【作者】 余秉琦；

【导师】 诸葛健；

【作者基本信息】 江南大学 ， 发酵工程， 2005， 博士

【摘要】 在过去四十多年氨基酸发酵工业的历史发展过程中,人们开展了不少有关谷氨酸棒杆菌的遗传和生理生化方面的研究。尽管如此,谷氨酸棒杆菌过量合成并分泌产生L-谷氨酸的机理仍然处于研究阶段,虽然存在一些相关的假说,但至今还没有统一的定论。 在L-谷氨酸发酵生产过程中,回补途径是谷氨酸棒杆菌过量合成L-谷氨酸的瓶颈之一。乙醛酸循环、磷酸烯醇式丙酮酸羧化酶PEPC和丙酮酸羧化酶PC是目前已经确认的三个具有回补功能的途径和酶。有关乙醛酸循环在L-谷氨酸生物合成中的作用的研究报道较少,并且彼此之间存在分歧。研究乙醛酸循环与谷氨酸棒杆菌菌体生长以及L-谷氨酸合成之间的关系,有助于阐明L-谷氨酸过量合成的机理,对L-谷氨酸菌种的选育具有指导意义。 本文围绕乙醛酸循环在L-谷氨酸生物合成中的作用开展了一系列研究工作,主要研究内容和结果如下: (1) 当以葡萄糖为唯一碳源时,Corynebacterium glutamicum细胞内的乙醛酸循环活性仍然存在,而且对L-谷氨酸的生物合成具有重要作用。因为异柠檬酸裂解酶活性受到部分抑制时,L-谷氨酸的分泌产生量有所下降;当异柠檬酸裂解酶编码基因被敲除后,L-谷氨酸的分泌产生量大幅下降;由此得出的进一步结论是在以生物素限量法诱导L-谷氨酸的过量合成时,菌体细胞更加需要乙醛酸循环的回补功能。可能原因是生物素限量供应时,以生物素为辅酶的丙酮酸羧化酶活性几乎为零,而另一条回补途径从磷酸烯醇式丙酮酸生成草酰乙酸的反应具有较强的逆向反应; (2) 增加异柠檬酸裂解酶编码基因的拷贝数,该酶活性增加近六倍,细胞的L-谷氨酸分泌产生量只是略有提高,可能是因为对于L-谷氨酸的生物合成来说,乙醛酸循环既是回补途径,同时也是“支流代谢”,L-谷氨酸的产生量并非随乙醛酸循环途径的增强而呈直线上升; (3) 虽然培养基中添加琥珀酸对异柠檬酸裂解酶具有抑制作用,但是,由于存在琥珀酸和琥珀酰辅酶A共同作用的综合效应(琥珀酰辅酶A对α-酮戊二酸脱氢酶具有抑制作用),L-谷氨酸产生量没有下降,基本上是随着琥珀酸的增加而上升,细胞生长减弱,残糖增加;更多还原

【Abstract】 Since the discovery of Corynebacterium glutamicum as an efficient glutamate-overproducing microorganism in 1957, the production of L-amino acids by the fermentative method has become one of the most important research-target of industrial microbiology over the last four decades. Nevertheless, the mechanism of L-glutamate production and excretion by the microorganism is still unclear.It has been suggested that metabolic flux through the anaplerotic pathways could be limiting for amino acids synthesis. The carbon flux supplying oxaloacetate is very complicated in coryneform bacteria because several potential anaplerotic enzymes / pathways exist: phophoenolpyruvate carboxylase (PEPC), pyruvate carboxylase (PC), and the glyoxylic cycle which all contribute to carbon flux through oxaloacetate. Less research reports about the relationship between the glyoxylate cycle and L-glutamate synthesis are available up to the present and their results are contradictory. It will help elucidate the mechanism of L-glutamate overproduction and guide the breeding of L-glutamate high yield strains to investigate the role played by the glyoxylate cycle during L-glutamate synthesis.A series of research work was carried out about the importance of the glyoxylate cycle during L-glutamate synthesis and main results are as follows:1. Since L-glutamate production decreased when isocitrate lyase was partially inhibited and much less L-glutamate was produced and excreted when the glyoxylate cycle was blocked, the glyoxylate cycle of Corynebacterium glutamicum was active and played an important role during L-glutamate production when grown on glucose as a sole carbon source. It was extremely possible that the glyoxylate cycle played an important anaplerotic function during L-glutamate overproduction especially under biotin limitation, because pyruvate carboxylase is less active when biotin concentration in the culture medium is limited.2. Although isocitrate lyase activity increased by six fold when ace A gene was overexpressed, L-glutamate production increased by 12.82%. It seemed that the glyoxylate cycle played a bifunctional role for L-glutamate production: anaplerotic function and divergent flux.3. Although succinate partially inhibited isocitrate lyase, L-glutamate production and residual glucose increased, cell growth decreased with the increase of succinate concentration in the fermentative broth. The reason for these may be thatsuccinyl coenzyme A inhibited the 2-ketoglutarate dehydrogenase.4. Addition of acetate (?￡ lOg/L when concentration of glucose was 50g/L) in the culture medium promoted cell growth but was disadvantageous to L-glutamate production. This effect might be due to the enhancement of TCA cycle and 2-ketoglutarate dehydrogenase by acetate and the decrease of flux into L-glutamate synthesis. However, both cell growth and L-glutamate were detrimentally affected with further addition of acetate. It might be because of the acetate function as an uncoupler of the transmembrane pH gradient.5. Simultaneous supplement of acetate and succinate promoted L-glutamate production. It seemed that TCA cycle was strengthened and succinyl coenzyme A inhibited the 2-ketoglutarate dehydrogenase, hence the metabolic flux was directed to L-glutamate synthesis.6. A temperature and lysozyme sensitive strain C.glutamicum WT^IL was constructed. It was induced to overproduce L-glutamate after a fermentative temperature shift from 30D to 38D under the presence of an excess of biotin. Simultaneous supplement of acetate and succinate were also effective during L-glutamate production by the temperature and lysozyme sensitive strain C.glutamicum WT A L.7. Isocitrate lyase, isocitrate dehydrogenase, 2-ketoglutarate dehydrogenase and glutamate dehydrogenase were analysed. The results indicated that the increase of the flux from citrate to 2-ketoglutarate was the main reason for the cells to produce more L-glutamate if the anaplerotic function was assured.更多还原