Coupling gene regulatory patterns to bioprocess conditions to optimize synthetic metabolic modules for improved sesquiterpene production in yeast

Abstract

BackgroundAssembly 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.ResultsHere, constitutive and inducible regulatory mechanisms were examined to optimize synthetic isoprenoid metabolic pathway modules for production of trans-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-inducible CUP1 promoter 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 the CUP1 promoter in multiple expression cassettes resulted in genetic instability. A diauxie-inducible expression system, based on an engineered GAL regulatory circuit and a set of four different GAL promoters, was characterized and employed to assemble nerolidol synthetic metabolic modules. Nerolidol production was further improved by 60% to 392 mg L−1 using this approach. Various carbon source systems were investigated in batch/fed-batch cultivation to regulate induction through the GAL system; final nerolidol titres of 4–5.5 g L−1 were achieved, depending on the conditions.ConclusionDirect 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0728-x) contains supplementary material, which is available to authorized users.

Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae

“…Two-phase flask cultivation was performed as described previously (Peng et al, 2017a;Peng et al, 2017b;Vickers et al, 2015b). Synthetic minimal (SM) medium (containing 6.7 g L -1 yeast nitrogen base, YNB, Sigma-Aldrich #Y0626; pH 6.0) with 20 g L -1 glucose as the carbon source was used (SM-glucose).…”

“…An HPLC method, modified from a previous study (Peng et al, 2017a), was used to analyse geraniol, linalool, trans,trans-farnesol, trans-nerolidol, geranylgeraniol, and geranyllinalool.…”

“…The promoters used to drivheterologous genes in the above constructs have decreased expression activities in the post-exponential phase (Peng et al, 2015), resulting in decreased productivities (Peng et al, 2017a). We have previously shown that sesquiterpene productivity can be improved by using diauxie-inducible GAL promoters in a gal80Δ background strain instead of constitutive promoters (Peng et al, 2017a). When GAL promoters were used, the resulting strain N391DA produced 400 mg L -1 nerolidol in flask cultivations (Peng et al, 2017a).…”

“…Previously, enzymes from the mevalonate (MVA) pathway were over-expressed to increase precursor flux for terpene synthesis (Figure 1) (Peng et al, 2017a;Peng et al, 2017b;Vickers et al, 2017). A dedicated GPP synthase has been used to increase the GPP pool for monoterpene production (Alonso-Gutierrez et al, 2013;Amiri et al, 2016;Cao et al, Liu et al, 2016;Rico et al, 2010;Sarria et al, 2014;Willrodt et al, 2014;Zhang et al, 2014).…”

“…This work pPMVAd41 pRS424: (-152,-1)::HIS3-T EFM1 <EfmvaS<P GAL1 -P GAL10 >ACS2>T ACS2 -P GAL2 > EfmvaE >T EBS1 -P GAL7 (Peng et al, 2017a) pdc5 (-31,94): Terpene-producing strains were constructed from S. cerevisiae CEN.PK2-1C by transforming plasmid DNA or PCR fragments using the LiAc/PEG/ssDNA method (Gietz and Schiestl, 2007). The URA3 genotype was generated by transforming the URA3 fragment amplified from CEN.PK112-7D genomic DNA.…”