title: Dynamic control of ERG9 expression for improved amorpha-4,11-diene production in Saccharomyces cerevisiae.
abstract: BACKGROUND
  To achieve high-level production of non-native isoprenoid products, it requires the metabolic flux to be diverted from the production of sterols to the heterologous metabolic reactions. However, there are limited tools for restricting metabolic flux towards ergosterol synthesis. In the present study, we explored dynamic control of ERG9 expression using different ergosterol-responsive promoters to improve the production of non-native isoprenoids.
  
  
  RESULTS
  Several ergosterol-responsive promoters were identified using quantitative real-time PCR (qRT-PCR) analysis in an engineered strain with relatively high mevalonate pathway activity. We found mRNA levels for ERG11, ERG2 and ERG3 expression were significantly lower in the engineered strain over the reference strain BY4742, indicating these genes are transcriptionally down-regulated when ergosterol is in excess. Further replacement of the native ERG9 promoter with these ergosterol-responsive promoters revealed that all engineered strains improved amorpha-4,11-diene by 2~5-fold over the reference strain with ERG9 under its native promoter. The best engineered strain with ERG9 under the control of P ERG1 produced amorpha-4,11-diene to a titer around 350 mg/L after 96 h cultivation in shake-flasks.
  
  
  CONCLUSIONS
  We envision dynamic control at the branching step using feedback regulation at transcriptional level could serve as a generalized approach for redirecting the metabolic flux towards product-of-interest.
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore. cheyuan@nus.edu.sg.|Synthetic Biology Research Consortium, National University of Singapore, 28 Medical Drive, Singapore, 117456, Singapore. cheyuan@nus.edu.sg.
authors.forename: Jifeng
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore. cheching@nus.edu.sg.|Synthetic Biology Research Consortium, National University of Singapore, 28 Medical Drive, Singapore, 117456, Singapore. cheching@nus.edu.sg.|Singapore Institute of Technology, 10 Dover Drive, Singapore, 138683, Singapore. cheching@nus.edu.sg.
authors.forename: Chi-Bun
authors.initials: CB
authors.lastname: Ching
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004875:Ergosterol / Q000378:metabolism; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics / Q000378:metabolism*; D025461:Feedback, Physiological; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D060847:Metabolic Engineering / Q000379:methods; D000081223:Polycyclic Sesquiterpenes; D011401:Promoter Regions, Genetic / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012717:Sesquiterpenes / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D000081223:Polycyclic Sesquiterpenes; D029701:Saccharomyces cerevisiae Proteins; D012717:Sesquiterpenes; C515348:amorpha-4,11-diene; D003577:Cytochrome P-450 Enzyme System; C526348:Erg11 protein, S cerevisiae; C496127:ERG9 protein, S cerevisiae; D013186:Farnesyl-Diphosphate Farnesyltransferase; D004875:Ergosterol