氧化葡萄糖酸杆菌DHA3-9的葡萄糖代谢酶系

Enzymatic system of glucose metabolism in Gluconobacter oxydans strain DHA3-9

研究氧化葡萄糖酸杆菌(Gluconobacter oxydans)对细胞内葡萄糖代谢除直接氧化途径外可能存在的其他途径.以G.oxydans DHA3-9菌株为研究对象,利用同源重组构建G.oxydans DHA3-9膜结合的葡萄糖脱氢酶(membrane-bound glucose dehydrogenase,mGDH)基因敲除的突变株,以阻断该菌的葡萄糖直接氧化途径,研究其在葡萄糖培养基中的生长速率、葡萄糖降解与葡萄糖酸转化、代谢产物成分等主要生理生化特性的变化.结果表明:实验成功地筛选到1株G.oxydans mgdh突变菌,该菌丧失了90%以上利用葡萄糖转化为葡萄糖酸的能力,其二羟基丙酮(dihydroxyacetone,DHA)转化量4倍于野生菌;同时,在该突变菌代谢产物中检测出丙酮酸和乙酸,且乙酸对野生菌和突变菌的生长抑制差异极显著.表明葡萄糖在G.oxydans DHA3-9△mgdh菌株细胞内依赖的代谢途径为糖酵解途径,而非2-酮-3-脱氧-6-磷酸葡萄糖酸裂解途径或磷酸戊糖途径.其中,丙酮酸作为糖酵解途径产物通过丙酮酸脱羧酶和乙醛脱氢酶转化为乙酸,而乙酸积累过多将对菌株生长产生抑制.

Summary Gluconobacter oxydans is widely used in industrial application for its dehydrogenase system locating on cell membrane.These dehydrogenases have a character to oxidize sugars and sugar alcohols incompletely. There are two potential pathways known for glucose oxidization in G.oxydans ：More than 90% of glucose is transformed into gluconate in the periplasmic space;only a minority of glucose（about 5%）is phosphorylated and taken into functional central metabolic pathways such as Entner-Doudoroff pathway（EDP）and pentose phosphate pathway （PPP） in the cytoplasmic compartment.In previous study,the Embden-Meyerhof-Parnas pathway （EMP） was found inactive in G.oxydans due to its lack of phosphofructokinase. In this study,a G.oxydans strain named DHA3-9 was screened which produced dihydroxyacetone （DHA） during glucose degradation.But DHA was not a product in EDP or PPP.A mutant strain of G.oxydans DHA3-9 lacking of glucose dehydrogenase in cell membrane was constructed to study the possibility of other pathway of glucose metabolism in G.oxydans . 〈br〉 A mgdh gene-disrupted mutant of G. oxydans DHA3-9 was constructed by the way of homologous recombination and its characteristic changes of the cells growth on glucose,glucose degradation,gluconate transformation,intermediate products and growth inhibition on acetate were studied. 〈br〉 The results indicated that the growth of mutant strain on glucose showed an obvious delay and pH dropped much slower than that of wild type.The mutant lost most of its ability of glucose degradation and produced little gluconate.Instead,DHA formation of the mutant was recorded four times as that of wild type.Pyruvate and acetate were detected in the products of mutant whereas none of such products were found in wild type culture. Under the condition with glucose as the sole carbon source,50 mmol/L acetate completely inhibited the growth of mutant,whereas this effect was remarkably low on wild type. 〈br〉 These results prove that in G. oxydans DHA3-9 mutant strain, glucose is utilized in cytoplasmic compartment primarily through EMP and acetate can be produced by activities of pyruvate decarboxylase and acetaldehyde dehydrogenase. In this study, a G. oxydans strain named DHA3-9 was screened which produced dihydroxyacetone （DHA） during glucose degradation. But DHA was not a product in EDP or PPP. A mutant strain of G. oxydans DHA3- 9 lacking of glucose dehydrogenase in cell membrane was constructed to study the possibility of other pathway of glucose metabolism in G. oxydans.