Engineering a Coenzyme A Detour To Expand the Product Scope and Enhance the Selectivity of the Ehrlich Pathway

• Published in: ACS synthetic biology Vol. 7; no. 12; pp. 2758 - 2764
• Main Authors: Black, William B, King, Edward, Wang, Yixi, Jenic, Ana, Rowley, Andrew T, Seki, Kosuke, Luo, Ray, Li, Han
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.12.2018
• Subjects: Aldehyde Dehydrogenase - genetics; Aldehyde Dehydrogenase - metabolism; Coenzyme A - metabolism; Escherichia coli - metabolism; Intramolecular Transferases - genetics; Intramolecular Transferases - metabolism; Lactobacillus brevis - enzymology; Metabolic Engineering - methods; Pentanols - chemistry; Pentanols - metabolism; Plasmids - genetics; Plasmids - metabolism; Xanthobacter - enzymology; Ehrlich pathway; metabolic engineering; tertiary branched-chemicals; higher alcohol; pivalyl-CoA mutase
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.8b00358

The Ehrlich pathway is a major route for the renewable production of higher alcohols. However, the product scope of the Ehrlich pathway is restricted, and the product selectivity is suboptimal. Here, we demonstrate that a Coenzyme A (CoA) detour, which involves conversion of the 2-keto acids into acyl-CoAs, expands the biological toolkit of reaction chemistries available in the Ehrlich pathway to include the gamut of CoA-dependent enzymes. As a proof-of-concept, we demonstrated the first biosynthesis of a tertiary branched-alcohol, pivalcohol, at a level of ∼10 mg/L from glucose in Escherichia coli, using a pivalyl-CoA mutase from Xanthobacter autotrophicus. Furthermore, engineering an enzyme in the CoA detour, the Lactobacillus brevis CoA-acylating aldehyde dehydrogenase, allowed stringent product selectivity. Targeted production of 3-methyl-1-butanol (3-MB) in E. coli mediated by the CoA detour showed a 3-MB:side-product (isobutanol) ratio of >20, an increase over the ratios previously achieved using the conventional Ehrlich pathway.