枯草芽孢桿菌基因修飾及木糖發(fā)酵生產(chǎn)核黃素的研究
發(fā)布時間：2018-12-31 11:36
【摘要】：核黃素是重要的B族維生素之一,作為醫(yī)藥、動物飼料營養(yǎng)強(qiáng)化劑和食物添加劑而具有普遍的應(yīng)用。本文以提高核黃素發(fā)酵菌株的生產(chǎn)性能為目的,研究了枯草芽孢桿菌中核黃素合成途徑及木糖代謝途徑相關(guān)基因的遺傳修飾,對核黃素發(fā)酵的影響;也考察了木糖/蔗糖共代謝進(jìn)行核黃素發(fā)酵的可行性。在枯草芽孢桿菌LX34的ribC基因編碼區(qū)中引入G596A堿基突變,構(gòu)建了重組菌LXZ-1。發(fā)酵結(jié)果顯示,G596A點(diǎn)突變使核黃素產(chǎn)量提高到0.24 g/L,相比于在該基因啟動子區(qū)域引入堿基突變(-35區(qū),T→C)的菌株LX36,產(chǎn)量提高了50%。而且啟動子-35區(qū)點(diǎn)突變與G596A突變不能同時存在。在LXZ-1菌株中過表達(dá)了ribA基因,得到重組菌株LXZ-2,核黃素產(chǎn)量提高到0.48 g/L,但會產(chǎn)生細(xì)胞自溶現(xiàn)象。共表達(dá)ribA-ribH基因所構(gòu)建的LXZ-3菌株,核黃素產(chǎn)量提高到0.9 g/L,且細(xì)胞自溶現(xiàn)象消失。共表達(dá)ribA-ribD基因的重組菌不能構(gòu)建。共表達(dá)ribA-ribE基因的重組菌,核黃素產(chǎn)量與出發(fā)菌相比基本保持不變。結(jié)果表明,核黃素合成途徑的中間代謝物合成5-氨基-6-(5'-磷酸核糖氨基)尿嘧啶可能具有細(xì)胞毒性。與其它碳源相比,木糖作為碳源時核黃素產(chǎn)量最高,但生物量有所降低;當(dāng)以木糖/蔗糖作為混合碳源進(jìn)行共代謝發(fā)酵時生物量水平基本保持正常,核黃素產(chǎn)量進(jìn)一步提高。結(jié)果說明,蔗糖與木糖共代謝,能夠改善前體物5-磷酸核酮糖供給,是進(jìn)一步促進(jìn)核黃素高產(chǎn)及收率的有效手段。以B.subtilis LXZ-3為出發(fā)菌,在染色體上整合過表達(dá)對木糖運(yùn)輸?shù)鞍拙幋a基因araE、表異構(gòu)酶編碼基因rpe以及原位組成型表達(dá)木糖操縱子xyl分別構(gòu)建了重組菌B.Subtilis LXZ-4、LXZ-5、LXZ-6。相比于出發(fā)菌,核黃素在這些重組菌的搖瓶發(fā)酵中產(chǎn)量都顯著降低。結(jié)果表明,在調(diào)節(jié)蛋白AraR缺失的前提下,以木糖作為主要碳源,B.subtilis固有的木糖吸收和代謝能力不再是限制因素來制約核黃素過量合成。在5L發(fā)酵罐中,以B.subtilis LXZ-3/pMX45為出發(fā)菌,進(jìn)行核黃素分批發(fā)酵。當(dāng)以8%的蔗糖作為發(fā)酵碳源時,核黃素最高產(chǎn)量為2 g/L。而當(dāng)以6.5%木糖+1.5%蔗糖作為發(fā)酵碳源進(jìn)行共代謝時,在發(fā)酵70 h后核黃素最高產(chǎn)量達(dá)到3.6 g/L,比蔗糖發(fā)酵提高了80%。結(jié)果表明,以木糖為主的木糖/蔗糖共代謝,對于提高核黃素發(fā)酵產(chǎn)量有顯著效應(yīng)。
[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.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：TQ466;TQ929
本文編號：2396517
[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.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：TQ466;TQ929
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