title: Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli.
abstract: Thraustochytriumsp. strain ATCC 26185 accumulates a high level of docosahexaenoic acid (DHA), a nutritionally important ω-3 very-long-chain polyunsaturated fatty acid (VLCPUFA) synthesized primarily by polyunsaturated fatty acid (PUFA) synthase, a type I polyketide synthase-like megaenzyme. The PUFA synthase in this species comprises three large subunits, each with multiple catalytic domains. It was hypothesized that among these domains, ketoacylsynthase (KS) domains might be critical for catalyzing the condensation of specific unsaturated acyl-acyl carrier proteins (ACPs) with malonyl-ACP, thereby retaining double bonds in an extended acyl chain. To investigate the functions of these putative KS domains, two segment sequences from subunit A (KS-A) and subunit B (KS-B) of the PUFA synthase were dissected and then expressed as stand-alone enzymes inEscherichia coliThe results showed that both KS-A and KS-B domains could complement the defective phenotypes of bothE. colifabBandfabFmutants. Overexpression of these domains in wild-typeE. coliled to increases in total fatty acid production. KS-B produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while KS-A could improve the overall production of fatty acids more effectively, particularly for the production of SFAs, implying that KS-A is more comparable to FabF, while KS-B is more similar to FabB in catalytic functions. Successful complementation and functional expression of the embedded KS domains inE. coliare the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs inThraustochytriumIMPORTANCEVery-long-chain polyunsaturated fatty acids (VLCPUFAs) are important for human health. They can be biosynthesized in either an aerobic pathway or an anaerobic pathway in nature. However, abundant VLCPUFAs in marine microorganisms are primarily synthesized by polyunsaturated fatty acid (PUFA) synthase, a megaenzyme with multiple subunits, each with multiple catalytic domains. Furthermore, the fundamental mechanism for this enzyme to synthesize these fatty acids still remains unknown. This report started with dissecting the embedded KS domains of the PUFA synthase from marine protistThraustochytriumsp. strain ATCC 26185 and then expressing them in wild-typeE. coliand mutants defective in condensation of acyl-ACP with malonyl-ACP. Successful complementation of the mutants and improved fatty acid production in the overexpression experiments indicate that these KS domains can effectively function as stand-alone enzymes inE. coliThis result has paved the way for further studying of molecular mechanisms of the PUFA synthase for the biosynthesis of VLCPUFAs.
authors.affiliation: Department of Food & Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Xi
authors.initials: X
authors.lastname: Xie
authors.affiliation: Department of Food & Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Dauenpen
authors.initials: D
authors.lastname: Meesapyodsuk
authors.affiliation: Department of Food & Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada xiao.qiu@usask.ca.
authors.forename: Xiao
authors.initials: X
authors.lastname: Qiu
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000172:deficiency / Q000235:genetics; D000123:Acetyltransferases / Q000172:deficiency / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D054889:Fatty Acid Synthase, Type II / Q000172:deficiency / Q000235:genetics; D064429:Fatty Acid Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D015870:Gene Expression; D005816:Genetic Complementation Test; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D058009:Stramenopiles / Q000201:enzymology* / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D005231:Fatty Acids, Unsaturated; D011994:Recombinant Proteins; D000123:Acetyltransferases; C557137:fabF protein, E coli; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; C490369:FabB protein, E coli; D064429:Fatty Acid Synthases; D054889:Fatty Acid Synthase, Type II
keywords: Escherichia coli; Thraustochytrium; docosahexaenoic acid; docosahexaenoic acid (DHA); fatty acid biosynthesis; ketoacylsynthase domain; polyunsaturated fatty acid synthase