Metabolic engineering of a synergistic pathway for n-butanol production in Saccharomyces cerevisiae.

Shuobo Shi,Hongfang Zhang,Zihe Liu,Huimin Zhao,Tong Si,Ee Lui Ang

doi:10.1038/srep25675

Abstract

n-Butanol has several favourable properties as an advanced fuel or a platform chemical. Bio-based production of n-butanol is becoming increasingly important for sustainable chemical industry. Synthesis of n-butanol can be achieved via more than one metabolic pathway. Here we report the metabolic engineering of Saccharomyces cerevisiae to produce n-butanol through a synergistic pathway: the endogenous threonine pathway and the introduced citramalate pathway. Firstly, we characterized and optimized the endogenous threonine pathway; then, a citramalate synthase (CimA) mediated pathway was introduced to construct the synergistic pathway; next, the synergistic pathway was optimized by additional overexpression of relevant genes identified previously; meanwhile, the n-butanol production was also improved by overexpression of keto-acid decarboxylases (KDC) and alcohol dehydrogenase (ADH). After combining these strategies with co-expression of LEU1 (two copies), LEU4, LEU2 (two copies), LEU5, CimA, NFS1, ADH7 and ARO10*, we achieved an n-butanol production of 835 mg/L in the final engineered strain, which is almost 7-fold increase compared to the initial strain. Furthermore, the production showed a 3-fold of the highest titer ever reported in yeast. Therefore, the engineered yeast strain represents a promising alternative platform for n-butanol production.

Highlights

The endogenous n-butanol production is dependent on the catabolism of threonine (Fig. 1A), and it was switched on by introduction of a single gene deletion adh1Δ with the n-butanol production at 120 mg/L using resting cells[18]
To increase the cellular supply of threonine, five genes encoding for the enzymes responsible for converting aspartic acid to threonine (i.e., HOM3, HOM2, HOM6, THR1 and THR4) were overexpressed on a multi-copy plasmid pRS426-THR to generate the strain THR5
It has been reported that the HOM3 gene which encodes an aspartate kinase, plays a crucial role in the regulation of the metabolic flux that leads to threonine biosynthesis[26,27]

Summary

Introduction

There are two pathways leading to the synthesis of the common intermediate α -ketobutyrate: the threonine pathway[18,19,20] and the citramalate pathway[20,21] Both pathways have been used in E. coli for the production of n-butanol and n-propanol[19,20,21], and there is no report about the adoption of the citramalate pathway in yeast. The production of n-butanol in this yeast is synthesized from two parallel pathways: threonine based keto-acid pathway and Clostridia based ABE pathway These studies highlighted the importance of ADH1 deletion and incorporation of synergy into the design principle of n-butanol producer. Subsequent overexpression of KDC and alcohol dehydrogenase (ADH) further increased n-butanol production By combining these modifications, the final engineered yeast strain produced the highest reported n-butanol titer in S. cerevisiae (835 mg/L) with a yield of 42 mg/g glucose in anaerobic glass tubes. When cultivated in a bioreactor, the strain could produce up to 1.05 g/L n-butanol

Methods

Results

Discussion

Conclusion