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Functional analysis of ketoacyl synthase and dehydratase domains from a PUFA synthase of Thraustochytrium in Escherichia coli and Arabidopsis thaliana

Date

2019-09-04

Journal Title

Journal ISSN

Volume Title

Publisher

ORCID

0000-0002-1048-0189

Type

Thesis

Degree Level

Doctoral

Abstract

Polyunsaturated fatty acid (PUFA) synthase in Thrasustochytrium comprises three subunits, each with multiple catalytic domains. Among these domains, ketoacyl synthase (KS) and dehydratase (DH) domains play critical roles in retaining and introducing double bonds during the biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFAs). To functionally analyze these domains, two putative KS domains (KS-A: KS domain from subunit-A and KS-B: KS domain from subunit-B) of the PUFA synthase were dissected and expressed as standalone enzymes in E. coli. The results showed that both KS-A and KS-B domains could complement defective phenotypes of E. coli FabB and FabF mutants, but the mutagenized ones where the active site cysteine residue was replaced by lysine could not. Overexpression of these domains individually in a wild type E. coli strain increased fatty acid production. Particularly, expression of KS-B domain produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while expression of KS-A improved the overall production of SFAs more effectively. These results imply that KS-A is more comparable to E. coli FabF, while KS-B is more similar to E. coli FabB in catalytic function. To further characterize the KS domain from the subunit-B of the PUFA synthase, plastidial expression of this domain in Arabidopsis thaliana was conducted. The results showed that it could functionally complement the defective growth phenotypes of AtKASI knockout mutant generated using CRISPR/Cas9. Expression of the KS domain under a seed specific promoter in wild type Arabidopsis could significantly enhance seed weight and seed oil, and promote seed germination and seedling growth. These results indicate that the main condensation process of fatty acid biosynthesis in plants might be a limiting step and overexpression of the KS domain from a PUFA synthase of microbial origin may offer a new strategy to increase oil production in oilseed crops. To functionally characterize the DH domains of the PUFA synthase, one DH domain from subunit-A (DH-A) and two DH domains from subunit-C (DH1-C and DH2-C) were dissected from the synthase and expressed as standalone proteins in both mutant and wild type E. coli strains. Expression of DH domains in a temperature sensitive mutant (FabA) defective in 3-hydroxyacyl-ACP dehydratase activity showed that all these DH domains could complement the defective growth phenotype. Overexpression of DH domains drastically altered the fatty acid production and the ratio of unsaturated to saturated fatty acids. These results indicate that the DH domain from subunit-A is comparable to DH domains of polyketide synthases, while the DH domains from subunit-C are more similar to E. coli FabA in catalytic function. Successful complementation and functional expression of the embedded KS and DH domains from the PUFA synthase in E.coli and Arabidopsis is an important step forwards for elucidating the molecular mechanism in the biosynthesis of VLCPUFAs catalyzed by the PUFA synthase in Thraustochytrium and offers strategies for metabolic engineering of the anaerobic pathway in heterologous systems to produce specialty fatty acids for nutritional and industrial uses.

Description

Keywords

KS domain, DH domain, PUFA synthase, Thraustochytrium, DHA, Escherichia coli, Fatty acid biosynthesis, Arabidopsis thaliana, Oil content

Citation

Degree

Doctor of Philosophy (Ph.D.)

Department

Food and Bioproduct Sciences

Program

Food Science

Advisor

Citation

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DOI

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