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首页> 外文期刊>Biotechnology Progress >Enhanced isoamyl acetate production upon manipulation of the acetyl-CoA node in Escherichia coli
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Enhanced isoamyl acetate production upon manipulation of the acetyl-CoA node in Escherichia coli

机译：通过操作大肠杆菌中的乙酰辅酶A节点提高乙酸异戊酯的产量

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摘要

Coenzyme A (CoA) and its thioester derivative acetyl-Coenzyme A (acetyl-CoA) participate in over 100 different reactions in intermediary metabolism of microorganisms. Earlier results indicated that overexpression of upstream rate-limiting enzyme pantothenate kinase with simultaneous supplementation of precursor pantothenic acid to the culture media increased intracellular CoA levels significantly (similar to10-fold). The acetyl-CoA levels also increased (similar to5-fold) but not as much as that of CoA, showing that the carbon flux from the pyruvate node is rate-limiting upon an increase in CoA levels. In this study, pyruvate dehydrogenase was overexpressed under elevated CoA levels to increase carbon flux from pyruvate to acetyl-CoA. This coexpression did not increase intracellular acetyl-CoA levels but increased the accumulation of extracellular acetate. The production of isoamyl. acetate, an industrially useful compound derived from acetyl-CoA, was used as a model reporter system to signify the beneficial effects of this metabolic engineering strategy. In addition, a strain was created in which the acetate production pathway was inactivated to relieve competition at the acetyl-CoA node and to efficiently channel the enhanced carbon flux to the ester production pathway. The synergistic effect of cofactor CoA manipulation and pyruvate dehydrogenase overexpression in the acetate pathway deletion mutant led to a 5-fold increase in isoamyl acetate production.. Under normal growth conditions the acetate pathway deletion mutant strains accumulate intracellular pyruvate, leading to excretion of pyruvate. However, upon enhancing the carbon flux from pyruvate to acetyl-CoA, the excretion of pyruvate was significantly reduced.
机译：辅酶A（CoA）及其硫酯衍生物乙酰辅酶A（乙酰CoA）参与微生物中间代谢的100多种不同反应。较早的结果表明，上游限速酶泛酸激酶的过表达，同时向培养基中补充前体泛酸，显着提高了细胞内CoA水平（约10倍）。乙酰辅酶A水平也增加（约5倍），但不如辅酶A增加那么多，这表明丙酮酸节点的碳通量限制了辅酶A水平的增加。在这项研究中，丙酮酸脱氢酶在升高的CoA水平下过表达，从而增加了从丙酮酸到乙酰辅酶A的碳通量。这种共表达不会增加细胞内乙酰辅酶A的水平，但会增加细胞外乙酸的积累。异戊基的生产。乙酸酯，一种工业上有用的衍生自乙酰辅酶A的化合物，被用作模型报告系统，以表明该代谢工程策略的有益作用。另外，产生了一种菌株，其中乙酸盐的生产途径被灭活以减轻在乙酰辅酶A节点上的竞争并且有效地将增强的碳通量引导至酯的生产途径。辅酶CoA操纵和丙酮酸脱氢酶过表达在乙酸盐途径缺失突变体中的协同作用导致乙酸异戊酯产量增加5倍。在正常生长条件下，乙酸盐途径缺失突变体菌株积累细胞内丙酮酸，导致丙酮酸排泄。然而，在增加从丙酮酸到乙酰辅酶A的碳通量后，丙酮酸的排泄显着减少。

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