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

Abstract

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.

Keywords: Ehrlich pathway; higher alcohol; metabolic engineering; pivalyl-CoA mutase; tertiary branched-chemicals.

Figure 1.

Coenzyme A (CoA) detour of the Ehrlich pathway. The conventional Ehrlich pathway converts 2-keto acids to alcohols via aldehydes. The CoA detour first converts 2-keto acids into acyl-CoAs, which allows the application of diverse CoA-dependent chemistries (The rearrangement chemistry explored in this study is highlighted). Finally, the CoA-derivatives are converted to aldehydes, re-entering the Ehrlich pathway. KDC, keto acid decarboxylase; KDHC, keto acid dehydrogenase complex; ALDH, CoA-acylating aldehyde dehydrogenase; ADH, alcohol dehydrogenase.

Figure 2.

CoA detour of the Ehrlich pathway enables the production of various higher alcohols in Escherichia coli. (A) The production pathways for linear and branched alcohols. (B) Choice of KDHC and overexpression of upstream genes altered the product distribution and titers. 2-KB, 2-ketobutyrate; 2-KV, 2-ketovalerate; 2-KC, 2-ketoisocaproate; KIV, ketoisovalerate; KIC, ketoisocaproate; 3-MB, 3-methyl-1-butanol. KDHC, keto acid dehydrogenase complex; ALDH, CoA-acylating aldehyde dehydrogenase; ADH, alcohol dehydrogenase. LeuA*, the feedback-resistant variant (G462D) of E. coli LeuA. Pp, Pseudomonas putida; Bs, Bacillus subtilis. Alcohol titers were quantified at 72 h. Error bars represent one standard deviation of three replicates (n = 3).

Figure 3.

Optimization of isobutanol and 3-MB production through the CoA detour of the Ehrlich pathway. 3-MB, 3-methyl-1-butanol. Alcohol titers were quantified at 72 h. Error bars represent one standard deviation of three replicates (n = 3).

Figure 4.

Targeted production of 3-methyl-1-butanol (3-MB) by the CoA detour of the Ehrlich pathway. (A) The native and engineered CoA acylating aldehyde dehydrogenases (ALDHs) showed different ratios of specific activities toward 3-methylbutyraldehyde and isobutryaldehyde (3-MBA:IBA). The specific activities were measured using enzyme assays in vitro with purified enzymes. (B,C) Computational structure modeling revealed the role of the T428A mutation in accommodating 3-MBA. Contours of the binding pocket are shown in black. The T428A mutation allows the bulky 3-MBA (green) to orient in a catalytic geometry, with the carbonyl carbon positioned for nucleophilic attack by Cys275 (cyan) and hydride transfer to NAD (orange). (D) Isobutanol and 3-MB production titers by WB108 strain. (E) Comparison of the 3-MB/isobutanol production ratio among different strains harboring different ALDHs. Alcohol titers were quantified at 72 h. Error bars represent one standard deviation of three replicates (n = 3).

Figure 5.

Production of pivalcohol using the CoA detour of the Ehrlich pathway. (A) The pivalcohol production pathway in Escherichia coli.(B) Conversion of isovaleryl-CoA to pivalyl-CoA in vitro by crude cell lysates containing the pivalyl-CoA mutase fusion protein (PCM-F) from Xanthobacter autotrophicus, and with its G-protein chaperone. (C) Typical gas chromatography-flame ionization detector (GC-FID) traces showing the absence of pivalcohol in the production medium of WB109 (which has the E. coli YqhD), and the presence of pivalcohol in that of WB110 (which has Ralstonia sp. ADH). (D) Pivalcohol titers of WB109 and WB110. (E) Specific activities of various CoA-acylating aldehyde dehydrogenases (ALDHs) toward pivaldehyde, as measured in vitro using purified proteins. (F) Specific activities of various alcohol dehydrogenases (ADHs) toward pivaldehyde, as measured in vitro using purified proteins. N.D. not detected. See text for the sources of ALDHs and ADHs. Alcohol titers were quantified at 72 h. Error bars represent one standard deviation of three replicates (n = 3).