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

Peng, Bingyin, Plan, Manuel R., Carpenter, Alexander, Nielsen, Lars K. and Vickers, Claudia E. (2017) Coupling gene regulatory patterns to bioprocess conditions to optimize synthetic metabolic modules for improved sesquiterpene production in yeast. Biotechnology for Biofuels, 10 1: 43. doi:10.1186/s13068-017-0728-x


Author Peng, Bingyin
Plan, Manuel R.
Carpenter, Alexander
Nielsen, Lars K.
Vickers, Claudia E.
Title Coupling gene regulatory patterns to bioprocess conditions to optimize synthetic metabolic modules for improved sesquiterpene production in yeast
Journal name Biotechnology for Biofuels   Check publisher's open access policy
ISSN 1754-6834
Publication date 2017-02-21
Year available 2017
Sub-type Article (original research)
DOI 10.1186/s13068-017-0728-x
Open Access Status DOI
Volume 10
Issue 1
Start page 43
Total pages 16
Place of publication London, United Kingdom
Publisher BioMed Central
Language eng
Subject 1305 Biotechnology
2402 Applied Microbiology and Biotechnology
2105 Renewable Energy, Sustainability and the Environment
2100 Energy
2308 Management, Monitoring, Policy and Law
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 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 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 were achieved, depending on the conditions. Conclusion: 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.
Formatted 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 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.

Conclusion: 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.
Keyword Saccharomyces cerevisiae
Sesquiterpene
Synthetic biology
Metabolic engineering
Microbial cell factories
Transcription regulation
Mevalonate pathway
Fed-batch cultivation
Overflow metabolism
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ
Additional Notes http://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-017-0728-x

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
Australian Institute for Bioengineering and Nanotechnology Publications
 
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Created: Wed, 01 Mar 2017, 11:23:34 EST by Chris Ende on behalf of Aust Institute for Bioengineering & Nanotechnology