【作者】 刘东风；

【导师】 刘双江；

【作者基本信息】 中国科学技术大学 ， 生物化学和分子生物学， 2014， 博士

【摘要】 以莽草酸为原料可以通过化学合成或微生物合成法生产抗禽流感病毒药物—GS4104(达菲)。莽草酸也是生物代谢途径中重要的中间体,是合成许多生物碱以及芳香氨基酸(L-Phe, L-Trp和L-Tyr)的原料。作为一个商业产品,莽草酸主要从八角茴香中提取。然而,利用微生物发酵方法生产莽草酸受到越来越多的关注。起始于PEP和E4P,莽草酸通过莽草酸途径的前四步反应合成而来。对于微生物生长来说,莽草酸途径是必须的,目前仍然没有关于微生物自然累积莽草酸的报道。目前,利用微生物生产莽草酸的工作主要集中在大肠杆菌,而利用枯草芽孢杆菌生产莽草酸鲜有报道。关于高产莽草酸菌株的代谢流工作前人没有开展。本研究中,我们利用代谢工程的方法对枯草芽孢杆菌进行改造并测定了莽草酸高产菌株的代谢流分布。B. subtilis1A474的莽草酸激酶基因(aroI)有一个位点发生突变,积累1.5g/L的莽草酸；B. subtilis1A229的EPSP合成酶基因(aroE)有一个位点发生突变,积累0.6g/L的莽草酸。在B. subtilis1A474中分别单独过表达aroA, aroB, aroC或aroD,结果表明过表达aroD莽草酸产量增长最多；在B. subtilis1A229中分别单独过表达aroA, aroB, aroC, aroD或arol,结果表明过表达aroA莽草酸产量增长最多。在B. subtilis1A47407或B. subtilis1A22911中同时过表达aroA和aroD,莽草酸产量最高,分别为3.2g/L或1.5g/L。B. subtilis BSSA47407被用于下‘步研究。蔗糖为其最适碳源；最适蔗糖初始浓度为80g/L; B. subtilis BSSA47407批式补料发酵莽草酸产量达到17.8g/L,其值为普通摇瓶发酵莽草酸产量的5.6倍。13C代谢流分析菌株BSSA474a和BSSA47407揭示以下结果：从葡萄糖到莽草酸途径的流量由BSSA474a中的4.4%增加到BSSA47407中的6.8%；从葡萄糖到莽草酸的流量由BSSA474a中的1.9%增加到BSSA47407中的4.6%；从葡萄糖到3-脱氢莽草酸的流量由BSSA474a中的2.5%减小到BSSA47407中的2.2%；经过TCA循环的流量减小；而通过磷酸戊糖途径和糖酵解途径的流量变化不大。基于代谢流量结果,两个代谢改造目标被提出。实验结果表明：敲除pyk曾加了莽草酸产量,而过表达tkt并没有增加莽草酸产量。本研究利用SLIC的方法一次将aroD, aroB, aroA和zwf个基因连接到质粒pHCMC04上。B. subtilis BSSA47416莽草酸产量达到4.35g/L,其值为对照菌株B.subtilis BSSA47407莽草酸产量的1.4倍。更多还原

【Abstract】 Shikimic acid (SA) is a key chiral starting molecule for the synthesis of the neuramidase inhibitor GS4104against viral influenza. It is a key metabolic intermediate of the shikimate pathway for biosynthesis of aromatic amino acids (L-Phe, L-Trp, and L-Tyr) and many alkaloids in plants and microorganisms. As a commercial product, SA has been extracted from the fruits of the Illicium plant. However, microbial fermentation as an alternative process for SA production has attracted more and more interests.Beginning with phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), SA is synthesized via first4reactions of the shikimate pathway. This shikimate pathway is essential to bacterial growth and there has been no report on excessive accumulation of SA by microorganisms. Microbial production of SA has been extensively investigated in Escherichia coli, and to a less extent in B.subtilis. However, metabolic flux of the high SA-producing strains has not been explored. In this study, we constructed with genetic manipulation and further determined metabolic flux with13C-labeling test of high SA-producing B. subtilis strains.B. subtilis1A474had a mutation in SA kinase gene (arol) and accumulated1.5g/L of SA. B. subtilis1A229had a mutation in EPSP synthase gene (aroE) and accumulated0.6g/L of SA. Overexpression of plasmid-encoded aroA, aroB, aroC or aroD in B. subtilis1A474revealed that aroD had the most significantly positive effects on SA production. Overexpression of plasmid-encoded aroA, aroB, aroC, aroD or arol in B. subtilis1A229revealed that aroA had the most significantly positive effects on SA production. Simultaneous overexpression of genes for3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroA) and SA dehydrogenase (aroD) in B. subtilis BSSA47407or BSSA22911resulted in SA production of3.2g/L or1.5g/L. B. subtilis BSSA47407was used for further research. Sucrose was the optimal initial carbon source and the optimal initial sucrose concentration was80g/L Fed-batch of B. subtilis BSSA47407gave the result of17.8g/1shikimic acid titer, which was5.6-fold higher than the corresponding value in a simple baffled flask.13C-Metabolic flux assay (MFA) on the two strains BSSA474a and BSSA47407indicated the overall conversion of glucose into shikimate pathway increased to6.8%in strain BSSA47407from4.4%in strain BSSA474a. Consequently, the metabolic flux to SA increased from1.9%in strain BSSA474a to4.6%in strain BSSA47407. Correspondingly, the metabolic flux from E4P and PEP into DHS decreased from 2.5%in the parent strain BSSA474a to2.2%in strain BSSA47407. The carbon flux through tricarboxylic acid cycle significantly reduced, while responses of the pentose phosphate pathway and the glycolysis to high SA production were rather weak, in the strain BSSA47407. Based on the results from MFA, two potential targets for further optimization of SA production were identified. Experiments on genetic deletion of phosphoenoylpyruvate kinase gene confirmed its positive influence on SA production, while the overexpression of the transketolase gene did not lead to increase in SA production.SLIC was applied for the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing. aroD, aroB, aroA and zwf were efficient and reproducible assembly into pHCMC04simultaneously by SLIC. B. subtilis BSSA47416gave the best results of4.35g/1shikimic acid titer, which was1.4-fold higher than the corresponding values in B. subtilis BSSA47407.更多还原