Effects Of Cofactor And Episomal Plasmids On The Production Of Vitamine C

In this thesis, the mixed culture of Ketogulonigenium vulgare WSH-001 and Bacillus megaterium WSH-002 were used to synthesis 2-keto-L-gluconic acid (2-KLG) in the production of vitamine C by means of two-step fermentation process. A series of experiments were performed to enhance the 2-KLG production and explore the relationship of the two strains, mainly including the effects of rare earth elements and episomal plasmids of B. megaterium on the 2-KLG production. The main results were described as follows:1.The effects of rare earth elements on the synthesis of 2-KLG were investigated by adding different rare earth ions and chelate complexes in the fermentation medium, a furture study of La3+ which had the most significant effect was performed. The results indicated that low concentrations≤5 mmol/L) of different rare earth ions had certain stimulation on the 2-KLG synthesis; EDTA-La and lanthanum citrate inhibited 2-KLG synthesis while lanthanum oxalate promoted the final production of 2-KLG by 6.4% at a concentration of 1 mmol/L. The cell growth conditions at the mid- and late- fermentation stages were remarkably improved with the addition of 5 mmol/L of La3+. At the same time, final 2-KLG output was increased and the fermentation time was reduced. The main reason of these results was that La3+inhibited the activity of 2-KLG reductase, thus decreased the degradation of 2-KLG. When 5 mmol/L of La3+ was fed to 3.6 L fermentor for batch fermentation, the L-sorbose consumption rate and the 2-KLG productivity were increased by 12.4% and 14.8 % compared with that of the control, respectively. Besides, the fermentation time was declined by 11.1%.2.Genome sequencing showed that there were three wild-type plasmdis in B. megaterium WSH-002, namely, pBM-001, pBM-002 and pBM-003. The three plasmids were 74,613 bp, 9,699 bp,7,006 bp and contained 69,11,14 ORFs, respectively. To investigate the role of wild-type episomal plasmids of B. megaterium WSH-002 on 2-KLG production, we performed plasmids elimination experiments on B. megaterium WSH-002. Two partly-eliminated derivatives D1 which had lost pBM-001 and D13 which had lost pBM-001 and pBM-003 were abtained by alternative treatment of temperature and SDS. A completely-eliminated derivative strain D123 was screened eventually by acridine orange treatment.3.The morphology and growth characteristics were compared among the three different plasmid-eliminated B. megaterium derivatives D1, D13, D123 and B. megaterium WSH-002 as well as the 2-KLG production when combined with K. vulgare. The results showed that the derivatives all had the same morphology and growth characteristics with the parent strain, suggesting that the plasmids were dispensable for the growth of B. megaterium. However, comparisons of the 2-KLG production indicated that the plasmids pBM-002 and pBM-003 of B. megaterium obviously affected the production of 2-KLG while the plasmid pBM-001 had no effect on it. COG functional classification and protein comparisons of all of the 96 ORFs on the three wild-type episomal plasmids were carried out according to the sequencing data. Many of the genes on the plasmids, especially pBM-002 and pBM-003 were of unknown functions or cannot be assigned to a specific metabolic pathway. These results indicated that the wild-type episomal plasmids of B. megaterium WSH-002 had some special features.