Xie Xi, Meesapyodsuk Dauenpen, Qiu Xiao
Department of Food & Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Department of Food & Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Appl Environ Microbiol. 2017 Apr 17;83(9). doi: 10.1128/AEM.03133-16. Print 2017 May 1.
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.
sp.菌株ATCC 26185积累了高水平的二十二碳六烯酸（DHA），这是一种营养上重要的ω-3超长链多不饱和脂肪酸（VLCPUFA），主要由多不饱和脂肪酸（PUFA）合酶合成，PUFA合酶是一种I型聚酮合酶样巨型酶。该物种中的PUFA合酶由三个大亚基组成，每个大亚基都有多个催化结构域。据推测，在这些结构域中，酮酰基合酶（KS）结构域可能对于催化特定不饱和酰基-酰基载体蛋白（ACP）与丙二酰-ACP的缩合至关重要，从而在延长的酰基链中保留双键。为了研究这些假定的KS结构域的功能，从PUFA合酶的亚基A（KS-A）和亚基B（KS-B）中分离出两个片段序列，然后在[具体宿主菌名称未给出]中作为独立酶进行表达。结果表明，KS-A和KS-B结构域都可以弥补[具体宿主菌名称未给出]和[具体突变体名称未给出]突变体的缺陷表型。在野生型[具体宿主菌名称未给出]中过表达这些结构域导致总脂肪酸产量增加。KS-B产生的不饱和脂肪酸（UFA）与饱和脂肪酸（SFA）的比例更高，而KS-A可以更有效地提高脂肪酸的总体产量，特别是对于SFA的产量，这意味着KS-A在催化功能上更类似于FabF，而KS-B更类似于FabB。在[具体宿主菌名称未给出]中成功互补嵌入的KS结构域并进行功能表达是研究PUFA合酶在[具体宿主菌名称未给出]中生物合成VLCPUFAs的分子机制的第一步。超长链多不饱和脂肪酸（VLCPUFAs）对人类健康很重要。它们在自然界中可以通过需氧途径或厌氧途径生物合成。然而，海洋微生物中丰富的VLCPUFAs主要由多不饱和脂肪酸（PUFA）合酶合成，PUFA合酶是一种具有多个亚基的巨型酶，每个亚基都有多个催化结构域。此外，这种酶合成这些脂肪酸的基本机制仍然未知。本报告首先从海洋原生生物sp.菌株ATCC 26185中剖析PUFA合酶的嵌入KS结构域，然后在野生型[具体宿主菌名称未给出]和酰基-ACP与丙二酰-ACP缩合有缺陷的突变体中表达它们。突变体的成功互补以及过表达实验中脂肪酸产量的提高表明，这些KS结构域可以在[具体宿主菌名称未给出]中有效地作为独立酶发挥作用。这一结果为进一步研究PUFA合酶生物合成VLCPUFAs的分子机制铺平了道路。
