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Metabolic engineering of Pseudomonas putida for production of docosahexaenoic acid based on a myxobacterial PUFA synthase

Paper ID Volume ID Publish Year Pages File Format Full-Text
31480 44800 2016 11 PDF Available
Title
Metabolic engineering of Pseudomonas putida for production of docosahexaenoic acid based on a myxobacterial PUFA synthase
Abstract

•Engineering of a myxobacterial polyunsaturated fatty acid biosynthetic pathway.•Heterologous expression of myxobacterial PUFA synthases in Escherichia coli and Pseudomonas putida.•First synthetic polyketide synthase-like pathway expressed in P. putida.•Production enhancement by genetic and metabolic engineering.

Long-chain polyunsaturated fatty acids (LC-PUFAs) can be produced de novo via polyketide synthase-like enzymes known as PUFA synthases, which are encoded by pfa biosynthetic gene clusters originally discovered from marine microorganisms. Recently similar gene clusters were detected and characterized in terrestrial myxobacteria revealing several striking differences. As the identified myxobacterial producers are difficult to handle genetically and grow very slowly we aimed to establish heterologous expression platforms for myxobacterial PUFA synthases. Here we report the heterologous expression of the pfa gene cluster from Aetherobacter fasciculatus (SBSr002) in the phylogenetically distant model host bacteria Escherichia coli and Pseudomonas putida. The latter host turned out to be the more promising PUFA producer revealing higher production rates of n-6 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). After several rounds of genetic engineering of expression plasmids combined with metabolic engineering of P. putida, DHA production yields were eventually increased more than threefold. Additionally, we applied synthetic biology approaches to redesign and construct artificial versions of the A. fasciculatus pfa gene cluster, which to the best of our knowledge represents the first example of a polyketide-like biosynthetic gene cluster modulated and synthesized for P. putida. Combination with the engineering efforts described above led to a further increase in LC-PUFA production yields. The established production platform based on synthetic DNA now sets the stage for flexible engineering of the complex PUFA synthase.

Keywords
Polyunsaturated fatty acids; Polyketide synthase; PUFA synthase; Heterologous expression; Synthetic gene cluster; Pathway engineering
First Page Preview
Metabolic engineering of Pseudomonas putida for production of docosahexaenoic acid based on a myxobacterial PUFA synthase
Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 33, January 2016, Pages 98–108
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering