Studies On Genes Related To Sorbitol Metabolism In Gluconobacter Oxydans

Objective: The production of Vitamin C has been treated as a pillar industrial procedure in medical field. One of the dominated method which has been carried out widespread is two-step fermentation, originated from China in the 1970s. The two-step method involves the two different fermentation associated with three different microbes. The major troubles of this method due to the complicated procedure as well as the contamination of microbial metabolism. Therefore, the mainstream of industrial production of Vitamin C tends to be simplified two-step fermentation into one-step fermentation. In our laboratory, one-step fermentation theoretically achieved succeefully in preliminary work. We managed to import 2-KGA-produce gene cluster into Gluconobacter oxydans so that the host bacteria could directly convert D-sorbitol to L-sorbose. However, the low converting rate of D-sorbitol to L-sorbose which was less than 10%. Thus, doing researches on sorbitol related genes and its metabolic pathway were placed on priority in this study. The excepted results were assumed through enhancing the major metabolic pathway and interdicting side pathway of metabolism when focusing on improving the converting rate from D-sorbitol to L-sorbose.Methods: (1) A promoter based on the report of Lulu Shi and four kinds of common promoters of Gluconobacter oxydans were screened, in the meantime,the other three promoters, whose activity ranked in the top three of transcription level were selected by sequencing the transcriptome of Gluconobacter oxydans.The availability and promoter strength of those promoters were evaluated, by insertion into the plasmid pBBR1MCS5 of expression of L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) of Gluconobacter oxydans. (2) According to the genome annotation variance and difference analysis transcriptome associating with literature retriving, there might be 12 types of metabolic enzymes were involved in sorbitol metabolism in total, including three different kinds of sorbose dehydrogenase (SDH),L-sorbose reductase (SR), xylitol dehydrogenase (XDH) and 2-keto gulonate reductase (HADH). Our project group set up a fast and high efficient homologous recombination method to delete the gene of related enzymes. After evaluating the growth level and metabolism level of sorbose and 2-KGA of the knockout strains with sorbitol and sorbose as carbon source. The gene of knockout strains with higher phenotypic variation were selected and were used for genetic complementation. The plasmid of expression of L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) was introduced into some of knockout strains, following by analyzing of the change of metabolism level of sorbose and 2-KGA. In order to establish markerless gene deletion system and achieve knocking-out the multiple related genes. The work of deletion and complementation of uracil phosphoribosyltransferases transferase (upp) was processed. In additional, the other unknown genes which were related to side metabolism had been screened by Tn5 transposon.Results: (1) The samples were cultured in tube and shake flask respectively,when compared with the production of 2-KGA from nature promoter, the increased proportion of production of 2-KGA from PcsbD promoter was 15%and was 19% in each equipments respectively. (2) Recently, 10 gene segments of Gluconobacter oxydans H24 and 11 gene segments of Gluconobacter oxydans 1.637 had been knocked-out respectively. H24?sr3 slowly growed in the early stage when treated with sugar, while 1.637?sr3 also showed a slow growth in both carbon source condition. The accumulation of L-sorbose had been increased in the mutant strains containing srl, sr2, sr3 of H24 and 1.637.Among these results, the accumulation of L-sorbose of 1.637?sr3 was more than the rest. Additionally, The results of complementation of 1.637?sr3 indicated the effect of gene sr3 existed in the growth of strains and ingestion of L-sorbose. After introducing the of expression of L-sorbose dehydrogenase(SDH) and L-sorbosone dehydrogenase (SNDH) into some of knockout strains,the product of 2-KGA of 1.637?sr3 strains was more than the product of 2-KGA of wild strains. (3) The work of deletion and complementation of uracil phosphoribosyltransferases transferase (upp) had been accomplished. A mutant library of pBBR1MCS5-sndhsdh/1.637 strains was constructed by random transposon Tn5 wherein 20 suspected strains which might contain stronger ability of producing 2-KGA were screened.Conclusions: (1) One strain containing PcsbD promoter whose ability of production of 2-KGA was higher than the nature promoter was selected, this can promote the conversion rate of D-sorbitol to 2-KGA and enhance the metabolism of main pathway. (2) Through evaluating the knockout stains, we assumed the gene sr3 is related to the growth and L-sorbose utilization. (3) The upp knockout strain was built, thereby confirming the gene upp can be treated as a negative selection maker.