Production of (R)-citramalate by engineered Saccharomyces cerevisiae

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
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引用次数: 0

Abstract

The budding yeast, Saccharomyces cerevisiae, has a high tolerance to organic acids and alcohols, and thus grows well under toxic concentrations of various compounds in the culture medium, potentially allowing for highly efficient compound production. (R)-citramalate is a raw material for methyl methacrylate and can be used as a metabolic intermediate in the biosynthesis of higher alcohols. (R)-citramalate is synthesized from pyruvate and acetyl-CoA. Unlike Escherichia coli, S. cerevisiae has organelles, and its intracellular metabolites are compartmentalized, preventing full use of intracellular acetyl-CoA. Therefore, in this study, to increase the amount of cytosolic acetyl-CoA for highly efficient production of (R)-citramalate, we inhibited the transport of cytosolic acetyl-CoA and pyruvate to the mitochondria. We also constructed a heterologous pathway to supply cytosolic acetyl-CoA. Additionally, we attempted to export (R)-citramalate from cells by expressing a heterologous dicarboxylate transporter gene. We evaluated the effects of these approaches on (R)-citramalate production and constructed a final strain by combining these positive approaches. The resulting strain produced 16.5 mM (R)-citramalate in batch culture flasks. This is the first report of (R)-citramalate production by recombinant S. cerevisiae, and the (R)-citramalate production by recombinant yeast achieved in this study was the highest reported to date.

利用工程酿酒酵母生产(R)-柠檬酸盐
芽殖酵母(Saccharomyces cerevisiae)对有机酸和酒精有很强的耐受性,因此在培养基中各种化合物浓度有毒的情况下也能很好地生长,从而有可能实现高效的化合物生产。(R)-柠檬醛酸酯是甲基丙烯酸甲酯的原料,可用作高级醇类生物合成的代谢中间体。(R)-柠檬醛酸由丙酮酸和乙酰-CoA 合成。与大肠杆菌不同,酿酒酵母具有细胞器,其胞内代谢物被分隔开来,无法充分利用胞内乙酰-CoA。因此,在本研究中,为了增加细胞内乙酰-CoA 的含量以高效生产(R)-柠檬酸,我们抑制了细胞内乙酰-CoA 和丙酮酸向线粒体的运输。我们还构建了一条异源途径来提供细胞质乙酰-CoA。此外,我们还尝试通过表达异源二羧酸盐转运体基因从细胞中输出 (R)-citramalate 。我们评估了这些方法对(R)-柠檬醛酸生产的影响,并结合这些积极的方法构建了最终菌株。由此产生的菌株在批量培养瓶中产生了 16.5 mM (R)-柠檬醛酸。这是重组酿酒酵母生产(R)-柠檬醛酸的首次报道,本研究中重组酵母的(R)-柠檬醛酸产量是迄今为止报道的最高产量。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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