Construction Of Alpha-Ketoglutarate Producing Strain And Regulation Of Fermentation Conditions

In the Krebs cycle, a-ketoglutarate (a-KG) is a key intermediate and it plays a pivotal role in the metabolism of carbon and nitrogen.That’s the reason why it plays a significant role in cell growth and metabolism. a-KG is widely used in medicine, food, cosmetics, chemical synthesis, agriculture and other fields. Chemically synthesized and produced by microbe are the primarily production method of a-KG. Currently, eukaryotic microbes are the major strain that studied in fermentation on the ketone derivative. In domestic and overseas, there are few reports on the research of a-KG with Corynebacterium glutamicum. In this paper, we studied the accumulation of a-KG in the aspects of molecular modification and controlling fermentation process with Corynebacterium glutamicum GDK-9△gdh, a strain with gene encoding glutamate dehydrogenase I knocked out.On the basis of the strain GDK-9△gdh, we knocked gene gdhA which encoding glutamate dehydrogenase II and successfully constructed strain GDK-9△gdh△gdhA. In shake flask and fermenter fermentation experiments, the L-glutamic acid content dropped by 5.72% and 15.78%, the amount of a-KG increased 10.99% and 7.49%, the conversion rate on glucose inproved by 7.13% and 7.36% of strain GDK-9△gdh△gdhA compared with the starting strain, respectively. Experimental results showed that the deleting of gene gdhA was helpful to the decrease accumulation of L-glutamate and improvement of the utilization of glucose.Alanine aminotransferase encoding by gene alaT catalyzeing alanine and a-KG to L-glutamate acid.To prevent the conversion of a-KG to L-glutamate, we got the alaT mutation strain GDK-9AgdhAalaT upon the original strain GDK-9△gdh with genetic engineering. The results of fermentation experiment showed that the growth and a-KG producing were inhibited while the synthesis of glutamate was reduced with the mutation strain compared to starting strain. It indicated that alanine aminotransferase was very important to the growth of bacteria and this enzyme was not the main reaction of L-glutamate accumulation upon the gdh mutation strain GDK-9Agdh.The by-product could also be synthesized with the glutamate synthase catalyzes a-KG and glutamine to L-glutamate. We knocked out the gene gogat which encoding glutamate synthase and got srtain GDK-9 △gdh△gdhA△gogat. The results of fermentation experiment indicated that the growth and a-KG producing were inhibited while the synthesis of glutamate was dropped with the mutation strain. It showed that the glutamate synthase was concerned with the regulation of bacterial growth factor and the massive accumulation of glutamate.Successfully, we constructed the aceA gene mutation strain GDK-9AgdhAaceA which could not synthesize isocitrate lyase upon the starting strain GDK-9△gdh. In the shake flask and fermenter fermentation experiments, the aceA deleted mutation resulted in an enhancement of a-KG producing by 17.57% and 4.07% and a higher of 14.39% and 9.09% of conversion rate on glucose compared with the original strain, respectively. The result indicated that the deleted of gene aceA could maximize carbon metabolic flow and rapidly guide to α-KG.To further improve the ability of bacterial cells accumulate α-KG， we overexpressed gene prpc that encoding citrate synthase with plasmid pXMJ19 and constructed strain GDK-9AgdhAaceApXMJl9prpc and GDK-9△gdh△aceApXMJ19. The fermentation experiments on shake flask and fermenter fermentation showed that the strain GDK-9△gdh△aceApXMJ19prpc had a higher 10.19% and 3.69% of a-KG producing and a more 7.68% and 6.51% conversion rate on glucose than the contrastive strain GDK-9△gdh△aceApXMJ19.We studied the accumulation of a-KG under the double phase pH and biotin control strategy with genetically engineered strain C.glutamicum GDK-9△gdh△gdhA. The result showed that compared with single phase pH control, double phase pH control got an L-glutamate dropped of 80.11%, a-KG increase of 67.31%, the conversion rate on glucose increase of 55.86%. With the condition of double phase pH control, we studied and determined the optimal biotin concentration (7 μg/L) in the initial fermentation medium. The most suitable biotin supplementary concentration was 5 μg/L during the middle and late fermentation (16h to 26h). In the final fermentation, the conversion rate on glucose increased by 7.82% with double phase pH and biotin control strategy compared to double phase pH control.