Metabolic engineering of Gluconobacter oxydans 621H for increased biomass yield
Metabolic engineering of Gluconobacter oxydans 621H for increased biomass yield
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DOI：
10.1007/s00253-017-8308-3
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发表时间：
2017-07-01
影响因子：
5
通讯作者：
Bott, Michael
中科院分区：
文献类型：
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作者：
Kiefler, Ines;Bringer, Stephanie;Bott, Michael
氧葡萄糖杆菌是一种必需的好氧乙酸细菌，它能不完全地在区域和立体选择性地氧化外周质中的碳源，因此在工业上被用于氧化生物转化，例如维生素C的生产。然而，它的生物质产量很低，因为氧化产物大部分留在培养基中，不能用于合成代谢。胞质碳代谢通过戊糖磷酸途径和enner - doudoroff途径进行，而糖酵解和三羧酸循环是不完整的。醋酸是通过丙酮酸脱羧酶和乙醛脱氢酶形成的最终产物。为了提高葡萄糖的产量，我们依次用巴氏醋酸杆菌琥珀酸脱氢酶和黄烷化因子SdhE基因（菌株IK001）替换了编码胞质nadp依赖性葡萄糖脱氢酶的gdhS基因，用编码ii型NADH脱氢酶的ndh基因（菌株IK002.1）替换了编码丙酮酸脱氢酶的pdc基因。（iii） gdhM编码膜结合pqq依赖的葡萄糖脱氢酶，由重氮养葡萄糖醋杆菌（glucconacetobacterium diazotrophicus）编码琥珀酰辅酶a合成酶（菌株IK003.1）的sucCD编码。在生物反应器中控制培养条件下对菌株进行分析，发现菌株IK003.1既不生成葡萄糖酸盐，也不生成2-酮葡萄糖酸盐，只生成5-酮葡萄糖酸盐。醋酸酯的生成被消除，取而代之的是相当数量的丙酮酸。与参考菌株相比，IK003.1的CO2生成量增加了一倍以上。IK003.1的生长受到抑制，但生物量提高了60%。IK003.1是氧化生物转化和进一步代谢工程的合适宿主。
The obligatory aerobic acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources regio- and stereoselectively in the periplasm and therefore is used industrially for oxidative biotransformations, e. g., in vitamin C production. However, it has a very low biomass yield as the oxidized products largely remain in the medium and cannot be used for anabolism. Cytoplasmic carbon metabolism occurs via the pentose phosphate pathway and the Entner-Doudoroff pathway, whereas glycolysis and the tricarboxylic acid cycle are incomplete. Acetate is formed as an end product via pyruvate decarboxylase and acetaldehyde dehydrogenase. In order to increase the biomass yield from glucose, we sequentially replaced (i) gdhS encoding the cytoplasmic NADP-dependent glucose dehydrogenase by the Acetobacter pasteurianus sdhCDABE genes for succinate dehydrogenase and the flavinylation factor SdhE (strain IK001), (ii) pdc encoding pyruvate decarboxylase by a second ndh gene encoding a type II NADH dehydrogenase (strain IK002.1), and (iii) gdhM encoding the membrane-bound PQQ-dependent glucose dehydrogenase by sucCD from Gluconacetobacter diazotrophicus encoding succinyl-CoA synthetase (strain IK003.1). Analysis of the strains under controlled cultivation conditions in bioreactors revealed for IK003.1 that neither gluconate nor 2-ketogluconate was formed, but some 5-ketogluconate. Acetate formation was eliminated, and comparable amounts of pyruvate were formed instead. CO2 formation by IK003.1 was more than doubled compared to the reference strain. Growth of IK003.1 was retarded, but the biomass yield of this strain was raised by 60%. IK003.1 serves as suitable host for oxidative biotransformations and for further metabolic engineering.