兽疫链球菌透明质酸代谢途径中基因功能研究-发酵工程专业论文.docxVIP

ABSTRACTWhile ABSTRACT While Streptococcus zooepidemicus is the main strain for industrial production of hyaluronic acid(HA)．the genes function of biosynthesis pathway of HA has not been svstematically studied．Recently，the complete genome sequences of several S zooepidemicus species have been released and the biosynthesis pathway of HA was elucidated．which provide the theoretical basis for the study on genes functions．In this paper．S zooepidemicus ATCC39920 was used．The main results are as follows： (1)All of the genes among biosynthesis pathway of HA were found in S zooepidemicus genome through sequences BLAST in NCBI and they were all expressed through RT—PCR．Analysis revealed that UDP—glucose一1-phosphate uridylyltransferase， phosph091ucomutase and bifunctional protein(Acetyltransferase and Uridyltransferase)are encoded by two genes，respectively,while the others has only one gene；five genes(hasA， hasB，hasCl，hasD，hasL3 are arranged in a contiguous fashion in has operon，while the others are arranged far away． (2)Functional characterization of the two different genes pgm and pgmA encoding phosphoglucomutase were studied．From the phenotype and fermentation analysis，we could see that the growth of△pgm was better while the growth of ApgmApgmA was weaker than that of wild type strain，and ApgmA and ApgmApgmA dispayed nonmucoid colony morphology and no HA were detected；furthermore，Pgm and PgmA were all expressed， purified and enzymatic detected in vitro，and the result showed that the affinity of glucose．．1．．phosphate with PgmA was better than that Pgm；the enzymatic analysis of PGM in vivo showed that the PGM activity of Apgm and ApgmA were 38．1％and 69．6％lower than that of wild type(1．534 U／mg)，respectively,while ApgmApgmA had negligible PGM ativity．All of me results indicated that both genes pgm and pgmA are required for PGM activity and pgmA plays a much more important role than pgm in S zooepidemicus· (3)Functional characterization of glmS encoding glucosamine-6一phophate