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

sp. 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 in The results showed that both KS-A and KS-B domains could complement the defective phenotypes of both and mutants. Overexpression of these domains in wild-type led 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 in are the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs in Very-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 protist sp. strain ATCC 26185 and then expressing them in wild-type and 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 in This result has paved the way for further studying of molecular mechanisms of the PUFA synthase for the biosynthesis of VLCPUFAs.

Distinct functions of two FabA‐like dehydratase domains of polyunsaturated fatty acid synthase in the biosynthesis of very long‐chain polyunsaturated fatty acids

“…However, the exact molecular mechanism of the PUFA synthase for intricately positioning multiple double bonds in VLCPUFAs has not been fully elucidated. There is evidence that KS and DH domains of a PUFA synthase play key roles in the biosynthesis of VLCPUFAs where DH domains catalyse the introduction of double bonds and KS domains are responsible for retaining the double bonds (Xie et al ., 2017, 2018; Hayashi et al ., 2019a; Hayashi et al ., 2019b). The PUFA synthase in Thraustochytrium possesses two different types of DH domains.…”

Enhancing oil production in Arabidopsis through expression of a ketoacyl-ACP synthase domain of the PUFA synthase from Thraustochytrium

“…However, the exact functions of these domains in the biosynthesis of these fatty acids are not well characterized. Our previous study indicated that a dissected KS domain from Subunit-B of the PUFA synthase from Thraustochytrium could complement the temperature-sensitive phenotype of E. coli β-ketoacyl-ACP synthase I ( FabB) mutant and enhance the total fatty acid production in a wild-type E. coli strain [17]. In the present study, this domain was further functionally analyzed in A. thaliana .…”

“…FAMEs were extracted with 2 mL of hexane and 1 mL of 0.9% NaCl. The total fatty acid profiles and quantity were analyzed on an Agilent 7890A system installed with a DB-23 column (30 m, 0.25 mm, 0.25 µm) [13, 17].…”

“…Our previous study showed ketoacyl-ACP synthase (KS) domains from the PUFA synthase could functionally complement the defective phenotype of two E. coli mutants of β-ketoacyl-ACP synthases. Particularly, overexpression of one KS domain from subunit-B (KS-B) resulted in accumulation of a high level of total fatty acids as well as unsaturated fatty acids in E. coli [17]. The aim of this study was to extend our previous research on this domain to model oilseed plant Arabidopsis for further functional analysis.…”

Functional Analysis of an Acyltransferase-Like Domain from Polyunsaturated Fatty Acid Synthase in Thraustochytrium

“…The PUFA synthase from Thraustochytrium comprises three large subunits, each with multiple catalytic domains. All these domains are predicted on the presence of characteristic catalytic motifs in the primary sequence, and the exact functions of most of them remain to be determined except for ketoacyl synthase and dehydratase domains of PUFA synthases [ 16 , 17 , 18 , 19 , 20 ]. The purpose of this study was to functionally analyze an acyltransferase (AT)-like domain from Subunit-B of a PUFA synthase in Thraustochytrium through complementation test, site-directed mutagenesis and in vitro assays in E. coli .…”