Genome-based metabolic engineering of Mannheimia succiniciproducens for succinic acid production
Genome-based metabolic engineering of Mannheimia succiniciproducens for succinic acid production
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DOI：
10.1128/aem.72.3.1939-1948.2006
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发表时间：
2006-03-01
影响因子：
4.4
通讯作者：
Lee, SY
中科院分区：
文献类型：
--
作者：
Lee, SJ;Song, H;Lee, SY
琥珀酸是一种四碳二羧酸，是厌氧代谢的发酵产物之一。基于一种嗜二氧化碳的产琥珀酸瘤胃细菌——产琥珀酸曼氏杆菌的全基因组序列，进行了基因敲除研究，以了解其厌氧发酵代谢，并进而开发一种能够在无副产物形成的情况下生产琥珀酸的代谢工程菌株。在由磷酸烯醇式丙酮酸（PEP）羧激酶、PEP羧化酶和苹果酸酶催化的三种不同的二氧化碳固定代谢反应中，PEP羧激酶对于产琥珀酸曼氏杆菌的厌氧生长和琥珀酸生产最为重要。发现由PEP羧化形成的草酰乙酸通过苹果酸脱氢酶、延胡索酸酶和延胡索酸还原酶催化的三个连续反应转化为琥珀酸。通过破坏ldhA、pflB、pta和ackA基因，成功去除了导致副产物形成的主要代谢途径。这种代谢工程化的LPK7菌株能够从20克/升葡萄糖中生产13.4克/升琥珀酸，几乎不形成乙酸、甲酸和乳酸，使得琥珀酸产率达到每摩尔葡萄糖0.97摩尔琥珀酸。产琥珀酸曼氏杆菌LPK7的补料分批培养，通过间歇性葡萄糖补料，可生产52.4克/升琥珀酸，琥珀酸产率为每摩尔葡萄糖1.16摩尔琥珀酸，琥珀酸生产率为1.8克/升/小时，这对于琥珀酸的工业生产应该是有用的。
Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO2-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.