重访恶臭假单胞菌 KT2440 中的 β-氧化-聚羟基链烷酸酯合成关系,Applied Microbiology and Biotechnology

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重访恶臭假单胞菌 KT2440 中的 β-氧化-聚羟基链烷酸酯合成关系
Applied Microbiology and Biotechnology ( IF 3.9 ) Pub Date : 2023-02-10 , DOI: 10.1007/s00253-023-12413-7
Si Liu _{1,

2} , Tanja Narancic _{1,

2} , Jia-Lynn Tham _{1,

2} , Kevin E O'Connor _{1,

2}

Affiliation

摘要

恶臭假单胞菌KT2440 是众所周知的中链长度 (mcl) 聚羟基脂肪酸酯 (PHA) 积累模型生物。( R )-特异性烯酰辅酶 A 水合酶 (PhaJ) 被认为是 PHA 合成单体的主要供应商，它通过将 β-氧化中间体，反式-2-烯酰辅酶 A 转化为 ( R )-3-羟酰基辅酶 A当使用脂肪酸 (FA) 时。三个 PhaJ 同系物 PhaJ1、PhaJ4 和 MaoC 在恶臭假单胞菌KT2440 中进行了注释。研究恶臭假单胞菌KT2440（一系列恶臭假单胞菌）中脂肪酸与 PHA 代谢的关系以及每个 PhaJ 在 PHA 生物合成中的作用获得了 KT2440 敲除。分析了不同生长条件下野生型 (WT) 和突变体的 PHA 含量和单体组成。PhaJ4 是 PHA 合成的主要单体供应商，以 FA 作为唯一的碳源和能源，优先选择 C8 和 C10 底物，而 PhaJ1 显示优先选择 C6 底物。然而，当所有三个 PhaJ 同源物都被删除时，突变体仍然积累了高达细胞干重 (CDW) 的 10.7% 的 PHA。删除 ( R)-3-hydroxydecanoyl-ACP:CoA transacylase (PhaG)，连接从头 FA 和 PHA 合成途径，同时导致 PHA 含量进一步降低 1.8 倍，但并未消除 PHA 积累。进一步的蛋白质组分析显示奎宁蛋白醇脱氢酶 PedE 和 PedH 作为潜在的单体供应商，但当这些被删除时，PHA 水平保持在 2.2-14.8% CDW，具体取决于使用的脂肪酸和是否应用氮限制。因此，很可能是其他一些非特异性脱氢酶为 PHA 合成提供了单体，证明了 PHA 代谢的冗余性。

关键点

• β-氧化中间体被恶臭假单胞菌KT2440 中的水合酶PhaJ1、PhaJ4 和MaoC 转化为PHA 单体。

•当删除这些时，PHA 水平会降低，但不会被废除。

• PHA 非特异性酶也有助于 KT2440 中的 PHA 代谢。

"点击查看英文标题和摘要"

β-oxidation–polyhydroxyalkanoates synthesis relationship in Pseudomonas putida KT2440 revisited

Abstract

Pseudomonas putida KT2440 is a well-known model organism for the medium-chain-length (mcl) polyhydroxyalkanoate (PHA) accumulation. (R)-Specific enoyl-coenzyme A hydratase (PhaJ) was considered to be the main supplier of monomers for PHA synthesis by converting the β-oxidation intermediate, trans-2-enoyl-CoA to (R)-3-hydroxyacyl-CoA when fatty acids (FA) are used. Three PhaJ homologues, PhaJ1, PhaJ4 and MaoC, are annotated in P. putida KT2440. To investigate the relationship of fatty acids–PHA metabolism and the role of each PhaJ in PHA biosynthesis in P. putida KT2440, a series of P. putida KT2440 knockouts was obtained. PHA content and monomer composition in wild type (WT) and mutants under different growth conditions were analysed. PhaJ4 was the main monomer supplier for PHA synthesis with FA as sole carbon and energy source, with preference towards C8 and C10 substrate, whereas PhaJ1 showed preference for the C6 substrate. However, when all three PhaJ homologues were deleted, the mutant still accumulated PHA up to 10.7% of the cell dry weight (CDW). The deletion of (R)-3-hydroxydecanoyl-ACP:CoA transacylase (PhaG), which connects de novo FA and PHA synthesis pathways, while causing a further 1.8-fold decrease in PHA content, did not abolish PHA accumulation. Further proteome analysis revealed quinoprotein alcohol dehydrogenases PedE and PedH as potential monomer suppliers, but when these were deleted, the PHA level remained at 2.2–14.8% CDW depending on the fatty acid used and whether nitrogen limitation was applied. Therefore, it is likely that some other non-specific dehydrogenases supply monomers for PHA synthesis, demonstrating the redundancy of PHA metabolism.