Directed Evolution of Methanococcus jannaschii Citramalate Synthase for Biosynthesis of 1-Propanol and 1-Butanol by Escherichia coli

• Published in: Applied and Environmental Microbiology Vol. 74; no. 24; pp. 7802 - 7808
• Main Authors: Atsumi, Shota, Liao, James C
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.12.2008; American Society for Microbiology (ASM)
• Subjects: 1-Butanol - metabolism; 1-Propanol - metabolism; Acetyltransferases; Acyltransferases - genetics; Acyltransferases - metabolism; Amino Acid Substitution; Biodiesel fuels; Biofuels; Biological and medical sciences; Biosynthetic Pathways - genetics; Biotechnology; Butyrates - metabolism; E coli; Enzyme Inhibitors - pharmacology; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genotype & phenotype; Glucose; Glucose - metabolism; Isoleucine - pharmacology; Methanococcus - enzymology; Methods. Procedures. Technologies; Microbiology; Mutagenesis; Mutation, Missense; Physiology and Biotechnology; Polymerase Chain Reaction; Protein engineering; Pyruvic Acid - metabolism; Temperature; Directed molecular evolution; Methanococcus; Archaeobacteria; Enzyme; Escherichia coli; Bacteria; Butanol; Biosynthesis; Methanococcaceae; Synthase; Enterobacteriaceae; Methanococcales
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.02046-08

Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we took advantage of the growth phenotype associated with 2-keto acid deficiency to construct a hyperproducer of 1-propanol and 1-butanol by evolving citramalate synthase (CimA) from Methanococcus jannaschii. This new pathway, which directly converts pyruvate to 2-ketobutyrate, bypasses threonine biosynthesis and represents the shortest keto acid-mediated pathway for producing 1-propanol and 1-butanol from glucose. Directed evolution of CimA enhanced the specific activity over a wide temperature range (30 to 70°C). The best CimA variant was found to be insensitive to feedback inhibition by isoleucine in addition to the improved activity. This CimA variant enabled 9- and 22-fold higher production levels of 1-propanol and 1-butanol, respectively, compared to the strain expressing the wild-type CimA. This work demonstrates (i) the first production of 1-propanol and 1-butanol using the citramalate pathway and (ii) the benefit of the 2-keto acid pathway that enables a growth-based evolutionary strategy to improve the production of non-growth-related products.