| As a basic amino acid of forming proteins,L-serine is widely used in medicine, food, cosmetics and many other industries, the market demand is increased. The current production methods of L-serine are protein hydrolysis, enzymatic conversion, chemical synthesis and microbial fermentation. With lower raw material prices, higher purity of products, easy to extract and so on, microbial fermentation becoming more promosing. Feedback-resistant serA was obtained by site-directed mutagenesis and the expression plasmid pSC-05 containing serA, serB, serC, pgk four genes was constructed, the flux of serine synthesis metabolic was increased. The random mutation screening was used and reduced specific activity of SHMT glyAm BJ-01 gene mutation strain was obtained from laboratory was choosed as a starting strain and sdaA gene of BJ-01 genomes was knocked out, so BJ-02 strain was constructed. Then the other four genes coding serine dehydration enzyme on BJ-02 genome were knocked out, build a series of L-Ser genetic engineering bacteria, the high-yield strains of L-Ser was obtained eventually. The main research contents and results were as follows:1. A feedback-resistant phosphoglycerate dehydrogenase(PDGH) encoding genes serA was obtained by site-directed mutagenesis, this mutant gene along with gene pgk encoding phosphoglycerate kinase(PGK), gene serC encoding phosphorus serine aminotransferase(PSAT), gene serB encoding phosphorus phosphatase(PSP) are all inserted into a low copy, temperature inducible expression plasmid pSC and the recombinant plasmid pSC-05 was constructed.2. By the method of red homologous recombination, the gene sdaA encoding serine dehydratase on E. coli BJ-01 genome was knocked out and E. coli BJ-02 was constructed. The plasmid pSC-05 was transformed into E. coli BJ-01 and E. coli BJ-02, the strains BJ-01/pSC-05 and BJ-02/pSC-05 was constructed. L-Ser production of BJ-01/pSC-05 and BJ-02 /pSC-05 was compared using fed-batch fermentation in 5L fermentor. The results show that BJ-02/pSC-05 in the maximum accumulation of L-Ser in the fermentation process is 11.4 g/L, it is showted that the capability of sdaA gene knocked out strains production L-Ser was significantly increased.3. E. coli BJ-02 genome except sdaA gene, other four genes sdaB, ilvA, tdcB and tdcG encoding serine dehydratase were knocked out separately through red homologous recombination. The recombinant plasmid pSC-05 has been transformed into four single knocked out gene of E. coli BJ-02 and four genetic engineerd strains were constructed. The four strains capability of L-Ser production were compared by fed-batch fermentation in 5 L fermentor. The results showed that the growth of strains was severely inhibited while gene ilvA was knocked out, and there is no accumulation of L-Ser was detected after the process of fermentation; When gene sdaB and tdcG were knocked out, cell growth were improved, but the difference was sdaB gene knocked out strain L-Ser yield further raised to 15.1 g/L, but the tdcG gene knocked out strain L-Ser yield decreased to 7.3 g/L; But cell growth and L-Ser yields were not significantly affected when gene tdcB was knocked out.4. The effects on different initial concentration of glucose to strain BJ-11/pSC-05 production and fermentation of L-Ser were also investigated. The results showed that with the initial concentration of glucose increasing, cell growth was increased gradually, when the initial concentration of glucose was 50 g/L, the maximum value of the cell OD600 reached to 64; In the case of the yield on L-Ser, when the initial concentration of glucose was 30 g/L, the yield of L-Ser up to 21.2 g/L; Secondly, when it was 50 g/L, the yield of L-Ser yield was 14.6 g/L; Therefore, the optimum initial concentration of glucose of BJ-11/pSC-05 yield of L-Ser was 30 g/L during fermentation process. |
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