title: Coupling gene regulatory patterns to bioprocess conditions to optimize synthetic metabolic modules for improved sesquiterpene production in yeast.
abstract: BACKGROUND
  Assembly of heterologous metabolic pathways is commonly required to generate microbial cell factories for industrial production of both commodity chemicals (including biofuels) and high-value chemicals. Promoter-mediated transcriptional regulation coordinates the expression of the individual components of these heterologous pathways. Expression patterns vary during culture as conditions change, and this can influence yeast physiology and productivity in both positive and negative ways. Well-characterized strategies are required for matching transcriptional regulation with desired output across changing culture conditions.
  
  
  RESULTS
  Here, constitutive and inducible regulatory mechanisms were examined to optimize synthetic isoprenoid metabolic pathway modules for production oftrans-nerolidol, an acyclic sesquiterpene alcohol, in yeast. The choice of regulatory system significantly affected physiological features (growth and productivity) over batch cultivation. Use of constitutive promoters resulted in poor growth during the exponential phase. Delaying expression of the assembled metabolic modules using the copper-inducibleCUP1promoter resulted in a 1.6-fold increase in the exponential-phase growth rate and a twofold increase in productivity in the post-exponential phase. However, repeated use of theCUP1promoter in multiple expression cassettes resulted in genetic instability. A diauxie-inducible expression system, based on an engineeredGALregulatory circuit and a set of four differentGALpromoters, was characterized and employed to assemble nerolidol synthetic metabolic modules. Nerolidol production was further improved by 60% to 392 mg L-1using this approach. Various carbon source systems were investigated in batch/fed-batch cultivation to regulate induction through theGALsystem; final nerolidol titres of 4-5.5 g L-1were achieved, depending on the conditions.
  
  
  CONCLUSIONS
  Direct comparison of different transcriptional regulatory mechanisms clearly demonstrated that coupling the output strength to the fermentation stage is important to optimize the growth fitness and overall productivities of engineered cells in industrially relevant processes. Applying different well-characterized promoters with the same induction behaviour mitigates against the risks of homologous sequence-mediated genetic instability. Using these approaches, we significantly improved sesquiterpene production in yeast.
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072 Australia.
authors.forename: Bingyin
authors.initials: B
authors.lastname: Peng
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072 Australia.|Metabolomics Australia (Queensland Node), The University of Queensland, St. Lucia, QLD 4072 Australia.
authors.forename: Manuel R
authors.initials: MR
authors.lastname: Plan
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072 Australia.
authors.forename: Alexander
authors.initials: A
authors.lastname: Carpenter
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072 Australia.
authors.forename: Lars K
authors.initials: LK
authors.lastname: Nielsen
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072 Australia.
authors.forename: Claudia E
authors.identifier: 0000-0002-0792-050X
authors.initials: CE
authors.lastname: Vickers
keywords: Fed-batch cultivation; Metabolic engineering; Mevalonate pathway; Microbial cell factories; Overflow metabolism; Saccharomyces cerevisiae; Sesquiterpene; Synthetic biology; Transcription regulation