effects of climate change, an urgent need exists to improve agricultural productivity to feed the ever-growing population. Plants interact with microorganisms at all trophic levels, adapting growth, developmental, and defense responses within a complicated network of community members. Endophytic fungi have been widely reported for their ability to aid in the defense of their host plants. Currently, many reports focus on the application of endophytic fungi with the capability to produce valuable bioactive molecules, while others focus on endophytic fungi as biocontrol agents. Plant responses upon endophytic fungi colonization are also good for the immune system of the plant. In this paper, the possible mechanisms between endophytic fungi and their hosts were reviewed. During long-term evolution, plants have acquired numerous beneficial strategies in response to endophytic fungi colonization. The interaction of endophytic fungi with plants modulates the relationship between plants and both biotic and abiotic stresses. It has previously been reported that this endophytic relationship confers additional defensive mechanisms on the modulation of the plant immune system, as the result of the manipulation of direct antimicrobial metabolites such as alkaloids to indirect phytohormones, jasmonic acid, or salicylic acid. Furthermore, plants have evolved to cope with combinations of stresses and experiments are required to address specific questions related to these multiple stresses. This review summarizes our current understanding of the intrinsic mechanism to better utilize these benefits for plant growth and disease resistance. It contributes new ideas to increase plant fitness and crop productivity.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Production of insulin-like growth factor 1 (IGF1) in 〈em〉Escherichia coli〈/em〉 mostly results in the formation of inclusion bodies. In the present study, IGF1 was fused to disulfide bond oxidoreductase A (DsbA) and expressed in 〈em〉SHuffle™ T7〈/em〉 strain, in order to obtain correctly folded protein. Soluble expression and IMAC purification of DsbA-IGF1 were optimized by applying the Box-Behnken design of response surface methodology. The optimization greatly increased concentration of soluble protein from 317 to 2600 mg/L, and IMAC yield from 400 to 1900 mg/L. Results of ANOVA showed induction OD〈sub〉600〈/sub〉 and temperature had significant effects on the soluble protein expression while isopropyl-β-d thiogalactoside, in the concentrations tested, displayed no significant effect. Moreover, the three parameters of the binding buffer including, pH, concentration of NaCl, and imidazole displayed significant effects on the IMAC yield. Then, purified DsbA-IGF1 was cleaved by human rhinovirus 3C protease, and authentic IGF1 was obtained in flow through of a subtractive IMAC. Final polishing of the protein by reversed-phase HPLC yielded IGF1 with purity of 96%. The quality attributes of purified IGF1 such as purity, identity, molecular size, molecular weight, secondary structure, and biological activity were assessed and showed to be comparable to the standard IGF1. The final yield of purified IGF1 was estimated to be 120 ± 18 mg from 1 L of the culture. Our results demonstrated a simple and easily scalable strategy for production of large amounts of bioactive IGF1 by rational designing soluble protein expression, and further optimization of expression and purification methods.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Pyoluteorin (Plt) is a PKS-NRPS hybrid antibiotic that is produced by 〈em〉Pseudomonas〈/em〉 spp. and shows strong antifungal and antibacterial activities. 〈em〉Pseudomonas protegens〈/em〉 H78, which was isolated from the rape rhizosphere in Shanghai, can produce a large array of secondary metabolites, including antibiotics and siderophores. Plt is produced at low levels in the H78 wild-type strain. This study aimed to improve Plt production through combinatory genetic engineered strategies. Plt production was significantly enhanced (by14.3-fold) in the strain engineered by the following steps: (1) deletion of the translational repressor gene 〈em〉rsmE〈/em〉 in the Gac/Rsm-RsmE pathway; (2) deletion of the ATP-dependent protease gene 〈em〉lon〈/em〉 that encodes a potential enzyme that degrades positive regulators; (3) deletion of the negative regulatory gene 〈em〉pltZ〈/em〉 of the Plt ABC-type transporter operon 〈em〉pltIJKNOP〈/em〉; (4) deletion of an inhibitory sequence within the operator of the transcriptional activator gene 〈em〉pltR〈/em〉; and (5) overexpression of the 〈em〉pltIJKNOP〈/em〉 transport operon. The Plt production of the final engineered strain was increased to 214 from 15 μg ml〈sup〉−1〈/sup〉 in the H78 wild-type strain. In addition, the 〈em〉pltA〈/em〉 gene in the 〈em〉pltLABCDEFG〈/em〉 biosynthetic operon was characterized as the gene encoding the rate-limiting enzyme in the Plt biosynthetic pathway of H78. However, overexpression of the rate-limiting enzyme gene 〈em〉pltA〈/em〉 or the transcriptional activator gene 〈em〉pltR〈/em〉 did not further improve Plt biosynthesis in the above multiple-gene knockout strains.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉In nature, cyclic dipeptide prenyltransferases catalyze regioselective Friedel-Crafts alkylations of tryptophan-containing cyclic dipeptides. This enzyme class, belonging to the dimethylallyl tryptophan synthase superfamily, is known to be flexible toward aromatic prenyl acceptors, while mostly retaining its typical regioselectivity. Ardeemin fumiquinazoline (FQ) (〈strong〉1〈/strong〉), a tryptophan-containing cyclic tripeptide derivative, is assembled in 〈em〉Aspergillus fischeri〈/em〉 by the non-ribosomal peptide synthetase ArdA and modified by the prenyltransferase ArdB, leading to the pharmaceutically active hexacyclic ardeemin. Therefore, 〈strong〉1〈/strong〉 and its enantiomer 〈em〉ent〈/em〉-ardeemin FQ (〈strong〉2〈/strong〉) constitute potential substrates for aromatic prenyltransferases. In this study, we investigated the acceptance of both enantiomers by two cyclic dipeptide 〈em〉C2〈/em〉-prenyltransferases BrePT and FtmPT1 and three 〈em〉C3〈/em〉-prenyltransferases CdpNPT, CdpC3PT, and AnaPT. LC-MS analysis of the incubation mixtures and NMR analysis of the isolated products revealed that the stereochemistry at C11 and C14 in 〈strong〉1〈/strong〉 and 〈strong〉2〈/strong〉 has a strong influence on their acceptance by these enzymes and the regioselectivity of the prenylation reactions. 〈strong〉1〈/strong〉 was very well accepted by BrePT, FtmPT1, and CdpNPT, with 〈em〉C2〈/em〉- or 〈em〉C3〈/em〉-prenylated derivatives as predominant products, which fills the prenylation gaps by tryptophan prenyltransferases reported in a previous study. 〈strong〉2〈/strong〉 was a poor substrate for all the enzymes and converted with low regioselectivity and mainly prenylated at C6 and C7 of the indole moiety.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Sandalwood oil is a valuable resource derived from 〈em〉Santalum album〈/em〉. It has antibacterial, cosmetic, and sedative effects. α-Santalene is the precursor of α-santalol, the main component of sandalwood oil. 〈em〉Yarrowia lipolytica〈/em〉 is an oleaginous yeast, which has been metabolically engineered to produce valuable compounds such as terpenoids and biofuel. This study presents a method for the heterologous synthesis of α-santalene by 〈em〉Y. lipolytica〈/em〉. Using 〈em〉Y. lipolytica〈/em〉 ATCC 201249, a codon-optimized plant-origin α-santalene synthase (STS) was integrated into the genome, and a yield of 5.19 mg/L α-santalene was obtained after fermentation. Upstream genes in the MVA pathway (〈em〉ERG8〈/em〉, 〈em〉ERG10〈/em〉, 〈em〉ERG12〈/em〉, 〈em〉ERG13〈/em〉, 〈em〉ERG19〈/em〉, 〈em〉ERG20〈/em〉, 〈em〉HMG1〈/em〉, and 〈em〉tHMG1〈/em〉) were overexpressed, and we found that the key genes 〈em〉ERG8〈/em〉, 〈em〉HMG1〈/em〉, and 〈em〉tHMG1〈/em〉 can increase the supply of FPP and the yield of α-santalene. 〈em〉ERG8〈/em〉 and 〈em〉HMG1〈/em〉 were overexpressed in multiple-copy formats, and the plasmid pERG8HMG1 and ERG8-HMG1 expression modules were optimized as single-copy and multiple-copy formats, respectively. The overexpression of single-copy plasmid pERG8HMG1 led to α-santalene yield of 13.31 mg/L. The optimal feeding strategy was determined by initial carbon source concentration optimizations and five feeding methods. Using 50 g/L glucose as the initial carbon source, maintaining the carbon source concentration at 5–20 g/L during the feeding process is most conducive to increased production. These results were verified in a 5-L fermenter by batch and fed-batch fermentation. The OD of fed-batch fermentation broth reached 79.09, and the production of α-santalene reached 27.92 mg/L; 5.38 times of the initial yield, without by-products farnesol and trans-α-bergamotene.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Diepoxy-containing compounds are widely distributed in nature. These metabolites are found in plants and marine organisms and are also produced by many microorganisms, fungi, or fungal endophytes. Many of these metabolites are antibiotics and exhibit a wide variety of biological activities. More than 80 α,β-diepoxy-containing compounds are presented in this article, which belong to different classes of chemical compounds including lipids, terpenoids, alkaloids, quinones, hydroquinones, and pyrones. The main activities that characterize α,β-diepoxy-containing compounds are antineoplastic with confidence up to 99%, antifungal with confidence up to 94%, antiinflammatory with confidence up to 92%, or antibacterial with confidence up to 78%. In addition, these metabolites can be used as a lipid metabolism regulator with a certainty of up to 81%, antiviral (〈em〉Arbovirus〈/em〉) activity with a certainty of up to 71%, or antiallergic activity with confidence up to 69%. These data on the biological activity of diepoxy-containing compounds are of considerable interest to pharmacologists, chemists, and medical professionals who are involved in phytomedicine and related areas of science and industry.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Biosynthesis of Ds(+)-threo-isocitric acid from ethanol in the 〈em〉Yarrowia lipolytica〈/em〉 batch and repeated-batch cultures was studied. Repeated-batch cultivation was found to provide for a good biosynthetic efficiency of the producer for as long as 748 h, probably due to maintenance of high activities of enzymes involved in the biosynthesis of isocitric acid. Under optimal repeated-batch cultivation conditions, the producer accumulated 109.6 g/L Ds(+)-threo-isocitric acid with a production rate of 1.346 g/L h. The monopotassium salt of isocitric acid isolated from the culture liquid and purified to 99.9% was found to remove neurointoxication, to restore memory, and to improve the learning of laboratory rats intoxicated with lead and molybdenum salts. Taking into account the fact that the neurotoxic effect of heavy metals is mainly determined by oxidative stress, the aforementioned favorable action of isocitric acid on the intoxicated rats can be explained by its antioxidant activity among other pharmacological effects.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The ungenerous release of metals from different industrial, agricultural, and anthropogenic sources has resulted in heavy metal pollution. Metals with a density larger than 5 g cm〈sup〉−3〈/sup〉 have been termed as heavy metals and have been stated to be potentially toxic to human and animals. Algae are known to be pioneer organisms with the potential to grow under extreme conditions including heavy metal-polluted sites. They have evolved efficient defense strategies to combat the toxic effects exerted by heavy metal ions. Most of the algal strains are reported to accumulate elevated metal ion concentration in cellular organelles. With respect to that, this review focuses on understanding the various strategies used by algal system for heavy metal resistance. Additionally, the application of this metal resistance in biosynthesis of metal nanoparticles and metal oxide nanoparticles has been investigated in details. We thereby conclude that algae serve as an excellent system for understanding metal uptake and accumulation. This thereby assists in the design and development of low-cost approaches for large-scale synthesis of nanoparticles and bioremediation approach, providing ample opportunities for future work.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉〈em〉Clostridium autoethanogenum〈/em〉 and 〈em〉Clostridium ljungdahlii〈/em〉 are physiologically and genetically very similar strict anaerobic acetogens capable of growth on carbon monoxide as sole carbon source. While exact nutritional requirements have not been reported, we observed that for growth, the addition of vitamins to media already containing yeast extract was required, an indication that these are fastidious microorganisms. Elimination of complex components and individual vitamins from the medium revealed that the only organic compounds required for growth were pantothenate, biotin and thiamine. Analysis of the genome sequences revealed that three genes were missing from pantothenate and thiamine biosynthetic pathways, and five genes were absent from the pathway for biotin biosynthesis. Prototrophy in 〈em〉C. autoethanogenum〈/em〉 and 〈em〉C. ljungdahlii〈/em〉 for pantothenate was obtained by the introduction of plasmids carrying the heterologous gene clusters 〈em〉panBCD〈/em〉 from 〈em〉Clostridium acetobutylicum〈/em〉, and for thiamine by the introduction of the 〈em〉thiC-purF〈/em〉 operon from 〈em〉Clostridium ragsdalei〈/em〉. Integration of 〈em〉panBCD〈/em〉 into the chromosome through allele-coupled exchange also conveyed prototrophy. 〈em〉C. autoethanogenum〈/em〉 was converted to biotin prototrophy with gene sets 〈em〉bioBDF〈/em〉 and 〈em〉bioHCA〈/em〉 from 〈em〉Desulfotomaculum nigrificans〈/em〉 strain CO-1-SRB, on plasmid and integrated in the chromosome. The genes could be used as auxotrophic selection markers in recombinant DNA technology. Additionally, transformation with a subset of the genes for pantothenate biosynthesis extended selection options with the pantothenate precursors pantolactone and/or beta-alanine. Similarly, growth was obtained with the biotin precursor pimelate combined with genes 〈em〉bioYDA〈/em〉 from 〈em〉C. acetobutylicum〈/em〉. The work raises questions whether alternative steps exist in biotin and thiamine biosynthesis pathways in these acetogens.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉〈em〉Candida albicans〈/em〉 is a fungal pathogen that is difficult to cure clinically. The current clinic 〈em〉C. albicans〈/em〉-inhibiting drugs are very harmful to humans. This study revealed the potential of iturin fractions from 〈em〉Bacillus subtilis〈/em〉 to inhibit 〈em〉C. albicans〈/em〉 in free status (MIC = 32 μg/mL) and natural biofilm in vitro. The inhibition mechanism was identified as an apoptosis pathway via the decrease of mitochondrial membrane potential, the increase of the reactive oxygen species (ROS) accumulation, and the induction of nuclear condensation. For in vivo experiments, the 〈em〉C. albicans〈/em〉 infection model was constructed via intraperitoneal injection of 1 × 10〈sup〉8〈/sup〉〈em〉C. albicans〈/em〉 cells into mice. One day after the infection, iturin was used to treat infected mice at different concentrations alone and in combination with amphotericin B (AmB) by intraperitoneal injection. The treatment with AmB alone could cause the death of infected mice, whereas treatment with 15 mg/kg iturin per day alone led to the survival of all infected mice throughout the study. After continuously treated for 6 days, all mice were sacrificed and analyzed. As results, the combination of 15 mg/kg iturin and AmB at a ratio of 2:1 had the most efficient effect to remove the fungal burden in the kidney and cure the infected mice by reversing the symptoms caused by 〈em〉C. albicans〈/em〉 infection, such as the loss of body weight, change of immunology cells in blood and cytokines in serum, and damage of organ structure and functions. Overall, iturin had potential in the development of efficient and safe drugs to cure 〈em〉C. albicans〈/em〉 infection.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Recent studies from our laboratory indicate that engineered silver nanoparticles can inhibit aflatoxin biosynthesis even at concentrations at which they do not demonstrate antifungal activities on the aflatoxin-producing fungus. Whether such inhibition can be modified by altering the nanoparticles’ physical properties remains unclear. In this study, we demonstrate that three differently sized citrated-coated silver nanoparticles denoted here as NP1, NP2, and NP3 (where, sizes of NP1 〈 NP2 〈 NP3) inhibit aflatoxin biosynthesis at different effective doses in 〈em〉Aspergillus parasiticus〈/em〉, the plant pathogenic filamentous fungus. Recapping NP2 with polyvinylpyrrolidone coating (denoted here as NP2p) also altered its ability to inhibit aflatoxin production. Dose-response experiments with NP concentrations ranging from 10 to 100 ng mL〈sup〉−1〈/sup〉 indicated a non-monotonic relationship between aflatoxin inhibition and NP concentration. The maximum inhibitory concentrations differed between the NP types. NP1 demonstrated maximum inhibition at 25 ng mL〈sup〉−1〈/sup〉. Both NP2 and NP3 showed maximum inhibition at 50 ng mL〈sup〉−1〈/sup〉, although NP2 resulted in a significantly higher inhibition than NP3. While both NP2 and NP2p demonstrated greater aflatoxin inhibition than NP1 and NP3, NP2p inhibited aflatoxin over a significantly wider concentration range as compared to NP2. Our results, therefore, suggest that nano-fungal interactions can be regulated by altering certain NP physical properties. This concept can be used to design NPs for mycotoxin prevention optimally.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Biosurfactants are amphiphilic molecules that interact with the surfaces of liquids leading to many useful applications. Most biosurfactants have been identified from cultured microbial sources, leaving a largely untapped resource of uncultured bacteria with potentially novel biosurfactant structures. To access the uncultured bacteria, a metagenomic library was constructed in 〈em〉Escherichia coli〈/em〉 from environmental DNA within an 〈em〉E. coli〈/em〉, 〈em〉Pseudomonas putida〈/em〉 and 〈em〉Streptomyces lividans〈/em〉 shuttle vector. Phenotypic screening of the library in 〈em〉E. coli〈/em〉 and 〈em〉P. putida〈/em〉 by the paraffin spray assay identified a 〈em〉P. putida〈/em〉 clone with biosurfactant activity. Sequence analysis and transposon mutagenesis confirmed that an ornithine acyl-ACP N-acyltransferase was responsible for the activity. Although the fosmid was not active in 〈em〉E. coli〈/em〉, overexpression of the 〈em〉olsB〈/em〉 gene could be achieved under the control of the inducible T7 promoter, resulting in lyso-ornithine lipid production and biosurfactant activity in the culture supernatants. Screening for activity in more than one host increases the range of sequences that can be identified through metagenomic, since 〈em〉olsB〈/em〉 would not have been identified if only 〈em〉E. coli〈/em〉 had been used as a host. The potential of lyso-ornithine lipids as a biosurfactant has not been fully explored. Here, we present several biosurfactant parameters of lyso-ornithine lipid to assess its suitability for industrial application.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Foot-and-mouth disease (FMD) is a highly contagious disease that affects all susceptible cloven-hoofed animals, resulting in considerable economic losses to animal industries worldwide. Numerous categories of enzyme-linked immunosorbent assays (ELISA) have been developed and widely used to evaluate herd immunity. Manufacturing inactivated FMD virus (FMDV) as a diagnostic antigen requires a facility with a high level of biosafety, but this requirement raises concern on viral leakage. In our previous study, bacterium-original FMD virus-like particles (VLPs) resemble the authentic FMDV and induce protective immunity against homologous viral challenges, thereby demonstrating that they are sufficiently safe without limitations on biosafety facilities and easily prepared. Herein, we developed a competitive ELISA (cELISA) based on FMDV-VLPs as a diagnostic antigen to evaluate herd immunity. The criterion of this cELISA was determined by detecting panels of positive sera with different antibody titers and negative sera. The working parameter of cELISA was optimized, and samples with a percentage inhibition of ≥ 50% were considered positive. The specificity of cELISA to test 277 serum samples with various antibody titers was 100%, and the sensitivity reached 96%. The coincidence rates of cELISA with a VDPro® FMDV and a PrioCHECK® FMDV type O antibody ELISA kit were 97.8% and 98.2%, respectively. Repeatability tests demonstrated that the coefficients of variation within and between runs were less than 7% and 14%, respectively. Our data demonstrated that cELISA based on bacterium-original VLPs had high specificity, sensitivity, and reproducibility. The cELISA could also be used for evaluating vaccination herd immunity effects, especially in developing countries.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The rumen microbiome is thought to play an important role in maintaining normal gastrointestinal metabolism and nutrient absorption in ruminants. The present study was designed to investigate the effect of heat stress on the rumen microbiome of goats using 16S rRNA sequencing technology. Six female goats were randomly allocated into two control metabolic chambers: A and B (in which the temperature and humidity could be precisely controlled with a precision deviation of ± 0.5 °C and ± 5%, with three goats/chamber). Dynamic changes in the rumen bacterial community were detected under 16 gradually increasing temperature and humidity indexes (THIs). Heat stress had no significant effect on alpha diversity but affected the main phyla and genera of the goat rumen microbiota. With a deeper level of heat stress, the TH groups formed a distinct cluster that differed from that of the control check (CK) group. The dominant phylum transitioned from 〈em〉Firmicutes〈/em〉 to 〈em〉Bacteroidetes〈/em〉, and co-exclusion occurred between these two phyla. With the increase in THI, the content of probiotics in the 〈em〉Lachnospiraceae〈/em〉_ND3007_group (〈em〉P〈/em〉 〈 0.05) decreased, and the abundance of pathogenic bacteria, such as 〈em〉Erysipelotrichaceae〈/em〉_UCG-004 and 〈em〉Treponema〈/em〉_2, increased; however, the difference between the groups was not significant (〈em〉P〈/em〉 〉 0.05). Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was used to predict bacterial function, and we found that the ambient environment significantly affected the balance between carbohydrate and energy metabolism (〈em〉P〈/em〉 〈 0.05). In conclusion, heat stress changed the composition of rumen microbes and affected metabolic function. This experiment provides a theoretical basis for exploring the effects of environmental factors on the rumen of goats.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Azoreductases reductively cleave azo linkages by using NAD(P)H as an electron donor. The enzymes are widely found in bacteria and act on numerous azo dyes, which allow various unique applications. This review describes primary amino acid sequences, structures, substrates, physiological roles, and biotechnological applications of bacterial azoreductases to discuss their remarkable diversification. According to primary sequences, azoreductases were classified phylogenetically into four main clades. Most members of clades I–III are flavoproteins, whereas clade IV members include flavin-free azoreductases. Clades I and II prefer NADPH and NADH, respectively, as electron donors, whereas other members generally use both. Several enzymes formed no clades; moreover, some bacteria produce azoreductases with longer primary structures than those hitherto identified, which implies further diversification of bacterial azoreductases. The crystal structures commonly reveal the Rossmann folds; however, ternary structures are moderately varied with different quaternary conformation. Although physiological roles are obscure, several azoreductases have been shown to act on metabolites such as flavins, quinones, and metal ions more efficiently than on azo dyes. Considering that many homologs exclusively act on these metabolites, it is possible that azoreductases are actually side activities of versatile reductases that act on various substrates with different specificities. In parallel, this idea raises the possibility that homologous enzymes, even if these are already defined as other types of reductases, widely harbor azoreductase activities. Although azoreductases for which their genes have been identified are not abundant, it may be simple to identify azoreductases of biotechnological importance that have novel substrate specificities.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The morbidity and mortality associated with bacterial infections have remained significant despite chemotherapeutic advances. With the emergence of drug-resistant bacterial strains, the situation has become a serious threat to the public health. Thus, there is an urgent need to identify novel antibacterials. The majority of antibiotics available in the market are produced by bacteria isolated from soil. However, the low-hanging fruit has been picked; hence, there is a need to mine bacteria from unusual sources. With this in mind, it is important to note that animals and pests such as cockroaches, snake, crocodiles, and water monitor lizard come across pathogenic bacteria regularly, yet flourish in contaminated environments. These species must have developed methods to defend themselves to counter pathogens. Although the immune system is known to possess antiinfective properties, gut bacteria of animals/pests may also offer a potential source of novel antibacterial agents, and it is the subject of this study. This paper discusses our current knowledge of bacteria isolated from land and marine animals with antibacterial properties and to propose untapped sources for the isolation of bacteria to mine potentially novel antibiotic molecules.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Owing to their unique potential to ferment carbohydrates, both homo- and heterofermentative lactic acid bacteria (LAB) are widely used in the food industry. Deciphering the genetic basis that determine the LAB fermentation type, and hence carbohydrate utilization, is paramount to optimize LAB industrial processes. Deep sequencing of 24 LAB species and comparison with 32 publicly available genome sequences provided a comparative data set including five major LAB genera for further analysis. Phylogenomic reconstruction confirmed 〈em〉Leuconostoc〈/em〉 and 〈em〉Pediococcus〈/em〉 species as independently emerging from the 〈em〉Lactobacillus〈/em〉 genus, within one of the three phylogenetic clades identified. These clades partially grouped LABs according to their fermentation types, suggesting that some metabolic capabilities were independently acquired during LAB evolution. In order to apply a genome-wide association study (GWAS) at the multigene family level, utilization of 49 carbohydrates was also profiled for these 56 LAB species. GWAS results indicated that obligately heterofermentative species lack 1-phosphofructokinase, required for 〈span〉d〈/span〉-mannose degradation in the homofermentative pathway. Heterofermentative species were found to often contain the 〈em〉araBAD〈/em〉 operon, involved in 〈span〉l〈/span〉-arabinose degradation, which is important for heterofermentation. Taken together, our results provide helpful insights into the genetic determinants of LAB carbohydrate metabolism, and opens for further experimental research, aiming at validating the role of these candidate genes for industrial applications.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Four cutinase genes are encoded in the genome of the saprophytic fungus 〈em〉Aspergillus nidulans〈/em〉, but only two of them have proven to codify for active cutinases. However, their overall roles in cutin degradation are unknown, and there is scarce information on the regulatory effectors of their expression. In this work, the expression of the cutinase genes was assayed by multiplex qRT-PCR in cultures grown in media containing both inducer and repressor carbon sources. The genes 〈em〉ancut1〈/em〉 and 〈em〉ancut2〈/em〉 were induced by cutin and its monomers, while 〈em〉ancut3〈/em〉 was constitutively expressed. Besides, cutin induced 〈em〉ancut4〈/em〉 only under oxidative stress conditions. An in silico analysis of the upstream regulatory sequences suggested binding regions for the lipid metabolism transcription factors (TF) FarA for 〈em〉ancut1〈/em〉 and 〈em〉ancut2〈/em〉 while FarB for 〈em〉ancut3〈/em〉. For 〈em〉ancut4〈/em〉, the analysis suggested binding to NapA (the stress response TF). These binding possibilities were experimentally tested by transcriptional analysis using the 〈em〉A〈/em〉. 〈em〉nidulans〈/em〉 mutants ANΔ〈em〉farA〈/em〉, ANΔ〈em〉farB〈/em〉, and ANΔnapA. Regarding cutin degradation, spectroscopic and chromatographic methods showed similar products from ANCUT1 and ANCUT3. In addition, ANCUT1 produced 9,10-dihydroxy hexadecanoic acid, suggesting an endo-cleavage action of this enzyme. Regarding ANCUT2 and ANCUT4, they produced omega fatty acids. Our results confirmed the cutinolytic activity of the four cutinases, allowed identification of their specific roles in the cutinolytic system and highlighted their differences in the regulatory mechanisms and affinity towards natural substrates. This information is expected to impact the cutinase production processes and broaden their current biotechnological applications.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Secondary metabolites (SM) produced by fungi and bacteria have long been of exceptional interest owing to their unique biomedical ramifications. The traditional discovery of new natural products that was mainly driven by bioactivity screening has now experienced a fresh new approach in the form of genome mining. Several bioinformatics tools have been continuously developed to detect potential biosynthetic gene clusters (BGCs) that are responsible for the production of SM. Although the principles underlying the computation of these tools have been discussed, the biological background is left underrated and ambiguous. In this review, we emphasize the biological hypotheses in BGC formation driven from the observations across genomes in bacteria and fungi, and provide a comprehensive list of updated algorithms/tools exclusively for BGC detection. Our review points to a direction that the biological hypotheses should be systematically incorporated into the BGC prediction and assist the prioritization of candidate BGC.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉This study described the production, characterization, and application of monoclonal antibodies (mAbs) against porcine circovirus type 2 (PCV2). Twelve stable hybridomas were produced by immunization with purified PCV2a/LG strain and characterized by immunoperoxidase monolayer assay (IPMA), Western blotting, and neutralization assays. All mAbs could react with the PCV2 Cap protein and neutralize PCV2a/LG strain. One of them, mAb 3A5, reacted to all PCV2 strains from PCV2a, PCV2b, and PCV2d and it could be applied to detect PCV2 antigen and antibodies. It was shown that the mAb 3A5 could be used to locate PCV2 antigen in PK15 cells and the inguinal lymph nodes of PCV2b/YJ stain-infected piglets. Furthermore, this mAb could immunoprecipitate the Cap protein in PCV2-infected PK15 cells. Meanwhile, a capture ELISA based on mAb 3A5 was developed and used to specifically test PCV2 antigen from cultures; a linear relationship was observed between the optical density at 405 nm of the ELISA and viral titers (200–12,800 TCID〈sub〉50〈/sub〉/mL), with a correlation coefficient of 0.9999. Finally, a competitive ELISA based on mAb 3A5 was developed to specifically detect antibodies in PCV2-infected and immunized pigs, and its sensitivity was higher than that of the blocking ELISA. This study suggested that the mAb 3A5 could be used in several convenient and efficient methods for PCV2 clinical and pathological studies, as well as surveillance in pigs and seroconversion monitoring in the vaccinated pigs.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Heat-killed probiotics or microbial autologous components show multiple activities on modulating host immune responses towards tolerance or vice versus aggressiveness. Gram-positive enhancer matrix particles (GEMs), the non-genetically modified particles which composed of the cell wall derived from 〈em〉Lactococcus lactis〈/em〉 (〈em〉L. lactis〈/em〉), were used as a typical microbial molecule to investigate the mechanism of opposite immune responses generated in disparate scenarios. The results of stool 16S rRNA Illumina sequencing suggested that the overwhelming number of mice pre-administered with GEMs showed the expansion of 〈em〉Bacteroidetes〈/em〉 but contraction of 〈em〉Verrucomicrobia〈/em〉. Co-administration GEMs and antibiotics could preserve the microbial diversity, even though the abundance of gut microbes was largely depleted by antibiotics. Additionally, dendritic cells (DCs) from mice receiving GEMs rather than DCs that in vitro treated with GEMs induced the expansion of regulatory T cells (Tregs), witnessing the critical role of gut flora alteration. Importantly, this alteration provided protection to alleviate dextran sulfate sodium (DSS)-induced intestinal inflammation. On the other hand, in the context of 〈em〉Helicobacter felis〈/em〉 (〈em〉H. felis〈/em〉) infection, the mice pre-administrated with GEMs exhibited a comparably potent gastric immunity with the elevated expression of IFN-γ, IL-17, and multiple anti-microbial factors, leading to the reduced burden of 〈em〉H. felis〈/em〉. However, tolerance for both DSS-induced intestinal inflammation and immunity against 〈em〉H. felis〈/em〉 was depleted in a mice model lacking of transforming growth factor-β1 (TGF-β1) in myeloid cells. These findings suggest that GEMs can modulate host immune responses bidirectionally according to context, and may serve as a supplement for antibiotic treatment.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Individual’s colonization of microbes in the gut is by birth, and there is a complex interaction between the gut microbiome and human. This interaction happens at various levels like genes, transcripts, proteins, and metabolites of different microbes present in the gut. The complete understanding of gut microflora can be studied using systems biology. Further, the contemporaneous information revealed by systems biology can be used for metabolic engineering of gut microbes. The engineered microbes having more pronounced activity helps to rejuvenate the gut microflora that plays a significant role in the management of various life-threatening diseases due to microbial imbalance. This review highlights various systems biology and metabolic engineering approaches. Moreover, this review can also emphasize on the different computational simulation models which can be further used in the efficient engineering of gut microbes. The genetically engineered models can help one to predict the significant pathways present in microbes that can be modified towards diseases treatments.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The ability of the multispecies biofilm membrane reactors (MBM reactors) to provide distinguished niches for aerobic and anaerobic microbes at the same time was used for the investigation of the consolidated bioprocessing of cellulose to short chain fatty acids (SCFAs). A consortium based consolidated bioprocess (CBP) was designed. The rumen microbiome was used as the converting microbial consortium, co-cultivated with selected individual aerobic fungi which formed a biofilm on the tubular membrane flushed with oxygen. The beneficial effect of the fungal biofilm on the process yields and productivities was attributed to the enhanced cellulolytic activities compared with those achieved by the rumen microbiome alone. At 30 °C, the MBM system with 〈em〉Trichoderma reesei〈/em〉 biofilm reached a concentration 39% higher (7.3 g/L SCFAs), than the rumen microbiome alone (5.1 g/L) using 15 g/L crystalline cellulose as the substrate. Fermentation temperature was crucial especially for the composition of the short chain fatty acids produced. The temperature increase resulted in shorter fatty acids produced. While a mixture of acetic, propionic, butyric, and caproic acids was produced at 30 °C with 〈em〉Trichoderma reesei〈/em〉 biofilm, butyric and caproic acids were not detected during the fermentations at 37.5 °C carried out with 〈em〉Coprinopsis cinerea〈/em〉 as the biofilm forming fungus. Apart from the presence of the fungal biofilm, no parameter studied had a significant impact on the total yield of organic acids produced, which reached 0.47 g of total SCFAs per g of cellulose (at 30 °C and at pH 6, with rumen inoculum to total volume ratio equal to 0.372).〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Biotechnologically produced (〈em〉R〈/em〉)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (〈em〉R〈/em〉)-3-hydroxybutyrate production in recombinant 〈em〉E. coli〈/em〉 consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (〈em〉R〈/em〉)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (〈em〉R〈/em〉)-3-hydroxybutyrate-CoA to (〈em〉R〈/em〉)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (〈em〉R〈/em〉)-3-hydroxybutyrate production by engineered 〈em〉E. coli〈/em〉 AF1000 containing a thiolase and reductase from 〈em〉Halomonas boliviensis〈/em〉. Deletion of 〈em〉yciA〈/em〉 decreased the (〈em〉R〈/em〉)-3-hydroxybutyrate yield by 43%, whereas deletion of 〈em〉tesB〈/em〉 and 〈em〉fadM〈/em〉 resulted in only minor decreases. Overexpression of 〈em〉yciA〈/em〉 resulted in doubling of (〈em〉R〈/em〉)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (〈em〉R〈/em〉)-3-hydroxybutyrate concentration in batch cultivations and in a final (〈em〉R〈/em〉)-3-hydroxybutyrate titer of 14.3 g L〈sup〉−1〈/sup〉 in fed-batch cultures. The positive impact of 〈em〉yciA〈/em〉 overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (〈em〉S〈/em〉)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (〈em〉R〈/em〉)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Halophilic fungi in hypersaline habitats require multiple cellular responses for high-salinity adaptation. However, the exact mechanisms behind these adaptation processes remain to be slightly known. The current study is aimed at elucidating the morphological, transcriptomic, and metabolomic changes of the halophilic fungus 〈em〉Aspergillus montevidensis〈/em〉 ZYD4 under hypersaline conditions. Under these conditions, the fungus promoted conidia formation and suppressed cleistothecium development. Furthermore, the fungus differentially expressed genes (〈em〉P〈/em〉 〈 0.0001) that controlled ion transport, amino acid transport and metabolism, soluble sugar accumulation, fatty acid β-oxidation, saturated fatty acid synthesis, electron transfer, and oxidative stress tolerance. Additionally, the hypersalinized mycelia widely accumulated metabolites, including amino acids, soluble sugars, saturated fatty acids, and other carbon- and nitrogen-containing compounds. The addition of metabolites—such as neohesperidin, biuret, aspartic acid, alanine, proline, and ornithine—significantly promoted the growth (〈em〉P〈/em〉 ≤ 0.05) and the morphological adaptations of 〈em〉A〈/em〉. 〈em〉montevidensis〈/em〉 ZYD4 grown in hypersaline environments. Our study demonstrated that morphological shifts, ion equilibrium, carbon and nitrogen metabolism for solute accumulation, and energy production are vital to halophilic fungi so that they can build tolerance to high-salinity environments.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The worldwide prevalence of type 2 diabetes mellitus (T2DM) is constantly increasing, and it has become a major concern, with several implications for public health, economy, and social well-being. It is well-known that several factors such as lifestyle, increased intake of fat and sugar-rich foods, and host genetics can lead to T2DM. Some recent studies have suggested that the composition of the intestinal microbiota can trigger T2DM. Since then, considerable effort has been made to understand the link between the composition of intestinal microbiota and T2DM, as well as the role of probiotics in modulation of intestinal microbiota. This mini-review summarizes the major findings and discusses the close relationship between intestinal microbiota, probiotics, and T2DM.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Pyruvic acid is an important chemical in the carboxylate platform. Obstacles for its implementation are the need for high energy in chemical synthesis and additives in fermentation leading to increased production costs. Here, pyruvic acid generation from direct conversion of lactic acid in fermentation broth by electrolysis method is presented. It was found that lactic acid could be converted to pyruvic acid in the electrolysis cell under alkaline conditions. Using 12.53 g/L lactic acid fermentation broth as anolyte, 7.01 g/L pyruvic acid could be produced and productivity to lactic acid was 57.66% at initial pH 11.74 and 5.0 V applied a voltage in the electrolysis cell. Meanwhile, 0.472 mol hydrogen was produced at the cathode. The electric energy efficiency was 76.18%. Lactic acid fermentation is relatively cheap and can be performed on many kinds of wastes and biomasses. The results suggest that pyruvic acid production from direct electrolysis of lactic acid fermentation broth can be economically feasible.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Microbial transglutaminase (mTGase) is commonly known in the food industry as meat glue due to its incredible ability to “glue” meat proteins together. Aside from being widely exploited in the meat processing industries, mTGase is also widely applied in other food and textile industries by catalysing the formation of isopeptide bonds between peptides or protein substrates. The advancement of technology has opened up new avenues for mTGase in the field of biomedical engineering. Efforts have been made to study the structural properties of mTGase in order to gain an in-depth understanding of the structure-function relationship. This review highlights the developments in mTGase engineering together with its role in biomedical applications including biomaterial fabrication for tissue engineering and biotherapeutics.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉A simple and stable immobilization of a laccase from 〈em〉Pleurotus ostreatus〈/em〉 was obtained through genetic fusion with a self-assembling and adhesive class I hydrophobin. The chimera protein was expressed in 〈em〉Pichia pastoris〈/em〉 and secreted into the culture medium. The crude culture supernatant was directly used for coatings of polystyrene multi-well plates without additional treatments, a procedure that resulted in a less time-consuming and chemicals reduction. Furthermore, the gene fusion yielded a positive effect with respect to the wild-type recombinant enzyme in terms of both immobilization and stability. The multi-well plate with the immobilized chimera was used to develop an optical biosensor to monitor two phenolic compounds: L-DOPA ((S)-2-amino-3-(3,4-dihydroxyphenyl) propanoic acid) and caffeic acid (3-(3,4-dihydroxyphenyl)-2-propenoic acid); the estimation of which is a matter of interest in the pharmaceutics and food field. The method was based on the use of the analytes as competing inhibitors of the laccase-mediated ABTS oxidation. The main advantages of the developed biosensor are the ease of preparation, the use of small sample volumes, and the simultaneous analysis of multiple samples on a single platform.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Cold-adapted microorganisms inhabiting permanently low-temperature environments were initially just a biological curiosity but have emerged as rich sources of numerous valuable tools for application in a broad spectrum of innovative technologies. To overcome the multiple challenges inherent to life in their cold habitats, these microorganisms have developed a diverse array of highly sophisticated synergistic adaptations at all levels within their cells: from cell envelope and enzyme adaptation, to cryoprotectant and chaperone production, and novel metabolic capabilities. Basic research has provided valuable insights into how these microorganisms can thrive in their challenging habitat conditions and into the mechanisms of action of the various adaptive features employed, and such insights have served as a foundation for the knowledge-based development of numerous novel biotechnological tools. In this review, we describe the current knowledge of the adaptation strategies of cold-adapted microorganisms and the biotechnological perspectives and commercial tools emerging from this knowledge. Adaptive features and, where possible, applications, in relation to membrane fatty acids, membrane pigments, the cell wall peptidoglycan layer, the lipopolysaccharide component of the outer cell membrane, compatible solutes, antifreeze and ice-nucleating proteins, extracellular polymeric substances, biosurfactants, chaperones, storage materials such as polyhydroxyalkanoates and cyanophycins and metabolic adjustments are presented and discussed.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Microbial contamination of alcoholic fermentation processes (e.g. winemaking and fuel-ethanol production) is a serious problem for the industry since it may render the product unacceptable and/or reduce its productivity, leading to large economic losses. 〈em〉Brettanomyces〈/em〉/〈em〉Dekkera bruxellensis〈/em〉 is one of the most dangerous microbial contaminant of ethanol industrial fermentations. In the case of wine, this yeast species can produce phenolic compounds that confer off-flavours to the final product. In fuel-ethanol fermentations, 〈em〉D. bruxellensis〈/em〉 is a persistent contaminant that affects ethanol yields and productivities. We recently found that 〈em〉Saccharomyces cerevisiae〈/em〉 secretes a biocide, which we named saccharomycin, composed of antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Saccharomycin is active against several wine-related yeast species, namely 〈em〉D. bruxellensis.〈/em〉 However, the levels of saccharomycin naturally secreted by 〈em〉S. cerevisiae〈/em〉 during alcoholic fermentation are not sufficient to ensure the complete death of 〈em〉D. bruxellensis〈/em〉. Therefore, the aim of the present work was to construct genetically modified 〈em〉S. cerevisiae〈/em〉 strains to overproduce these GAPDH-derived AMPs. The expression levels of the nucleotides sequences encoding the AMPs were evaluated in the modified 〈em〉S. cerevisiae〈/em〉 strains by RT-qPCR, confirming the success of the recombinant approach. Furthermore, we confirmed by immunological tests that the modified 〈em〉S. cerevisiae〈/em〉 strains secreted higher amounts of the AMPs by comparison with the non-modified strain, inducing total death of 〈em〉D. bruxellensis〈/em〉 during alcoholic fermentations.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The cycloalkanes, comprising up to 45% of the hydrocarbon fraction, occur in crude oil or refined oil products (e.g., gasoline) mainly as alkylated cyclohexane derivatives and have been increasingly found in environmental samples of soil and water. Furthermore, short-chain alkylated cycloalkanes are components of the so-called volatile organic compounds (VOCs). This study highlights the biotransformation of methyl- and ethylcyclohexane by the alkane-assimilating yeast 〈em〉Candida maltosa〈/em〉 and the phenol- and benzoate-utilizing yeast 〈em〉Trichosporon mucoides〈/em〉 under laboratory conditions. In the course of this biotransformation, we detected 25 different metabolites, which were analyzed by HPLC and GC-MS. The biotransformation process of methylcyclohexane in both yeasts involve (A) ring hydroxylation at different positions (C2, C3, and C4) and subsequent oxidation to ketones as well as (B) oxidation of the alkyl side chain to hydroxylated and acid products. The yeast 〈em〉T. mucoides〈/em〉 additionally performs ring hydroxylation at the C1-position and (C) oxidative decarboxylation and (D) aromatization of cyclohexanecarboxylic acid. Both yeasts also oxidized the saturated ring system and the side chain of ethylcyclohexane. However, the cyclohexylacetic acid, which was formed, seemed not to be substrate for aromatization. This is the first report of several new transformation reactions of alkylated cycloalkanes for eukaryotic microorganisms.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Homologous recombination-based recombineering is a widely used DNA cloning and modification technique; recombineering efficiency improvement would be helpful for high-throughput DNA manipulation. 〈em〉Escherichia coli〈/em〉 primase DnaG variants, such as DnaG Q576A and DnaG K580A, increase the recombineering efficiency via impairment of the interaction between primase and the replisome and boost the loading of more ssDNA on the replication fork. Bacterial adaptive immunity origin CRISPR-Cas9 is emerging as a powerful genome editing strategy. In this study, ssDNA recombineering and CRISPR-Cas9 were combined for the generation of DnaG variants. The tightly regulated Red operon expression cassette and tightly regulated Cas9 expression cassette were integrated into one chloroamphenicol resistance, p15A replicon-based vector. A self-curing, kanamycin resistance, p15A replicon-based plasmid was applied for the plasmid elimination after genome editing. The genome editing efficiency was as high as 100%. The recombineering efficiency of the strains harboring the DnaG variants was assayed via the kanamycin resistance gene repair as well as the chromosomal gene deletion experiments. The established genome editing strategy will expedite the DnaG structure and function relationship study as well as the metabolic engineering and synthetic biology applications.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Strain development is critical for microbial production of bio-based chemicals. The stereo-complex form of polylactic acid, a complex of poly-〈span〉l〈/span〉- and poly-〈span〉d〈/span〉-lactic acid, is a promising polymer candidate due to its high thermotolerance. Here, we developed 〈em〉Corynebacterium glutamicum〈/em〉 strains producing high amounts of 〈span〉l〈/span〉- and 〈span〉d〈/span〉-lactic acid through intensive metabolic engineering. Chromosomal overexpression of genes encoding the glycolytic enzymes, glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, triosephosphate isomerase, and enolase, increased 〈span〉l〈/span〉- and 〈span〉d〈/span〉-lactic acid concentration by 146% and 56%, respectively. Chromosomal integration of two genes involved in the Entner–Doudoroff pathway (6-phosphogluconate dehydratase and 2-dehydro-3-deoxyphosphogluconate aldolase), together with a gene encoding glucose-6-phosphate dehydrogenase from 〈em〉Zymomonas mobilis〈/em〉, to bypass the carbon flow from glucose, further increased 〈span〉l〈/span〉- and 〈span〉d〈/span〉-lactic acid concentration by 11% and 44%, respectively. Finally, additional chromosomal overexpression of a gene encoding NADH dehydrogenase to modulate the redox balance resulted in the production of 212 g/L 〈span〉l〈/span〉-lactic acid with a 97.9% yield and 264 g/L 〈span〉d〈/span〉-lactic acid with a 95.0% yield. The optical purity of both 〈span〉l〈/span〉- and 〈span〉d〈/span〉-lactic acid was 99.9%. Because the constructed metabolically engineered strains were devoid of plasmids and antibiotic resistance genes and were cultivated in mineral salts medium, these strains could contribute to the cost-effective production of the stereo-complex form of polylactic acid in practical scale.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The variant strains of porcine epidemic diarrhea virus (PEDV) severely threaten the pig industry worldwide and cause up to 100% mortality in suckling piglets. It is critically important and urgent to develop tools for detection of PEDV infection. In this study, we developed six monoclonal antibodies (mAbs) targeting N protein of PEDV and analyzed their applications on enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), western blot assay, and flow cytometry assay. The results demonstrated that all these six mAbs were IgG1 isotype and κ chain. Among these six mAbs, 3F12 recognizes a linear epitope (VAAVKDALKSLGI) while the other five mAbs recognize different conformational epitopes formed by a specific peptide fragment or the full length of N protein. The functional analysis showed that all these six mAbs were applicable to ELISA, western blot, IFA, and flow cytometry assay. In conclusion, we developed six mAbs against PEDV-N protein to facilitate the early detection of PEDV infection using ELISA, western blot, IFA, and flow cytometry.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉〈em〉Corynebacterium glutamicum〈/em〉 was only examined in the early 2000s as a possible microorganism for the production of the polyamide cyanophycin (multi-〈span〉l〈/span〉-arginyl-poly-[〈span〉l〈/span〉-aspartic acid], CGP). CGP is a potential precursor for the synthesis of polyaspartic acid and CGP-derived dipeptides which may be of use in peptide-based clinical diets, as dietary supplements, or in livestock feeds. In the past, 〈em〉C. glutamicum〈/em〉 was disregarded for CGP production due to low CGP contents and difficulties in isolating the polymer. However, considering recent advances in CGP research, the capabilities of this organism were revisited. In this study, several cyanophycin synthetases (CphA) as well as expression vectors and cultivation conditions were evaluated. The ability of 〈em〉C. glutamicum〈/em〉 to incorporate additional amino acids such as lysine and glutamic acid was also examined. The strains 〈em〉C. glutamicum〈/em〉 pVWEx1::〈em〉cphA〈/em〉〈sub〉Δ1〈/sub〉 and 〈em〉C. glutamicum〈/em〉 pVWEx1::〈em〉cphA〈/em〉〈sub〉BP1〈/sub〉 accumulated up to 14% of their dry weight CGP, including soluble CGP containing more than 40 mol% of the alternative side-chain amino acid lysine. The soluble, lysine-rich form of the polymer was not detected in 〈em〉C. glutamicum〈/em〉 in previous studies. Additionally, an incorporation of up to 6 mol% of glutamic acid into the backbone of CGP synthesized by 〈em〉C. glutamicum〈/em〉 pVWEx1::〈em〉cphA〈/em〉〈sub〉Dh〈/sub〉 was detected. The strain accumulated up to 17% of its dry weight in soluble CGP. Although glutamic acid had previously been found to replace arginine in the side chain, this is the first time that glutamic acid was found to substitute aspartic acid in the backbone.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Teicoplanin is a frontline glycopeptide antibiotic produced by 〈em〉Actinoplanes teichomyceticus〈/em〉. It is used to treat complicated cases of infection, including pediatric ones, caused by Gram-positive pathogens. There is a steady interest in elucidating the genetic mechanisms determining teicoplanin production, as they would help overproduce known teicoplanins and discover novel glycopeptides. Herein, we investigate the transcriptional organization of the 〈em〉tei〈/em〉 biosynthetic gene cluster and the roles of the cluster-situated regulatory genes in controlling teicoplanin production and self-resistance in 〈em〉A. teichomyceticus.〈/em〉 We demonstrate that the 〈em〉tei〈/em〉 cluster is organized into nine polygenic and nine monogenic transcriptional units. Most of 〈em〉tei〈/em〉 biosynthetic genes are subjected to StrR-like Tei15* control, which, in turn, appears to be regulated by LuxR-type Tei16*. Expression of the genes conferring teicoplanin self-resistance in 〈em〉A. teichomyceticus〈/em〉 is not co-regulated with antibiotic production. The gene 〈em〉tei31*〈/em〉, coding for a putative DNA binding protein, is not expressed under teicoplanin producing conditions and is dispensable for antibiotic production. Finally, phylogenesis reconstruction of the glycopeptide cluster-encoded regulators reveals two main clades of StrR-like regulators. Tei15* and close orthologues form one of these clades; the second clade is composed by orthologues of Bbr and Dbv4, governing the biosynthesis of balhimycin and teicoplanin-like A40926, respectively. In addition, the LuxR-type Tei16* appears unrelated to the LuxR-like Dbv3, which is controlling A40926 biosynthesis. Our results shed new light on teicoplanin biosynthesis regulation and on the evolution of novel and old glycopeptide biosynthetic gene clusters.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉〈span〉d〈/span〉-Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidic 〈span〉d〈/span〉-amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus, 〈em〉Thermomyces dupontii〈/em〉, and characterized the recombinant enzyme expressed in 〈em〉Escherichia coli〈/em〉. In addition, we generated a variant that has a higher substrate-binding affinity. The recombinant TdDDO expressed in 〈em〉E. coli〈/em〉 exhibited oxidase activity toward acidic 〈span〉d〈/span〉-amino acids and a neutral 〈span〉d〈/span〉-amino acid, 〈span〉d〈/span〉-Gln, with the highest activity toward 〈span〉d〈/span〉-Glu. The 〈em〉K〈/em〉〈sub〉m〈/sub〉 and 〈em〉k〈/em〉〈sub〉cat〈/sub〉 values for 〈span〉d〈/span〉-Glu were 2.16 mM and 217 s〈sup〉−1〈/sup〉, respectively. The enzyme had an optimum pH and temperature 8.0 and 60 °C, respectively, and was stable between pH 5.0 and 10.0, with a 〈em〉T〈/em〉〈sub〉50〈/sub〉 of ca. 51 °C, which was much higher than that in DDOs from other origins. Enzyme stability decreased following a decrease in protein concentration, and externally added FAD could not repress the destabilization. The mutation of Phe248, potentially located in the active site of TdDDO, to Tyr residue, conserved in DDOs and 〈span〉d〈/span〉-amino acid oxidases, markedly increased substrate-binding affinity. The results showed the great potential of TdDDO and the variant for practical applications.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉High-level biosynthesis of desired metabolites is challenging due to complexity of metabolic networks. Here, we report that platform chemical 3-hydroxypropionic acid (3-HP) can be overproduced through promoter engineering and growth-sustaining cultivation, two parallel strategies relying on RNA polymerases (RNAPs). First, we screened a promoter library and revealed that IPTG-inducible 〈em〉tac〈/em〉 promoter was most effective for overexpression of PuuC, an endogenous aldehyde dehydrogenase catalyzing 3-HP biosynthesis in 〈em〉Klebsiella pneumoniae〈/em〉. Next, tandem repetitive 〈em〉tac〈/em〉 promoters were harnessed to accommodate adequate RNAPs. When three tandem repetitive 〈em〉tac〈/em〉 promoters were recruited to overexpress PuuC, up to 102.61 g/L 3-HP was produced. This is the highest 3-HP titer reported so far. In addition, lactic acid completely vanished during the late stage of fermentation. The backflow of lactic acid to pyruvic acid saves the trouble of downstream separation of lactic acid from 3-HP, which has long been a mission impossible because they are small-molecule isomers. Furthermore, timely removal of acid stress and replenishment of nitrogen source are crucial for 3-HP biosynthesis. A mathematical model shows that RNAPs modulate the tradeoff between bacterial growth and 3-HP formation. Overall, promoter engineering and growth-promoting cultivation jointly leverage RNAPs to maximize 3-HP. This study provides a paradigm for maximizing growth-coupled metabolites.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉A growing world population and a growing number of applications for vegetable oils are generating an increasing demand for these oils, causing serious environmental problems. A sustainable lipid production is then fundamental to address these problems. Oleaginous yeasts are a promising solution for sustainable lipid production, but, with the current knowledge and technology, they are still not a serious alternative in the market. In this review, the potential of these yeasts is highlighted and a discussion is made mainly focused on the economics of the oleaginous yeast oil production and identification of the key points to be improved to achieve lower production costs and higher income. Three main stages of the production process, where costs are higher, were identified. To render economically feasible the production of oils using oleaginous yeasts, a reduction in production costs must occur in all stages, lipid yields and productivities must be improved, and production must be targeted to high-value product applications.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The scientific community’s interest in magnetotactic bacteria has increased substantially in recent decades. These prokaryotes have the particularity of synthesizing nanomagnets, called magnetosomes. The majority of research is based on several scientific questions. Where do magnetotactic bacteria live, what are their characteristics, and why are they magnetic? What are the molecular phenomena of magnetosome biomineralization and what are the physical characteristics of magnetosomes? In addition to scientific curiosity to better understand these stunning organisms, there are biotechnological opportunities to consider. Magnetotactic bacteria, as well as magnetosomes, are used in medical applications, for example cancer treatment, or in environmental ones, for example bioremediation. In this mini-review, we investigated all the aspects mentioned above and summarized the currently available knowledge.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Few studies have investigated the effect of environment on the root-associated microbiome, especially for woody plants in their native environment. The roots and rhizosphere soils of a native woody species (〈em〉Broussonetia papyrifera〈/em〉) sampled across four different climate types in China were used to elucidate the influence of environment on the root-associated microbiome. Our results showed that the 〈em〉B. papyrifera〈/em〉 root-associated microbiome contained abundant 〈em〉Proteobacteria〈/em〉 and 〈em〉Basidiomycota〈/em〉, especially 〈em〉Pseudomonas〈/em〉 and 〈em〉Rhizobium〈/em〉. The root-associated microbiomes were found to be significantly different under different climate types except for the bacterial community in the rhizosphere, and the proportion of bacterial operational taxonomic units (OTUs) shared among different climate types was lower than that of fungi. More than 50% of the total variance between microbiomes could be explained by 15 environmental factors, six of which, especially soil concentration phosphate and nitrate, had a significant effect. This study provided a comprehensive understanding of the root-associated microbiome of 〈em〉B. papyrifera〈/em〉 and further confirmed the effect of environment on the root-associated microbiome of 〈em〉B. papyrifera〈/em〉 under different climate types, with some exceptions in the rhizobacterial community and fungal OTUs. Our findings advanced knowledge of the effect of environment through an exploration of environmental factors and found that the nitrogen and phosphorus content represented the key factors.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The expression of transgenes in the nucleus is an attractive alternative for the expression of recombinant proteins in the green microalga 〈em〉Chlamydomonas reinhardtii〈/em〉. For this purpose, a strong inducible promoter that allows protein accumulation without possible negative effects on cell multiplication and biomass accumulation is desirable. A previous study at our laboratory identified that the 〈em〉CrGPDH3〈/em〉 gene from 〈em〉C. reinhardtii〈/em〉 was inducible under NaCl treatments. In this study, we cloned and characterized a 3012 bp sequence upstream of the start codon of the 〈em〉CrGPDH3〈/em〉 gene, including the 285 bp 5′ untranslated region. This region was identified as the full-length promoter and named 〈em〉PromA〈/em〉 (− 2727 to + 285). Deletion analysis of 〈em〉PromA〈/em〉 using 〈em〉GUSPlus〈/em〉 as a reporter gene enabled us to identify 〈em〉PromC〈/em〉 (− 653 to + 285) as the core promoter, displaying basal expression. A region named RIA1 (− 2727 to − 1672) was suggested to contain the NaCl response elements. Moreover, deletion analysis of RIA1 enabled us to identify a region of 577 bp named RIA3 (− 2727 to − 2150) that, when cloned upstream of 〈em〉PromC〈/em〉, was able to drive the expression of 〈em〉GUSPlus〈/em〉 in response to 5 and 100 mM NaCl, and 100 mM KCl, similar to the native 〈em〉CrGPDH3〈/em〉 promoter. These results expand our understanding of the transcriptional mechanism of 〈em〉CrGPDH3〈/em〉 and clearly show that 〈em〉CrGPDH3〈/em〉 promoter and its chimeric forms are highly salt-inducible and can be used as inducible promoters for the overexpression of transgenes in 〈em〉C. reinhardtii〈/em〉.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Many recent studies have shown that flagellin fused to heterologous antigens can induce significantly enhanced humoral and cellular immune responses through its adjuvant activity. Therefore, in this study, two key B cell epitopes and a truncated VP1 (ΔVP1) protein from foot-and-mouth disease virus (FMDV) were expressed as flagellin fusion proteins in different patterns. Specifically, ΔVP1 and two duplicates of two key B cell epitopes (2×B1B2) were fused separately to the C-terminus of flagellin with a universal exogenous T cell epitope to construct FT (Flagellin-Truncated VP1) and FME (Flagellin-Multiple Epitopes). In addition, the D3 domain of flagellin was replaced by ΔVP1 in FME, yielding FTME (Flagellin-Truncated VP1-Multiple Epitopes). The immunogenicity and protective efficacy of the three fusion proteins as novel FMDV vaccine candidates were evaluated. The results showed that FT, FME, and FTME elicited significant FMDV-specific IgG responses at 10 μg/dose compared with the mock group (〈em〉P〈/em〉 〈 0.05), with FTME producing the highest response. No significant differences in the antibody response to FTME were observed between different immunization routes or among adjuvants (ISA-206, poly(I·C), MPLA, and CpG-ODN) in mice. In addition, at 30 μg/dose, all three fusion proteins significantly induced neutralizing antibody production and upregulated the levels of some cytokines, including TNF-α, IFN-γ, and IL-12, in guinea pigs. Importantly, all three fusion proteins provided effective protective immunity against FMDV challenge in guinea pigs, though different protection rates were found. The results presented in this study indicate that the FTME fusion protein is a promising novel vaccine candidate for the future prevention and control of foot-and-mouth disease.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Bioengineering of probiotics allows the improvement of their beneficial characteristics. In this work, we develop a molecular tool that would allow the activation of desirable traits in probiotics once they reach the intestine. The activity of upstream regions of bile-inducible genes of 〈em〉Lactobacillus casei〈/em〉 BL23 and 〈em〉Lactobacillus plantarum〈/em〉 WCFS1 was analyzed using plasmids encoding an anaerobic fluorescent protein as reporter. The promoter P16090 from 〈em〉Lb. casei〈/em〉 BL23 was selected and its bile induction confirmed in 〈em〉Lb. casei〈/em〉 BL23, 〈em〉Lb. plantarum〈/em〉 WCFS1, and in 〈em〉Lactobacillus rhamnosus〈/em〉 and 〈em〉Lactobacillus reuteri〈/em〉 strains. However, the induction did not occur in 〈em〉Lactococcus lactis〈/em〉 MG1363 or 〈em〉Bifidobacterium〈/em〉 strains. Studies with different bile compounds revealed the importance of cholic acid in the bile induction process. Induction of fluorescence was also confirmed for transformed 〈em〉Lb. casei〈/em〉 BL23 under simulated colonic conditions and in the presence of intestinal microbiota. The developed vector, pNZ:16090-aFP, constitutes a promising tool suitable for the expression of genes of interest under intestinal conditions in probiotic strains of the species 〈em〉Lb. casei〈/em〉, 〈em〉Lb. plantarum〈/em〉, 〈em〉Lb. rhamnosus〈/em〉, and 〈em〉Lb. reuteri〈/em〉.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Antigen-binding fragments (Fabs) are an important part of monoclonal antibody (mAb) therapeutics and can be cost-effectively produced using an 〈em〉Escherichia coli〈/em〉 (〈em〉E. coli〈/em〉) expression system. However, Fabs tend to form undesirable aggregates when expressed in the cytoplasm of 〈em〉E. coli〈/em〉, substantially reducing the yield of correctly folded proteins. To solve this problem, in this study, we used five Fab fragments targeting IGF1R, Her2, VEGF, RANKL, and PD-1 to develop a novel system employing the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence to facilitate the efficient expression and extracellular secretion of Fabs. Following phosphate starvation, all five Fab fragments were expressed in BL21(DE3), were largely secreted into the culture medium, and then, were further purified by affinity chromatography specific to the constant region of the light chain. The purified Fab products were evaluated and were found to have high purity, antigen-binding affinity, and in vitro bioactivity. The mechanism experiments revealed that (1) BL21(DE3) had significantly higher productivity than the K-12 strains investigated; (2) the secretion ability of the PhoA promoter was superior to that of the T7 promoter; and (3) signal peptide, STII, showed higher extracellular secretion efficiency than pelB. Our findings strongly suggested that the phoA-STII-facilitated extracellular production platform is highly promising for application in the manufacturing of Fab fragments for both academic and industrial purposes.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Microalgae are arguably the most abundant single-celled eukaryotes and are widely distributed in oceans and freshwater lakes. Moreover, microalgae are widely used in biotechnology to produce bioenergy and high-value products such as polyunsaturated fatty acids (PUFAs), bioactive peptides, proteins, antioxidants and so on. In general, genetic editing techniques were adapted to increase the production of microalgal metabolites. The main genome editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system. Due to its high genome editing efficiency, the CRISPR/Cas system is emerging as the most important genome editing method. In this review, we summarized the available literature on the application of CRISPR/Cas in microalgal genetic engineering, including transformation methods, strategies for the expression of Cas9 and sgRNA, the CRISPR/Cas9-mediated gene knock-in/knock-out strategies, and CRISPR interference expression modification strategies.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Over 470 prototype Type II restriction endonucleases (REases) are currently known. Most recognise specific DNA sequences 4–8 bp long, with very few exceptions cleaving DNA more frequently. 〈em〉Tso〈/em〉I is a thermostable Type IIC enzyme that recognises the DNA sequence TARCCA (R = A or G) and cleaves downstream at N11/N9. The enzyme exhibits extensive top-strand nicking of the supercoiled single-site DNA substrate. The second DNA strand of such substrate is specifically cleaved only in the presence of duplex oligonucleotides containing a cognate site. We have previously shown that some Type IIC/IIG/IIS enzymes from the 〈em〉Thermus〈/em〉-family exhibit ‘affinity star’ activity, which can be induced by the S-adenosyl-L-methionine (SAM) cofactor analogue—sinefungin (SIN). Here, we define a novel type of inherently built-in ‘star’ activity, exemplified by 〈em〉Tso〈/em〉I. The 〈em〉Tso〈/em〉I ‘star’ activity cannot be described under the definition of the classic ‘star’ activity as it is independent of the reaction conditions used and cannot be separated from the cognate specificity. Therefore, we define this phenomenon as Secondary-Cognate-Specificity (SCS). The 〈em〉Tso〈/em〉I SCS comprises several degenerated variants of the cognate site. Although the efficiency of 〈em〉Tso〈/em〉I SCS cleavage is lower in comparison to the cognate 〈em〉Tso〈/em〉I recognition sequence, it can be stimulated by S-adenosyl-L-cysteine (SAC). We present a new route for the chemical synthesis of SAC. The 〈em〉Tso〈/em〉I/SAC REase may serve as a novel tool for DNA manipulation.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The planting of transgenic rice has aroused ongoing controversy, due to the public anxiety surrounding the potential risk of transgenic rice to health and the environment. The soil microbial community plays an important environmental role in the plant-soil-microbe system; however, few studies have focused on the effect of transgenic rice on the soil rhizospheric microbiome. We labeled transgenic gene rice (〈em〉TT51〈/em〉, transformed with Cry1Ab/1Ac gene), able to produce the 〈em〉Bt〈/em〉 (〈em〉Bacillus thuringiensis〈/em〉) toxin, its parental variety (〈em〉Minghui 63〈/em〉), and a non-parental variety (9931) with 〈sup〉13〈/sup〉CO〈sub〉2〈/sub〉. The DNA of the associated soil rhizospheric microbes was extracted, subjected to density gradient centrifugation, followed by high-throughput sequencing of bacterial 16S rRNA gene. Unweighted unifrac analysis of the sequencing showed that transgenic rice did not significantly change the soil bacterial community structure compared with its parental variety. The order 〈em〉Opitutales〈/em〉, affiliated to phylum 〈em〉Verrucomicrobia〈/em〉 and order 〈em〉Sphingobacteriales〈/em〉, was the main group of labeled bacteria in soil planted with the transgenic and parental varieties, while the orders 〈em〉Pedosphaerales〈/em〉, 〈em〉Chthoniobacteraceae〈/em〉, also affiliated to 〈em〉Verrucomicrobia〈/em〉, and the genus 〈em〉Geobacter〈/em〉, affiliated to class 〈em〉Deltaproteobacteria〈/em〉, dominated in the soil of the non-parental rice variety. The non-significant difference in soil bacterial community structure of labeled microbes between the transgenic and parental varieties, but the comparatively large difference with the non-parental variety, suggests a limited effect of planting transgenic 〈em〉Bt〈/em〉 rice on the soil microbiome.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Only a few cobalamin-producing bacterial species are known which are suitable for food fermentations. The strain of 〈em〉Acetobacter pasteurianus〈/em〉 DSM 3509 was found to have the capability to synthesize cobalamin. A survival test and a preliminary genetic study of the gene of uroporphyrinogen-III synthase indicated the ability to synthesize cobalamin. By a modified microbiological assay based on 〈em〉Lactobacillus delbrueckii〈/em〉 ssp〈em〉. lactis〈/em〉 DSM 20355, 4.57 ng/mL of cyanocorrinoids and 0.75 ng/mL of noncorrinoid growth factors were detected. The product extracted and isolated by immunoaffinity chromatography in its cyanide form had the similar UV spectrum as standard cyanocobalamin and Coα-[α-(7-adenyl)]-(Coβ-cyano) cobamide also known as pseudovitamin B〈sub〉12〈/sub〉 produced by 〈em〉Lactobacillus reuteri〈/em〉 DSM 20016. The chromatographically separated product of 〈em〉A. pasteurianus〈/em〉 was subjected to mass spectrometrical analysis. There, its fragmentation pattern turned out to be equivalent to that of cyanocobalamin also produced by 〈em〉Propionibacterium freudenreichii〈/em〉 ssp. 〈em〉freudenreichii〈/em〉 DSM 20271 and clearly differs from pseudovitamin B〈sub〉12〈/sub〉. Due to the presence of this species in several food applications, there might be cobalamin residues in food fermented with these bacteria.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Rebeccamycin is an antibiotic and antitumor substance isolated from the filamentous bacterium 〈em〉Lentzea aerocolonigenes〈/em〉. After its discovery, investigations of rebeccamycin focused on elucidating its structure, biological activity, and biosynthetic pathway. For potential medical application, a sufficient drug supply has to be ensured, meaning that the production process of rebeccamycin plays a major role. In addition to the natural production of rebeccamycin in 〈em〉L. aerocolonigenes〈/em〉, where the complex cell morphology is an important factor for a sufficient production, rebeccamycin can also be heterologously produced or chemically synthesized. Each of these production processes has its own challenges, and first approaches to production often lead to low final product concentrations, which is why process optimizations are performed. This review provides an overview of the production of rebeccamycin and the different approaches used for rebeccamycin formation including process optimizations.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Enzyme-mediated synthesis of pharmaceutical compounds is a ‘green’ alternative to traditional synthetic chemistry, and microbial engineering opens up the possibility of using whole cells as mini-factories. Whole-cell biocatalysis reduces cost by eliminating expensive enzyme purification and cofactor addition steps, as well as resulting in increased enzyme stability. 〈em〉Haloferax volcanii〈/em〉 is a model halophilic archaeon encoding highly salt and organic solvent tolerant enzymes such as alcohol dehydrogenase (〈em〉Hv〈/em〉ADH2), which catalyses the reduction of aldehydes and ketone in the presence of NADPH/NADH cofactor. A 〈em〉H. volcanii〈/em〉 strain for constitutive 〈em〉Hv〈/em〉ADH2 expression was generated using a strong synthetic promoter (p.〈em〉syn〈/em〉). The strain was immobilised in calcium alginate beads and repeatedly used as a whole-cell biocatalyst. The reduction of acetophenone, used as test substrate, was very successful and high yields were detected from immobilised whole cells over repeated biotransformation cycles. The immobilised 〈em〉H. volcanii〈/em〉 retained stability and high product yields after 1 month of storage at room temperature. This newly developed system offers halophilic enzyme expression in its native environment, high product yield, stability and reusability without the addition of any expensive NADPH/NADH cofactor. This is the first report of whole cell–mediated biocatalysis by the halophilic archaeon 〈em〉H. volcanii〈/em〉.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Arabinofuranosidase plays an essential role in the process of hydrolysis of arabinoxylan (AX). Thermostable, versatile, and efficient arabinofuranosidase is thus of great interest for the biorefinery industry. A GH51 arabinofuranosidase, Abf51, from 〈em〉Hungateiclostridium clariflavum〈/em〉 DSM 19732 was heterogeneously expressed in 〈em〉Escherichia coli〈/em〉. Abf51 was found to have an optimal pH and temperature of 6.5 and 60 °C, respectively, with very high thermostability. At the optimal working temperature (60 °C), Abf51 retained over 90% activity after a 2-day incubation and over 60% activity after a 6-day incubation. Abf51 could effectively remove the arabinofuranosyls from three kinds of AX oligosaccharides [2〈sup〉3〈/sup〉-α-〈span〉l〈/span〉-arabinofuranosyl-xylotriose (A〈sup〉2〈/sup〉XX), 3〈sup〉2〈/sup〉-α-〈span〉l〈/span〉-arabinofuranosyl-xylobiose (A〈sup〉3〈/sup〉X), and 2〈sup〉3〈/sup〉3〈sup〉3〈/sup〉-di-α-〈span〉l〈/span〉-arabinofuranosyl-xylotriose (A〈sup〉2 + 3〈/sup〉XX)], which characterized as either single substitution or double substitution by arabinofuranosyls on terminal xylopyranosyl units. The maximal catalytic efficiency (〈em〉K〈/em〉〈sub〉cat〈/sub〉/〈em〉K〈/em〉〈sub〉m〈/sub〉) was observed using 〈em〉p〈/em〉-nitrophenyl-α-〈span〉l〈/span〉-arabinofuranoside (pNPAF) as a substrate (205.0 s〈sup〉−1〈/sup〉 mM〈sup〉−1〈/sup〉), followed by using A〈sup〉3〈/sup〉X (22.8 s〈sup〉−1〈/sup〉 mM〈sup〉−1〈/sup〉), A〈sup〉2〈/sup〉XX (6.9 s〈sup〉−1〈/sup〉 mM〈sup〉−1〈/sup〉), and A〈sup〉2 + 3〈/sup〉XX (0.5 s〈sup〉−1〈/sup〉 mM〈sup〉−1〈/sup〉) as substrates. Moreover, the presence of Abf51 significantly stimulated the saccharification level of AX (18.5 g L〈sup〉−1〈/sup〉) up to six times along with a β-xylanase as well as a β-xylosidase. Interestingly, in our survey of top thermostable arabinofuranosidases, most members were found from GH51, probably due to their owning of (β/α)〈sub〉8〈/sub〉-barrel architectures. Our results suggested the great importance of GH51s as candidates for thermostable, versatile, and efficient arabinofuranosidases toward industry application.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Methods for heterologous protein production in 〈em〉Escherichia coli〈/em〉 have revolutionized biotechnology and the bioindustry. It is ultimately important to increase the amount of protein product from bacteria. To this end, a variety of tools, such as effective promoters, have been developed. Here, we present a versatile molecular tool based on a phenomenon termed “translation enhancement by a 〈em〉Dictyostelium〈/em〉 gene sequence” (“TED”) in 〈em〉E. coli〈/em〉. We found that protein expression was increased when a gene sequence of 〈em〉Dictyostelium discoideum〈/em〉 was placed upstream of the Shine–Dalgarno sequence located between the promoter and the initiation codon of a target gene. The most effective sequence among the genes examined was 〈em〉mlcR〈/em〉, which encodes the myosin regulatory light chain, a subunit of myosin II. Serial deletion analysis revealed that at least 10 bases of the 3′ end of the 〈em〉mlcR〈/em〉 gene enhanced the production of green fluorescent protein in cells. We applied this tool to a T7 expression system and found that the expression level of the proteins tested was increased when compared with the conventional method. Thus, current protein production systems can be improved by combination with TED.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉A vast array of plant-based compounds has enriched red biotechnology to serve the human health and food. A peculiar medicinal plant which was an element of traditional Chinese medicine for centuries as a liver and kidney tonic, for life longevity and hair blackening, is 〈em〉Polygonum multiflorum〈/em〉 Thunb. (PM) which is popularly known as “He shou wu” or “Fo-ti” and is rich in chemical components like stilbenes, quinones, and flavonoids which have been used as anti-aging, anti-alopecia, anti-cancer, anti-oxidative, anti-bacterial, anti-hyperlipidemia, anti-atherosclerosis, and immunomodulating and hepatoprotective agents in the modern medicine. The health benefits from PM are attained since long through commercial products such as PM root powder, extract, capsules, tincture, shampoo, and body sprays in the market. Currently, the production of these pharmaceuticals and functional foods possessing stilbenes, quinones, and flavonoids is through cell and organ cultures to meet the commercial demand. However, hepatotoxic effects of PM-based products are the stumbling blocks for its long-term usage. The current review encompasses a comprehensive account of bioactive compounds of PM roots, their biological activities as well as efficacy and toxicity issues of PM ingredients and future perspectives.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The biosynthetic pathway of 2,3-butanediol (2,3-BDO) production from pyruvate under anaerobic conditions includes three enzymes: acetolactate synthase (ALS), acetolactate decarboxylase (ALDC), and acetoin reductase (AR). Recently, in anaerobic hyperthermophilic 〈em〉Pyrococcus furiosus〈/em〉, it has been reported that acetoin, a precursor of 2,3-BDO, is produced from pyruvate by ALS through a temperature-dependent metabolic switch. In this study, we first attempted to produce 2,3-BDO from 〈em〉Thermococcus onnurineus〈/em〉 NA1 using a simple biosynthetic pathway by two enzymes (ALS and AR) at a high temperature. Two heterologous genes, acetolactate synthase (〈em〉alsS〈/em〉) from 〈em〉Pyrococcus〈/em〉 sp. NA2 and alcohol dehydrogenase (〈em〉adh〈/em〉) from 〈em〉T. guaymacensis〈/em〉, were introduced and expressed in 〈em〉T. onnurineus〈/em〉 NA1. The mutant strain produced approximately 3.3 mM 2,3-BDO at 80 °C. An acetyl-CoA synthetase IIIα (TON_1001) was further deleted to enhance 2,3-BDO production, and the mutant strain showed a 25% increase in the specific production of 2,3-BDO. Furthermore, when carbon monoxide (CO) gas was added as a reductant, specific production of 2,3-BDO increased by 45%. These results suggest a new biosynthetic pathway for 2,3-BDO and demonstrate the possibility of 〈em〉T. onnurineus〈/em〉 NA1 as a platform strain for 2,3-BDO production at high temperatures.〈/p〉