Understanding and Harnessing the Microaerobic Metabolism of Glycerol in Escherichia coli
Understanding and Harnessing the Microaerobic Metabolism of Glycerol in Escherichia coli
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DOI：
10.1002/bit.22246
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发表时间：
2009-05-01
影响因子：
3.8
通讯作者：
Gonzalez, Ramon
中科院分区：
文献类型：
--
作者：
Durnin, Guyton;Clomburg, James;Gonzalez, Ramon
鉴于其可用性、低廉的价格和高度的还原性，甘油已成为生产还原化合物的理想原料。大肠杆菌对甘油的厌氧发酵可能是实现此目的的绝佳平台，但它需要昂贵的营养物质，例如胰蛋白胨和酵母提取物。在这项工作中，微氧条件被用作消除对丰富营养素的需求的手段。少量氧气的存在可以实现氧化还原平衡，同时保留合成还原产物的能力。发酵平衡分析显示碳和还原当量回收率接近 95%。使用不同的遗传和生化方法鉴定了参与甘油异化的途径。呼吸途径（GlpK-GlpD/GlpABC）和发酵途径（GldA-DhaKLM）介导甘油向糖酵解中间体的转化。尽管丙酮酸甲酸裂解酶（PFL）和丙酮酸脱氢酶有助于从丙酮酸合成乙酰辅酶A，但大部分碳通量通过PFL进行。甘油的有效利用需要介导乙酸和乙醇合成的途径。工程菌株利用甘油的微氧代谢来联产乙醇和氢气 (EH05 [pZSKLMgIdA]) 以及乙醇和甲酸盐 (EF06 [pZSKLMgIdA])。通过基因操作减少副产物琥珀酸盐、乙酸盐和乳酸盐的合成，实现了高乙醇产量。氢气的共生产需要使用酸性 PH，而甲酸的共生产则通过甲酸氢裂合酶的失活来促进。通过过度表达甘油脱氢酶 (GldA) 和二羟基丙酮激酶 (DhaKLM) 实现了高产物合成率。工程菌株在没有丰富补充剂的情况下，在基本培养基中有效地从甘油产生乙醇和氢气以及乙醇和甲酸盐。
Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock for the production of reduced compounds. The anaerobic fermentation of glycerol by Escherichia coli could be an excellent platform for this purpose but it requires expensive nutrients such as tryptone and yeast extract. In this work, microaerobic conditions were used as a means of eliminating the need for rich nutrients. Availability of low amounts of oxygen enabled redox balance while preserving the ability to synthesize reduced products. A fermentation balance analysis showed similar to 95% recovery of carbon and reducing equivalents. The pathways involved in glycerol dissimilation were identified using different genetic and biochemical approaches. Respiratory (GlpK-GlpD/GlpABC) and fermentative (GldA-DhaKLM) routes mediated the conversion of glycerol to glycolytic intermediates. Although pyruvate formate-lyase (PFL) and pyruvate dehydrogenase contributed to the synthesis of acetyl-CoA from pyruvate, most of the carbon flux proceeded through PFL. The pathways mediating the synthesis of acetate and ethanol were required for the efficient utilization of glycerol. The microaerobic metabolism of glycerol was harnessed by engineering strains for the co-production of ethanol and hydrogen (EH05 [pZSKLMgIdA]), and ethanol and formate (EF06 [pZSKLMgIdA]). High ethanol yields were achieved by genetic manipulations that reduced the synthesis of by-products succinate, acetate, and lactate. Co-production of hydrogen required the use of acidic PH while formate co-production was facilitated by inactivation of the enzyme formate-hydrogen lyase. High rates of product synthesis were realized by overexpressing glycerol dehydrogenase (GldA) and dihydroxyacetone kinase (DhaKLM). Engineered strains efficiently produced ethanol and hydrogen and ethanol and formate from glycerol in a minimal medium without rich supplements.