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Engineering Escherichia coli for production of C12–C14 polyhydroxyalkanoate from glucose

Paper ID Volume ID Publish Year Pages File Format Full-Text
31720 44832 2012 9 PDF Available
Title
Engineering Escherichia coli for production of C12–C14 polyhydroxyalkanoate from glucose
Abstract

Demand for sustainable materials motivates the development of microorganisms capable of synthesizing products from renewable substrates. A challenge to commercial production of polyhydroxyalkanoates (PHA), microbially derived polyesters, is engineering metabolic pathways to produce a polymer with the desired monomer composition from an unrelated and renewable source. Here, we demonstrate a metabolic pathway for converting glucose into medium-chain-length (mcl)-PHA composed primarily of 3-hydroxydodecanoate monomers. This pathway combines fatty acid biosynthesis, an acyl-ACP thioesterase to generate desired C12 and C14 fatty acids, β-oxidation for conversion of fatty acids to (R)-3-hydroxyacyl-CoAs, and a PHA polymerase. A key finding is that Escherichia coli expresses multiple copies of enzymes involved in β-oxidation under aerobic conditions. To produce polyhydroxydodecanoate, an acyl-ACP thioesterase (BTE), an enoyl-CoA hydratase (phaJ3), and mcl-PHA polymerase (phaC2) were overexpressed in E. coli ΔfadRABIJ. Yields were improved through expression of an acyl-CoA synthetase resulting in production over 15% CDW – the highest reported production of mcl-PHA of a defined composition from an unrelated carbon source.

► Characterization of a β-oxidation mutant library under aerobic conditions. ► Characterization of six PHA biosynthetic enzymes from Pseudomonas aeruginosa PAO1. ► Metabolic engineering of Escherichia coli to produce 15% CDW mcl-PHA with monomer profile that matches the thioesterase product profile. ► Demonstration that CoA-synthetase activity is a limiting step in the pathway.

Keywords
(mcl)-PHA, (medium-chain-length)-polyhydroxyalkanoate; LB, Lysogeny Broth; PCR, polymerase chain reaction; BTE, California Bay Laurel (Umbellularia californica) Thioesterase; PBS, phosphate buffered saline; FAME, fatty acid methyl ester; GC/MS, gas chroma
First Page Preview
Engineering Escherichia coli for production of C12–C14 polyhydroxyalkanoate from glucose
Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 14, Issue 6, November 2012, Pages 705–713
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering