Abstract
Due to their high secretion capacity, Gram-positive bacteria from the genus Bacillus are important expression hosts for the high-yield production of enzymes in industrial biotechnology; however, to date, strains from only few Bacillus species are used for enzyme production at industrial scale. Herein, we introduce Paenibacillus polymyxa DSM 292, a member of a different genus, as a novel host for secretory protein production. The model gene cel8A from Clostridium thermocellum was chosen as an easily detectable reporter gene with industrial relevance to demonstrate heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A protein was increased by optimizing the expression medium and testing several promoter sequences in the expression plasmid pBACOV. Quantitative mass spectrometry was used to analyze the secretome in order to identify promising new promoter sequences from the P. polymyxa genome itself. The most abundantly secreted host proteins were identified, and the promoters regulating the expression of their corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from Bacillus subtilis and Bacillus megaterium. The best result was achieved with the promoter for the hypothetical protein PPOLYM_03468 from P. polymyxa. In combination with the optimized expression medium, this promoter enabled the production of 5475 U/l of Cel8A, which represents a 6.2-fold increase compared to the reference promoter PaprE. The set of promoters described in this work covers a broad range of promoter strengths useful for heterologous expression in the new host P. polymyxa.
Similar content being viewed by others
References
Ahmann D, Dorgan JR (2007) Bioengineering for pollution prevention through development of biobased energy and materials state of the science report. Ind Biotechnol 3:218–259. https://doi.org/10.1089/ind.2007.3.218
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Alzari PM, Souchon H, Dominguez R (1996) The crystal structure of endoglucanase CelA, a family 8 glycosyl hydrolase from Clostridium thermocellum. Structure 4:265–275
Bayer EA, Lamed R, Himmel ME (2007) The potential of cellulases and cellulosomes for cellulosic waste management. Curr Opin Biotechnol 18:237–245. https://doi.org/10.1016/j.copbio.2007.04.004
Bertani G (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300
Bien TLT, Tsuji S, Tanaka K, Takenaka S, Yoshida K (2014) Secretion of heterologous thermostable cellulases in Bacillus subtilis. J Gen Appl Microbiol 60:175–182. https://doi.org/10.2323/jgam.60.175
Brockmeier U, Caspers M, Freudl R, Jockwer A, Noll T, Eggert T (2006) Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. J Mol Biol 362:393–402. https://doi.org/10.1016/j.jmb.2006.07.034
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372. https://doi.org/10.1038/nbt.1511
Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10:1794–1805. https://doi.org/10.1021/pr101065j
Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M (2014) Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 13:2513–2526. https://doi.org/10.1074/mcp.M113.031591
Creecy JP, Conway T (2015) Quantitative bacterial transcriptomics with RNA-seq. Curr Opin Microbiol 23:133–140. https://doi.org/10.1016/J.MIB.2014.11.011
Dahl MK, Schmiedel D, Hillen W (1995) Glucose and glucose-6-phosphate interaction with Xyl repressor proteins from Bacillus spp. may contribute to regulation of xylose utilization. J Bacteriol 177:5467–5472
Degering C, Eggert T, Puls M, Bongaerts J, Evers S, Maurer K-H, Jaeger K-E (2010) Optimization of protease secretion in Bacillus subtilis and Bacillus licheniformis by screening of homologous and heterologous signal peptides. Appl Environ Microbiol 76:6370–6376. https://doi.org/10.1128/AEM.01146-10
Fang TJ, Liao B-C, Lee S-C (2010) Enhanced production of xylanase by Aspergillus carneus M34 in solid-state fermentation with agricultural waste using statistical approach. New Biotechnol 27:25–32. https://doi.org/10.1016/J.NBT.2009.09.008
Farhat-Khemakhem A, Farhat MB, Boukhris I, Bejar W, Bouchaala K, Kammoun R, Maguin E, Bejar S, Chouayekh H (2012) Heterologous expression and optimization using experimental designs allowed highly efficient production of the PHY US417 phytase in Bacillus subtilis 168. AMB Express 2:10. https://doi.org/10.1186/2191-0855-2-10
Ferrari E, Henner DJ, Perego M, Hoch JA (1988) Transcription of Bacillus subtilis subtilisin and expression of subtilisin in sporulation mutants. J Bacteriol 170:289–295
Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345. https://doi.org/10.1038/nmeth.1318
Görke B, Stülke J (2008) Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol 6:613–624. https://doi.org/10.1038/nrmicro1932
Grady EN, MacDonald J, Liu L, Richman A, Yuan Z-C (2016) Current knowledge and perspectives of Paenibacillus: a review. Microb Cell Factories 15:203. https://doi.org/10.1186/s12934-016-0603-7
Harwood CR (1992) Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends Biotechnol 10:247–256. https://doi.org/10.1016/0167-7799(92)90233-L
Häßler T, Schieder D, Pfaller R, Faulstich M, Sieber V (2012) Enhanced fed-batch fermentation of 2,3-butanediol by Paenibacillus polymyxa DSM 365. Bioresour Technol 124:237–244. https://doi.org/10.1016/j.biortech.2012.08.047
Heinze S, Kornberger P, Graetz C, Schwarz WH, Zverlov VV, Liebl W (2018) Transmating: conjugative transfer of a new broad host range expression vector to various Bacillus species using a single protocol. BMC Microbiol 18:56. https://doi.org/10.1186/s12866-018-1198-4
Jan J, Valle F, Bolivar F, Merino E (2000) Characterization of the 5′ subtilisin (aprE) regulatory region from Bacillus subtilis. FEMS Microbiol Lett 183:9–14. https://doi.org/10.1111/j.1574-6968.2000.tb08926.x
Joliff G, Edelman A, Klier A, Rapoport G (1989) Inducible secretion of a cellulase from Clostridium thermocellum in Bacillus subtilis. Appl Environ Microbiol 55:2739–2744
Küppers T, Steffen V, Hellmuth H, O’Connell T, Bongaerts J, Maurer K-H, Wiechert W (2014) Developing a new production host from a blueprint: Bacillus pumilus as an industrial enzyme producer. Microb Cell Factories 13:46. https://doi.org/10.1186/1475-2859-13-46
Lee S-J, Pan J-G, Park S-H, Choi S-K (2010) Development of a stationary phase-specific autoinducible expression system in Bacillus subtilis. J Biotechnol 149:16–20. https://doi.org/10.1016/j.jbiotec.2010.06.021
Leis B, Held C, Bergkemper F, Dennemarck K, Steinbauer R, Reiter A, Mechelke M, Moerch M, Graubner S, Liebl W, Schwarz WH, Zverlov VV (2017) Comparative characterization of all cellulosomal cellulases from Clostridium thermocellum reveals high diversity in endoglucanase product formation essential for complex activity. Biotechnol Biofuels 10:240. https://doi.org/10.1186/s13068-017-0928-4
Malten M, Hollmann R, Deckwer W-D, Jahn D (2005) Production and secretion of recombinant Leuconostoc mesenteroides dextransucrase DsrS in Bacillus megaterium. Biotechnol Bioeng 89:206–218. https://doi.org/10.1002/bit.20341
Narasimhan A, Shivakumar S (2012) Optimization of chitinase produced by a biocontrol strain of Bacillus subtilis using Plackett–Burman design. Eur J Exp Biol 2:861–865
Nicolas P, Mader U, Dervyn E, Rochat T, Leduc A, Pigeonneau N, Bidnenko E, Marchadier E, Hoebeke M, Aymerich S, Becher D, Bisicchia P, Botella E, Delumeau O, Doherty G, Denham EL, Fogg MJ, Fromion V, Goelzer A, Hansen A, Hartig E, Harwood CR, Homuth G, Jarmer H, Jules M, Klipp E, Le Chat L, Lecointe F, Lewis P, Liebermeister W, March A, Mars RAT, Nannapaneni P, Noone D, Pohl S, Rinn B, Rugheimer F, Sappa PK, Samson F, Schaffer M, Schwikowski B, Steil L, Stulke J, Wiegert T, Devine KM, Wilkinson AJ, Maarten van Dijl J, Hecker M, Volker U, Bessieres P, Noirot P (2012) Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis. Science 335:1103–1106. https://doi.org/10.1126/science.1206848
Nijland R, Lindner C, van Hartskamp M, Hamoen LW, Kuipers OP (2007) Heterologous production and secretion of Clostridium perfringens β-toxoid in closely related Gram-positive hosts. J Biotechnol 127:361–372. https://doi.org/10.1016/j.jbiotec.2006.07.014
Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786. https://doi.org/10.1038/nmeth.1701
Rütering M, Cress BF, Schilling M, Rühmann B, Koffas MAG, Sieber V, Schmid J (2017) Tailor-made exopolysaccharides—CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa. Synth Biol 2:ysx007. https://doi.org/10.1093/synbio/ysx007
Rygus T, Hillen W (1991) Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon. Appl Microbiol Biotechnol 35:594–599. https://doi.org/10.1007/BF00169622
Sawhney N, Crooks C, Chow V, Preston JF, St John FJ (2016) Genomic and transcriptomic analysis of carbohydrate utilization by Paenibacillus sp. JDR-2: systems for bioprocessing plant polysaccharides. BMC Genomics 17:131. https://doi.org/10.1186/s12864-016-2436-5
Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50:1–17
Schumann W (2007) Production of recombinant proteins in Bacillus subtilis. Adv Appl Microbiol 62:137–189. https://doi.org/10.1016/S0065-2164(07)62006-1
Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M (2011) Global quantification of mammalian gene expression control. Nature 473:337–342. https://doi.org/10.1038/nature10098
Schwarz WH (2001) The cellulosome and cellulose degradation by anaerobic bacteria. Appl Microbiol Biotechnol 56:634–649
Schwarz WH, Gräbnitz F, Staudenbauer WL (1986) Properties of a Clostridium thermocellum endoglucanase produced in Escherichia coli. Appl Environ Microbiol 51:1293–1299
Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860. https://doi.org/10.1038/nprot.2006.468
Shoham Y, Lamed R, Bayer EA (1999) The cellulosome concept as an efficient microbial strategy for the degradation of insoluble polysaccharides. Trends Microbiol 7:275–281
Singh V, Haque S, Niwas R, Srivastava A, Pasupuleti M, Tripathi CKM (2017) Strategies for fermentation medium optimization: an in-depth review. Front Microbiol 7:2087. https://doi.org/10.3389/fmicb.2016.02087
Solovyev V, Salamov A (2011) Automatic annotation of microbial genomes and metagenomic sequences. In: Li RW (ed) Metagenomics and its applications in agriculture, biomedicine and environmental studies. Nova Science Publishers, New York, pp 61–78
Song B-H, Neuhard J (1989) Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase. MGG Mol Gen Genet 216:462–468. https://doi.org/10.1007/BF00334391
Song Y, Nikoloff JM, Zhang D (2015) Improving protein production on the level of regulation of both expression and secretion pathways in Bacillus subtilis. J Microbiol Biotechnol 25:963–977. https://doi.org/10.4014/jmb.1501.01028
Song Y, Nikoloff JM, Fu G, Chen J, Li Q, Xie N, Zheng P, Sun J, Zhang D (2016) Promoter screening from Bacillus subtilis in various conditions hunting for synthetic biology and industrial applications. PLoS One 11:e0158447. https://doi.org/10.1371/journal.pone.0158447
Soutschek-Bauer E, Staudenbauer WL (1987) Synthesis and secretion of a heat-stable carboxymethylcellulase from Clostridium thermocellum in Bacillus subtilis and Bacillus stearothermophilus. Mol Gen Genet 208:537–541
Stammen S, Müller BK, Korneli C, Biedendieck R, Gamer M, Franco-Lara E, Jahn D (2010) High-yield intra- and extracellular protein production using Bacillus megaterium. Appl Environ Microbiol 76:4037–4046. https://doi.org/10.1128/AEM.00431-10
Valle F, Ferrari E (1989) Subtilisin: a redundantly temporally regulated gene? In: Smith I, Slepecky RA, Setlow P (eds) Regulation of procaryotic development. American Society for Microbiology, Washington, DC, pp 131–146
Wang P-Z, Doi RH (1984) Overlapping promoters transcribed by Bacillus subtilis sigma55 and sigma37 RNA polymerase holoenzymes during growth and stationary phases. J Biol Chem 259:8619–8625
Wood TM, Bhat KM (1988) Methods for measuring cellulase activities. Methods Enzymol 160:87–112. https://doi.org/10.1016/0076-6879(88)60109-1
Yin Y, Mao X, Yang J, Chen X, Mao F, Xu Y (2012) dbCAN: a web resource for automated carbohydrate-active enzyme annotation. Nucleic Acids Res 40:W445–W451. https://doi.org/10.1093/nar/gks479
Yu X, Xu J, Liu X, Chu X, Wang P, Tian J, Wu N, Fan Y (2015) Identification of a highly efficient stationary phase promoter in Bacillus subtilis. Sci Rep 5:18405. https://doi.org/10.1038/srep18405
Zverlov VV, Schwarz WH (2004) The Clostridium thermocellum cellulosome -the paradigm of a multienzyme complex. In: Ohmiya K, Sakka K, Karita S, Kimura T, Sakka M, YO (eds) Biotechnology of lignocellulose degradation and biomass utilization. Uni Pub. Co. Ltd., Tokyo, pp 137–147
Acknowledgments
The authors thank Patricia Krähe and Benedikt Leis for preparing and providing the sample of Cel8A, produced in E. coli, which was used to establish the reference curve for the Azo-CMC assay. We would like to thank Hermine Kienberger for her excellent technical assistance in proteomic sample preparation.
Funding
This work was funded by the German Federal Ministry of Education and Research (FKZ 031A555, Bioeconomy International 2014).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Rights and permissions
About this article
Cite this article
Heinze, S., Zimmermann, K., Ludwig, C. et al. Evaluation of promoter sequences for the secretory production of a Clostridium thermocellum cellulase in Paenibacillus polymyxa. Appl Microbiol Biotechnol 102, 10147–10159 (2018). https://doi.org/10.1007/s00253-018-9369-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-018-9369-7