Dynamic control of ERG9 expression for improved amorpha-4,11-diene production in Saccharomyces cerevisiae.

Jifeng Yuan,Chi-Bun Ching

doi:10.1186/s12934-015-0220-x

Jifeng Yuan, Chi-Bun Ching

Open Access

https://doi.org/10.1186/s12934-015-0220-x

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Abstract

BackgroundTo 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.ResultsSeveral 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 PERG1 produced amorpha-4,11-diene to a titer around 350 mg/L after 96 h cultivation in shake-flasks.ConclusionsWe 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.

Highlights

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
In order to produce these molecules at industrial levels, pathway genes involved in the synthesis of these molecules must be expressed at appropriately balanced levels, to avoid the accumulation of toxic intermediates or bottlenecks that result in growth inhibition or suboptimal yields [1,2,3]
We found ERG11, ERG2 and ERG3 were transcriptionally down-regulated in our engineered strain when compared to the reference strain of BY4742, whereas mRNA level of ERG1 gene in both strains remained relatively low

Summary

Introduction

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. We explored dynamic control of ERG9 expression using different ergosterol-responsive promoters to improve the production of non-native isoprenoids. High-level production of non-native isoprenoid products requires the metabolic flux to be diverted from the production of. As ergosterol fulfills several essential functions and each requires optimal sterol concentrations, synthesis of sterols in yeast is tightly regulated. In budding yeast, it requires thirteen-enzymatic steps to synthesize ergosterol from farnesyl diphosphate – a precursor from the mevalonate pathway (Figure 1). ERG9 expression in yeast was reported to be positively and negatively regulated by diverse factors such as the heme activator

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