【作者】 张晋宇；

【导师】 陈国强；

【作者基本信息】 清华大学 ， 生物学， 2005， 硕士

【摘要】 透明质酸是一种在医药及化妆品等领域有着广泛应用的生物高分子物质,其具有优良的保湿性,生物相容性等性质,现阶段透明质酸的生产主要依靠 C类链球菌的发酵,具有成本低,质量好的优点,但是在发酵工业中,碳源向透明质酸转化率低,乳酸大量生成,是制约发酵产量的主要问题。本文为解决发酵工业中的相关问题,获得更高的透明质酸生产效率进行了相关的研究,主要研究成果如下:克隆了 C 类链球菌兽疫链球菌透明质酸合成相关基因 hasB,其编码 UDP-葡萄糖脱氢酶,通过 T7 启动子在大肠杆菌中进行表达具有正确的活性,其蛋白序列与Streptococcus pyogene和Streptococcus uberis的HasB序列分别有着63.1%和 70.6%的相似性,而 hasB 在基因进化树中处于一个相对比较独立的位置,这些结果表明兽疫链球菌的 hasB 具有比较特殊的性质,这可能与 C 类链球菌具有更高的透明质酸合成能力是相关的。在大肠杆菌中同时表达了聚羟基丁酸 PHB 合成基因 phbCAB 和透明质酸合成基因 hasABC,希望使两条代谢途径在辅酶(NAD(P)/NAD(P)H)水平上偶联起来,结果显示虽然积累了大量的 PHB,但是几乎不能检测到透明质酸的合成,可以认为大肠杆菌不适合作为透明质酸生产的宿主菌。以阳性菌表达质粒 pEU308 为载体,将 Wautersia eutropha 的 PHB 合成基因phbCAB 在兽疫链球菌中进行表达,酶活测定表明在链球菌中 PhbA,PhbB 具有正确的活性,而 PhbC 没有活性,因此没有 PHB 的积累。PhbB 催化的反应中,伴随着 NADPH 向 NADP 的转化,因此 PhbAB 蛋白的作用,为细胞提供了一条氧化力的再生途径。在摇瓶培养中,细菌处于一个氧气较为充足的环境中,具有利用氧气氧化 NADH 和乳酸合成两条途径来获得氧化力,而且前一条途径所占比例较大,因此乳酸合成所受的影响不大;在发酵罐培养中,乳酸合成途径几乎是唯一的氧化力再生途径,因此 phbCAB 基因的导入,将乳酸合成从 64 g/L降低到 41 g/L,同时由于细胞内碳流的改变,透明质酸产量也提高了 1 g/L 左右。此结果表明W. eutropha的PHB合成途径能够作为一个有效的调节细胞氧化还原力水平,影响产物合成的工具。更多还原

【Abstract】 Hyaluronic acid (HA) is a high molecuLar weight polysaccharide, whichis widely used in medicine and cosmetics. It has good ability of keeping waterand biocompatibility. These years, hyaluronic acid is industrially produced byfermentation of Streptococci. It has the advantage of low cost and goodquality. But in the fermentation process, large part of carbon source wastransformed to lactate and the productivity of HA is low. This study was tosolve these problems and make some improvements on HA production.Following results were achieved:hasB, which encodes the UDP-glucose dehydrogenase, was cloned fromthe genome of Steptococcus zooepidemicus (S. zooepidemicus). It wasexpressed in E.coli under T7 promoter and displayed the right activity. Theprotein sequence has 63.1% and 70.6% similarity to HasB of S. pyogene and Suberis, respectively. It had a special position in the phylogenetic tree based onUDP-glucose dehydrogenase gene sequence. These results means that thehasB of S. zooepidemicus has special characteristics, which is correlated withthe high HA synthesis ability of group C Streptococci.The PHB synthesis gene of Wautersia eutropha, phbCAB, wascoexpressed with the HA synthesis gene, hasABC, in E. coli. These twopathways could be coupled at the level of coenzyme. Although there was largeamount of PHB accumulation, no HA was synthesized. It means that E. coli isnot a suitable host for the production of HA.phbCAB gene was expressed in S. zooepidemicus harboring plasmidpEU308, an expression vector in Gram-positive bacteria. The activities ofPhbA and PhbB were, respectively, 3.13 U/mg and 1.23 U/mg. But no PhbCactivity was detected. And as a result, there was no PHB accumulation yet.NADPH was transformed to NADP in the reaction catalyzed by PhbB. Theexpression of phbAB provided an oxidation potential regeneration pathway. Inflask fermentation, the cells were in an oxygen-rich enviroment. There weretwo pathways for the bacteria to regenerate oxdation potential: one was thatNADH was oxidized to NAD by oxygen, the other was the lactate pathway.The first pathway could provide most part of oxidation potential. There wasno obvious influence on lactate formation. While in fermentor study, lactatepathway was almost the only way to regenerate oxidation potential, theintroduction of phbCAB gene reduced the lactate formation from 64 g/L to 41g/L. At the same time, the change of celluLar carbon flux led to about 1 g/Lincrease on HA production. These results indicated that the PHB synthesispathway could act as a global regulator to adjust the celluLaroxidation/reduction potential.更多还原