ALBERT

Description: Abstract Programmable transcriptional regulation is a powerful tool to study gene functions. Current methods to selectively regulate target genes are mainly based on promoter exchange or on overexpressing transcriptional activators. To expand the discovery toolbox, we designed a dCas9-based RNA-guided synthetic transcription activation system for Aspergillus nidulans that uses enzymatically disabled “dead” Cas9 fused to three consecutive activation domains (VPR-dCas9). The dCas9-encoding gene is under the control of an estrogen-responsive promoter to allow induction timing and to avoid possible negative effects by strong constitutive expression of the highly active VPR domains. Especially in silent genomic regions, facultative heterochromatin and strictly positioned nucleosomes can constitute a relevant obstacle to the transcriptional machinery. To avoid this negative impact and to facilitate optimal positioning of RNA-guided VPR-dCas9 to targeted promoters, we have created a genome-wide nucleosome map from actively growing cells and stationary cultures to identify the cognate nucleosome-free regions (NFRs). Based on these maps, different single-guide RNAs (sgRNAs) were designed and tested for their targeting and activation potential. Our results demonstrate that the system can be used to regulate several genes in parallel and, depending on the VPR-dCas9 positioning, expression can be pushed to very high levels. We have used the system to turn on individual genes within two different biosynthetic gene clusters (BGCs) which are silent under normal growth conditions. This method also opens opportunities to stepwise activate individual genes in a cluster to decipher the correlated biosynthetic pathway. Keypoints • An inducible RNA-guided transcriptional regulator based on VPR-dCas9 was established in Aspergillus nidulans. • Genome-wide nucleosome positioning maps were created that facilitate sgRNA positioning. • The system was successfully applied to activate genes within two silent biosynthetic gene clusters.

Description: Coenzyme A (CoA) and its derivatives such as acetyl-CoA are essential metabolites for several biosynthetic reactions. In the yeast S. cerevisiae, five enzymes (encoded by essential genes CAB1-CAB5; coenzyme A biosynthesis) are required to perform CoA biosynthesis from pantothenate, cysteine, and ATP. Similar to enzymes from other eukaryotes, yeast pantothenate kinase (PanK, encoded by CAB1) turned out to be inhibited by acetyl-CoA. By genetic selection of intragenic suppressors of a temperature-sensitive cab1 mutant combined with rationale mutagenesis of the presumed acetyl-CoA binding site within PanK, we were able to identify the variant CAB1 W331R, encoding a hyperactive PanK completely insensitive to inhibition by acetyl-CoA. Using a versatile gene integration cassette containing the TPI1 promoter, we constructed strains overexpressing CAB1 W331R in combination with additional genes of CoA biosynthesis (CAB2, CAB3, HAL3, CAB4, and CAB5). In these strains, the level of CoA nucleotides was 15-fold increased, compared to a reference strain without additional CAB genes. Overexpression of wild-type CAB1 instead of CAB1 W331R turned out as substantially less effective (fourfold increase of CoA nucleotides). Supplementation of overproducing strains with additional pantothenate could further elevate the level of CoA (2.3-fold). Minor increases were observed after overexpression of FEN2 (encoding a pantothenate permease) and deletion of PCD1 (CoA-specific phosphatase). We conclude that the strategy described in this work may improve the efficiency of biotechnological applications depending on acetyl-CoA.Key points• A gene encoding a hyperactive yeast pantothenate kinase (PanK) was constructed.• Overexpression of CoA biosynthetic genes elevated CoA nucleotides 15-fold.• Supplementation with pantothenate further increased the level of CoA nucleotides.