ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production

Ahmed Zahoor,Katrin Messerschmidt,Simon Boecker,Steffen Klamt

doi:10.1186/s13068-020-01822-9

Abstract

BackgroundEnforced ATP wasting has been recognized as a promising metabolic engineering strategy to enhance the microbial production of metabolites that are coupled to ATP generation. It also appears to be a suitable approach to improve production of ethanol by Saccharomyces cerevisiae. In the present study, we constructed different S. cerevisiae strains with heterologous expression of genes of the ATP-hydrolyzing F1-part of the ATPase enzyme to induce enforced ATP wasting and quantify the resulting effect on biomass and ethanol formation.ResultsIn contrast to genomic integration, we found that episomal expression of the αβγ subunits of the F1-ATPase genes of Escherichia coli in S. cerevisiae resulted in significantly increased ATPase activity, while neither genomic integration nor episomal expression of the β subunit from Trichoderma reesei could enhance ATPase activity. When grown in minimal medium under anaerobic growth-coupled conditions, the strains expressing E. coli’s F1-ATPase genes showed significantly improved ethanol yield (increase of 10% compared to the control strain). However, elevated product formation reduces biomass formation and, therefore, volumetric productivity. We demonstrate that this negative effect can be overcome under growth-decoupled (nitrogen-starved) operation with high and constant biomass concentration. Under these conditions, which mimic the second (production) phase of a two-stage fermentation process, the ATPase-expressing strains showed significant improvement in volumetric productivity (up to 111%) compared to the control strain.ConclusionsOur study shows that expression of genes of the F1-portion of E. coli’s ATPase induces ATPase activity in S. cerevisiae and can be a promising way to improve ethanol production. This ATP-wasting strategy can be easily applied to other metabolites of interest, whose formation is coupled to ATP generation.

Highlights

Enforced ATP wasting has been recognized as a promising metabolic engineering strategy to enhance the microbial production of metabolites that are coupled to ATP generation
Heterologous expression of ATPase genes in S. cerevisiae The objective of this study was to implement ATP wasting in S. cerevisiae by heterologous expression of genes of an ATPase to improve ethanol production by the engineered strains
We have successfully constructed yeast strains overexpressing ATPase genes and showed that the resulting increased ATPase activity leads to beneficial effects on ethanol yield or volumetric ethanol production, depending on the chosen process type

Summary

Introduction

Enforced ATP wasting has been recognized as a promising metabolic engineering strategy to enhance the microbial production of metabolites that are coupled to ATP generation. Saccharomyces cerevisiae is the predominant organism used for bioethanol production and there is great interest in generating superior production strains [3, 4] since merely a 1% increase in ethanol yield can already save the industry millions of dollars annually [5]. Several approaches have been implemented to generate improved ethanol-producing S. cerevisiae strains. Many of these strategies seek to either reduce production of ATP or to increase ATP turnover (enforced ATP wasting) (Table 1). The rationale behind this approach is that ethanol synthesis and ATP production are coupled in both directions (ethanol synthesis delivers ATP and (balanced) ATP synthesis implies synthesis of ethanol) and a loss of ATP must be counter-balanced by the cell with increased synthesis of ATP and of ethanol. One of the two enzymes required for this pathway (6-phosphogluconate dehydratase) failed to show activity despite several attempts to improve the iron–sulfur-cluster assembly which is critical for functioning of the enzyme [6]

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