A Futile Metabolic Cycle of Fatty Acyl Coenzyme A (Acyl-CoA) Hydrolysis and Resynthesis in Corynebacterium glutamicum and Its Disruption Leading to Fatty Acid Production
A Futile Metabolic Cycle of Fatty Acyl Coenzyme A (Acyl-CoA) Hydrolysis and Resynthesis in Corynebacterium glutamicum and Its Disruption Leading to Fatty Acid Production
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DOI：
10.1128/aem.02469-20
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发表时间：
2021-02-01
影响因子：
4.4
通讯作者：
Takeno, Seiki
中科院分区：
文献类型：
--
作者：
Ikeda, Masato;Takahashi, Keisuke;Takeno, Seiki
脂肪酰基辅酶A（酰基 - CoA）硫酯酶（Tes）和酰基 - CoA合成酶（FadD）催化酰基 - CoA和游离脂肪酸之间的相反反应。在谷氨酸棒杆菌的基因组中，存在几种编码每种酶的候选基因，尽管它们的功能尚不清楚。在脂肪酸生产菌WTΔfasR中对候选基因进行修饰表达，从而鉴定出一个tes基因（tesA）和两个fadD基因（fadD5和fadD15），它们分别在脂肪酸生产中起正向和负向作用。遗传分析表明，fadD5和fadD15负责外源脂肪酸的利用，而tesA在为外层成分——分枝菌酸的合成提供脂肪酸方面发挥作用。酶活性测定和表达分析显示，tesA、fadD5和fadD15共表达，在酰基 - CoA和脂肪酸之间形成一个循环途径。当在野生型谷氨酸棒杆菌中破坏fadD5或fadD15时，两种突变体在生长过程中都会分泌脂肪酸。对这些基因进行双敲除导致产量协同增加。对tesA进行额外敲除显示出对产量的抵消作用。这些结果表明，FadD通常将TesA产生的多余脂肪酸分流回酰基 - CoA用于脂质生物合成，而阻断这种分流会促使细胞过量生产脂肪酸。当将这种策略应用于一种高产脂肪酸的菌株时，所得的fadD被破坏且tesA被扩增的菌株相对于其亲本产量提高了72%，并且从1%的葡萄糖中每升可生产出以克为单位的脂肪酸，主要包括油酸、棕榈酸和硬脂酸。
重要性：工业氨基酸生产菌谷氨酸棒杆菌已经发展成为一种潜在的脂肪酸生产主力。在这种生物中，我们获得的证据表明存在一种独特的脂质稳态机制，即分别由酰基 - CoA硫酯酶（Tes）和酰基 - CoA合成酶（FadD）介导的酰基 - CoA水解和再合成的无效循环的形成。Tes和FadD偶联的生物学作用是为外层成分——分枝菌酸的合成提供游离脂肪酸，并将多余的脂肪酸循环回酰基 - CoA用于膜脂质合成。我们进一步证明，在一种高产脂肪酸的菌株中对该循环进行工程改造可显著提高产量，这为在这种具有工业重要性的微生物中生产脂肪酸提供了一种有用的工程策略。
Fatty acyl coenzyme A (acyl-CoA) thioesterase (Tes) and acyl-CoA synthetase (FadD) catalyze opposing reactions between acyl-CoAs and free fatty acids. Within the genome of Corynebacterium glutamicum, several candidate genes for each enzyme are present, although their functions remain unknown. Modified expression of the candidate genes in the fatty acid producer WT Delta fasR led to identification of one tes gene (tesA) and two fadD genes (fadD5 and fadD15), which functioned positively and negatively in fatty acid production, respectively. Genetic analysis showed that fadD5 and fadD15 are responsible for utilization of exogenous fatty acids and that tesA plays a role in supplying fatty acids for synthesis of the outer layer components mycolic acids. Enzyme assays and expression analysis revealed that tesA, fadD5, and fadD15 were coexpressed to create a cyclic route between acyl-CoAs and fatty acids. When fadD5 or fadD15 was disrupted in wild-type C. glutamicum, both disruptants excreted fatty acids during growth. Double disruption of these genes resulted in a synergistic increase in production. Additional disruption of tesA revealed a canceling effect on production. These results indicate that the FadDs normally shunt the surplus of TesA-generated fatty acids back to acyl-CoAs for lipid biosynthesis and that interception of this shunt provokes cells to overproduce fatty acids. When this strategy was applied to a high-fatty-acid producer, the resulting fadD-disrupted and tesA-amplified strain exhibited a 72% yield increase relative to its parent and produced fatty acids, which consisted mainly of oleic acid, palmitic acid, and stearic acid, on the gram scale per liter from 1% glucose.IMPORTANCE The industrial amino acid producer Corynebacterium glutamicum has evolved into a potential workhorse for fatty acid production. In this organism, we obtained evidence showing the presence of a unique mechanism of lipid homeostasis, namely, formation of a futile cycle of acyl-CoA hydrolysis and resynthesis mediated by acyl-CoA thioesterase (Tes) and acyl-CoA synthetase (FadD), respectively. The biological role of the coupling of Tes and FadD would be to supply free fatty acids for synthesis of the outer layer components mycolic acids and to recycle their excess to acyl-CoAs for membrane lipid synthesis. We further demonstrated that engineering of the cycle in a high-fatty-acid producer led to dramatically improved production, which provides a useful engineering strategy for fatty acid production in this industrially important microorganism.