title: Metabolic engineering of Pseudomonas putida for production of docosahexaenoic acid based on a myxobacterial PUFA synthase.
abstract: 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.
authors.affiliation: Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, D-66123 Saarbrücken, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Gemperlein
authors.affiliation: ATG:biosynthetics GmbH, D-79249 Merzhausen, Germany.
authors.forename: Gregor
authors.initials: G
authors.lastname: Zipf
authors.affiliation: ATG:biosynthetics GmbH, D-79249 Merzhausen, Germany.
authors.forename: Hubert S
authors.initials: HS
authors.lastname: Bernauer
authors.affiliation: Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, D-66123 Saarbrücken, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Müller
authors.affiliation: Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, D-66123 Saarbrücken, Germany. Electronic address: silke.wenzel@helmholtz-hzi.de.
authors.forename: Silke C
authors.initials: SC
authors.lastname: Wenzel
mesh_terms: D019732:Amide Synthases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004281:Docosahexaenoic Acids / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005231:Fatty Acids, Unsaturated / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D009231:Myxococcales / Q000201:enzymology* / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D005231:Fatty Acids, Unsaturated; D011994:Recombinant Proteins; D004281:Docosahexaenoic Acids; D019732:Amide Synthases
keywords: Heterologous expression; PUFA synthase; Pathway engineering; Polyketide synthase; Polyunsaturated fatty acids; Synthetic gene cluster