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Multi‐factorial engineering of heterologous polyketide production in Escherichia coli reveals complex pathway interactions
Author(s) -
Boghigian Brett A.,
Zhang Haoran,
Pfeifer Blaine A.
Publication year - 2011
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.23069
Subject(s) - heterologous , polyketide , biochemistry , heterologous expression , metabolic engineering , propionate , escherichia coli , biosynthesis , chemistry , enzyme , acetyl coa , biology , stereochemistry , recombinant dna , gene
Polyketides represent a significant fraction of all natural products. Many possess pharmacological activity which makes them attractive drug candidates. The production of the parent macrocyclic aglycones is catalyzed by multi‐modular polyketide synthases utilizing short‐chain acyl‐CoA monomers. When producing polyketides through heterologous hosts, one must not only functionally express the synthase itself, but activate the machinery used to generate the required substrate acyl‐CoA's. As a result, metabolic engineering of these pathways is necessary for high‐level production of heterologous polyketides. In this study, we over‐express three different pathways for provision of the two substrates (propionyl‐CoA and (2 S )‐methylmalonyl‐CoA) utilized for the biosynthesis of 6‐deoxyerythronolide B (6‐dEB; the macrolactone precursor of erythromycin): (1) a propionate → propionyl‐CoA → (2 S )‐methylmalonyl‐CoA pathway, (2) a methylmalonate → methylmalonyl‐CoA → propionyl‐CoA pathway, and (3) a succinate → succinyl‐CoA → (2 R )‐methylmalonyl‐CoA → (2 S )‐methylmalonyl‐CoA → propionyl‐CoA pathway. The current study revealed that propionate is a necessary component for greater than 5 mg L −1 titers. Deletion of the propionyl‐CoA:succinate CoA transferase ( ygfH ) or over‐expression of the transcriptional activator of short chain fatty acid uptake improved titer to over 100 mg L −1 , while the combination of the two improved titer to over 130 mg L −1 . The addition of exogenous methylmalonate could also improve titer to over 100 mg L −1 . Expression of a Streptomyces coelicolor A3(2) methylmalonyl‐CoA epimerase, in conjunction with over‐expression of Escherichia coli 's native methylmalonyl‐CoA mutase, allowed for the incorporation of exogenously fed succinate into the 6‐dEB core. Biotechnol. Bioeng. 2011; 108:1360–1371. © 2011 Wiley Periodicals, Inc.