【作者】 李健；

【导师】 班睿； 李琳；

【作者基本信息】 天津大学 ， 生物工程， 2018， 硕士

【摘要】 核黄素是B族维生素之一（维生素B₂）,作为动物饲料添加剂、食品添加剂和药物有重要的应用价值。迄今为止,核黄素的工业生产完全采用微生物发酵生产工艺,其中重组Bacillis subtilis是主要的核黄素工业生产菌株。尽管重组Bacillis subtilis发酵生产核黄素的工艺已经有超过20年的工业化历史,但是,重组菌的性能还有较大的提升空间;影响核黄素过量合成的某些代谢和分子遗传学机制还没有被完全揭示。本文在前期工作基础上,研究氮代谢、黄嘌呤分解、核黄素转化、糖异生和糖酵解代谢与核黄素过量合成的关系。在Bacillis subtilis LXZ7中过表达ure ABC操纵子,构建脲酶组成型表达菌株LR1。过表达tnr A基因,构建全局氮调节蛋白组成型表达菌株LR2和LR3。结果显示,促进细胞对尿素的利用不能提高核黄素产量。在菌株LXZ7中敲除puc ABCDE操纵子,构建黄嘌呤脱氢酶缺陷株LR01。进而敲除rib R基因,构建黄素激酶缺陷株LR11。结果显示,阻断黄嘌呤分解和核黄素的转化,能够部分解除速效有机氮源对核黄素合成的抑制作用。敲除LR11的ccp N和pck A基因,构建gap B基因去调控菌株LR22。进而分别用强启动子P_cdd和P_{lyt R}整合表达gap B基因,构建gap B基因过表达菌株LR23和LR24。菌株LR22的核黄素产量提高44.8%,达到3.33 g/L;菌株LR23提高53.9%,达到3.54 g/L;LR24下降31.5%,为2.18 g/L。敲除以上菌株的sr1基因,衍生菌株LR32、LR33和LR34,其中LR33的核黄素产量提高至3.78 g/L。在LR11中过表达fbp基因,构建果糖-1,6-二磷酸酶过表达菌株LR25、LR26、LR27和LR28。结果,fbp基因过表达的LR28核黄素产量提高至2.82 g/L。修饰LR11的gap A基因,得到3-磷酸甘油醛脱氢酶表达水平不同的菌株LR35、LR36、LR37、LR38、LR39和LR40。过度降低gap A基因表达水平,导致细胞生长缺陷;轻度降低gap A基因表达水平,对核黄素产量无明显提高。在Bacillis subtilis LR34中过表达gap A基因,构建3-磷酸甘油醛脱氢酶不同程度过表达菌株LR41和LR42,gap A基因表达水平较低的LR42菌株,核黄素产量略高于LR34,达到3.19 g/L。过表达gap B基因提高了3-磷酸甘油醛脱氢酶催化的糖异生反应,造成NADPH的额外消耗,进而导致磷酸戊糖途径代谢通量增加,改善了核黄素合成前体物的供给,最终使核黄素产量显著提高。更多还原

【Abstract】 Riboflavin（vitamin B2）is an important vitamin not only for pharmaceuticals but also for animal feed or food additives.The riboflavin produced by microbiological fermentation process,in which recombinant B.subtilis is the main strain.Although the study of riboflavin fermentation by B.subtilis over 20 years,the performance of recombinant bacteria could get better.Some metabolic and molecular genetic mechanisms affecting the synthesis of riboflavin have not been fully revealed.Based on the previous work,this article studies the relationship of nitrogen metabolism,xanthine decomposition,riboflavin conversion,gluconeogenesis and glycolytic metabolism to excessive synthesis of riboflavin.The ure ABC operon was overexpressed in B.subtilis LXZ7,get LR1.Overexpress the tnr A gene in LXZ7,the global nitrogen regulatory protein constitutive expression strains LR2 and LR3 were constructed.The results show that these two change disadvantage to synthesis of riboflavin.The puc ABCDE operon was knocked out in B.subtilis LXZ7,get LR01.The rib R was knocked out in LR01,construct strain LR11.The results showed that blocked the decomposition of xanthine and the conversion of riboflavin,which partly release the inhibitory effect of organic nitrogen sources on the synthesis of riboflavin.Knockout ccp N and pck A in B.subtilis LR11,construct strains LR22.Overexpress gap B by P_cdd and P_{lyt R} respectively,construct LR23 and LR24.The riboflavin yield of strain LR22 increased to 3.33 g/L.The riboflavin yield of strain LR23 is 3.54 g/L,and the yield of LR24 was 2.18 g/L,which decreased by 31.5%.Knock out the sr1 of the above strains and construct LR32,LR33 and LR34 strains.The yield of riboflavin in strain LR33 was the highest,reaching 3.78 g/L.Overexpress fbp in LR11,construct different expression level of fbp strains:LR25,LR26,LR27 and LR28.Riboflavin of LR28 increase to 2.82 g/L.Modify gap A of B.subtilis LR11,construct LR35,LR36,LR37,LR38,LR39 and LR40 with different gap A expression levels.The results showed that too low expression of gap A affected cell growth and decreased riboflavin production.Mild reduction of gap A gene expression slightly affect cell growth and riboflavin biosynthesis.Overexpress gap A gene in B.subtilis LR34,construct different gap A expression level:LR41 and LR42.LR42 gap A expression level lower than LR41,whose riboflavin yield was slightly higher than LR34,reaching 3.19 g/L.Therefore,maybe the 3-phospho glyceraldehyde dehydrogenase catalyzed by the gluconeogenesis consumed an additional NADPH,lead to the high metabolic flux of pentose phosphate pathway and the improvement of the supply of riboflavin precursors and the high production of riboflavin.更多还原