枯草芽孢杆菌基因修饰及木糖发酵生产核黄素的研究

发布时间：2018-12-31 11:36

【摘要】：核黄素是重要的B族维生素之一,作为医药、动物饲料营养强化剂和食物添加剂而具有普遍的应用。本文以提高核黄素发酵菌株的生产性能为目的,研究了枯草芽孢杆菌中核黄素合成途径及木糖代谢途径相关基因的遗传修饰,对核黄素发酵的影响;也考察了木糖/蔗糖共代谢进行核黄素发酵的可行性。在枯草芽孢杆菌LX34的ribC基因编码区中引入G596A碱基突变,构建了重组菌LXZ-1。发酵结果显示,G596A点突变使核黄素产量提高到0.24 g/L,相比于在该基因启动子区域引入碱基突变(-35区,T→C)的菌株LX36,产量提高了50%。而且启动子-35区点突变与G596A突变不能同时存在。在LXZ-1菌株中过表达了ribA基因,得到重组菌株LXZ-2,核黄素产量提高到0.48 g/L,但会产生细胞自溶现象。共表达ribA-ribH基因所构建的LXZ-3菌株,核黄素产量提高到0.9 g/L,且细胞自溶现象消失。共表达ribA-ribD基因的重组菌不能构建。共表达ribA-ribE基因的重组菌,核黄素产量与出发菌相比基本保持不变。结果表明,核黄素合成途径的中间代谢物合成5-氨基-6-(5'-磷酸核糖氨基)尿嘧啶可能具有细胞毒性。与其它碳源相比,木糖作为碳源时核黄素产量最高,但生物量有所降低;当以木糖/蔗糖作为混合碳源进行共代谢发酵时生物量水平基本保持正常,核黄素产量进一步提高。结果说明,蔗糖与木糖共代谢,能够改善前体物5-磷酸核酮糖供给,是进一步促进核黄素高产及收率的有效手段。以B.subtilis LXZ-3为出发菌,在染色体上整合过表达对木糖运输蛋白编码基因araE、表异构酶编码基因rpe以及原位组成型表达木糖操纵子xyl分别构建了重组菌B.Subtilis LXZ-4、LXZ-5、LXZ-6。相比于出发菌,核黄素在这些重组菌的摇瓶发酵中产量都显著降低。结果表明,在调节蛋白AraR缺失的前提下,以木糖作为主要碳源,B.subtilis固有的木糖吸收和代谢能力不再是限制因素来制约核黄素过量合成。在5L发酵罐中,以B.subtilis LXZ-3/pMX45为出发菌,进行核黄素分批发酵。当以8%的蔗糖作为发酵碳源时,核黄素最高产量为2 g/L。而当以6.5%木糖+1.5%蔗糖作为发酵碳源进行共代谢时,在发酵70 h后核黄素最高产量达到3.6 g/L,比蔗糖发酵提高了80%。结果表明,以木糖为主的木糖/蔗糖共代谢,对于提高核黄素发酵产量有显著效应。
[Abstract]:Riboflavin is one of the important B vitamins, which is widely used as medicine, animal feed nutrition fortifier and food additive. In order to improve the production performance of riboflavin fermentation strain, the genetic modification of riboflavin synthesis pathway and xylose metabolic pathway gene in Bacillus subtilis was studied, and the effect of riboflavin fermentation on riboflavin fermentation was studied. The feasibility of riboflavin fermentation by xylose / sucrose co-metabolism was also investigated. The G596A base mutation was introduced into the coding region of ribC gene of Bacillus subtilis LX34, and the recombinant strain LXZ-1. was constructed. The results of fermentation showed that point mutation of G596A increased riboflavin production to 0.24 g / L, which was 50% higher than that of the strain that introduced base mutation (-35 region, T TOC) into the promoter region of the gene. Moreover, the point mutation of promoter-35 and the mutation of G 596A could not exist at the same time. After overexpression of ribA gene in LXZ-1 strain, the recombinant strain LXZ-2, riboflavin production was increased to 0.48 g / L, but cell autolysis was produced. The riboflavin production of the LXZ-3 strain co-expressed with ribA-ribH gene was increased to 0.9 g / L, and the phenomenon of cell autolysis disappeared. The recombinant strain which co-expressed ribA-ribD gene could not be constructed. The production of riboflavin in the recombinant strain co-expressing ribA-ribE gene remained basically unchanged compared with that of the original strain. The results showed that the intermediate metabolites of riboflavin synthesis pathway may be cytotoxic to 5-amino-6- (5-phosphoribose amino) uracil. Compared with other carbon sources, the yield of riboflavin was the highest when xylose was used as carbon source, but the biomass decreased, and when xylose / sucrose was used as mixed carbon source for co-metabolic fermentation, the biomass level remained basically normal, and the yield of riboflavin increased further. The results showed that the co-metabolism of sucrose and xylose could improve the supply of 5-phosphate, which was an effective method to promote the yield and yield of riboflavin. The recombinant strain B.Subtilis LXZ-4, was constructed by using B.subtilis LXZ-3 as the starting strain, the rpe gene encoding xylose transport protein encoding gene araE, epiisomerase and the in situ expressed xylose operon xyl, which were over-expressed on the chromosomes. LXZ-5,LXZ-6. The yield of riboflavin in shaking flask fermentation of these recombinant bacteria was significantly lower than that of original bacteria. The results showed that under the premise of regulating the deletion of AraR, xylose was the main carbon source, and the inherent xylose absorption and metabolism ability of B.subtilis was no longer a limiting factor to restrict riboflavin excess synthesis. In 5 L fermenter, riboflavin was fermented in batches with B.subtilis LXZ-3/pMX45 as starter strain. When 8% sucrose was used as carbon source, the highest yield of riboflavin was 2 g / L. However, when 6.5% xylose 1.5% sucrose was used as carbon source for fermentation, the highest yield of riboflavin reached 3.6 g / L after 70 h fermentation, which was 80% higher than that of sucrose fermentation. The results showed that xylose / sucrose co-metabolism had a significant effect on the increase of riboflavin fermentation yield.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TQ466;TQ929

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