{"title":"Production of (R)-citramalate by engineered Saccharomyces cerevisiae","authors":"Ryosuke Mitsui , Akihiko Kondo , Tomokazu Shirai","doi":"10.1016/j.mec.2024.e00247","DOIUrl":null,"url":null,"abstract":"<div><p>The budding yeast, <em>Saccharomyces cerevisiae</em>, 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. (<em>R</em>)-citramalate is a raw material for methyl methacrylate and can be used as a metabolic intermediate in the biosynthesis of higher alcohols. (<em>R</em>)-citramalate is synthesized from pyruvate and acetyl-CoA. Unlike <em>Escherichia coli</em>, <em>S. cerevisiae</em> 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 (<em>R</em>)-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 (<em>R</em>)-citramalate from cells by expressing a heterologous dicarboxylate transporter gene. We evaluated the effects of these approaches on (<em>R</em>)-citramalate production and constructed a final strain by combining these positive approaches. The resulting strain produced 16.5 mM (<em>R</em>)-citramalate in batch culture flasks. This is the first report of (<em>R</em>)-citramalate production by recombinant <em>S. cerevisiae</em>, and the (<em>R</em>)-citramalate production by recombinant yeast achieved in this study was the highest reported to date.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"19 ","pages":"Article e00247"},"PeriodicalIF":3.7000,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000166/pdfft?md5=8e77960467f6df90982ae565f50fc7ce&pid=1-s2.0-S2214030124000166-main.pdf","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Metabolic Engineering Communications","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2214030124000166","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
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.
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.