【作者】 高弘；

【导师】 曹竹安；

【作者基本信息】 清华大学 ， 化学工程与技术， 2005， 博士

【摘要】 长链α,ω-二元酸是一种重要的精细化学产品。热带假丝酵母(Candida tropicalis)可以利用烷烃和脂肪酸通过生物氧化来合成从C11到C18的长链二元酸,代谢过程中二元酸的过度β-氧化会造成产品得率下降。本文以热带假丝酵母生产十三碳二元酸过程中的β-氧化途径为研究对象,对代谢工程的应用进行了探索研究,首次通过调节β-氧化与三羧酸循环之间的乙酰辅酶A转运系统,达到调节β-氧化代谢流量和提高烷烃转化率的目的,丰富了真核细胞传输系统的改造方法和工业微生物的技术手段;还从提供辅助能源取代β-氧化提供能量的角度出发,提出了有效提高产酸水平的方法。改造乙酰辅酶A转运系统的关键酶——肉毒碱乙酰转移酶(CAT),构建了CAT基因单拷贝缺失菌CZ-15,其生长和形态与原始菌F10-1相似。二元酸生产发酵实验中,CZ-15比原始菌F10-1的二元酸终浓度提高了18.3%,烷烃转化率提高了12.0 %,其遗传特性能够稳定传代。利用热带假丝酵母代谢网络对发酵数据进行分析,在CZ-15中由CAT转运进入三羧酸循环的乙酰辅酶A降为F10-1中的50 %左右,十三碳二元酸进入β-氧化的流量也明显降低,说明CAT酶活的下降有效减少了脂肪酸在β-氧化中的无谓消耗。构建了CAT基因双拷贝缺失菌CKC-11。在CKC-11中检测不到CAT酶活。当以烷烃为碳源时,CKC-11无法生长和产酸,说明CAT酶的完全阻断对细胞的能源供应系统造成了严重的影响。根据这一结果,进一步完善了热带假丝酵母中的二元酸代谢途径。尝试在热带假丝酵母的产酸期分别补加NaAc和蔗糖。结果表明,通过提供辅助能源的方式也可以提高热带假丝酵母的产酸和烷烃转化率。更多还原

【Abstract】 Long chainα,ω-dicarboxylic acids (DCA) are a series of important fine chemicals. Candida tropicalis can be used for oxidizing n-alkanes or fatty acids with carbon chain length from 11 to 18 to produce the corresponding DCA, in which excessiveβ-oxidation of products will decrease the productivity. Theβ-oxidation pathway in bioproduction of DCA13 by Candida tropicalis was investigated. The traditional application of metabolic engineering for reconstruction of the key enzymes was broken through, and new application was explored. To further increase the conversion of DCA13 with respect to alkane, the transportation system of acetyl-CoA, which linksβ-oxidation and TCA cycle together, was engineered, to regulate the metabolic fluxes inβ-oxidation. This method can be served as a new way for reconstructing the transportation system in eucaryote and industrial microbiological technologies. From the view of providing assistant energy source as a substitute ofβ-oxidation, fed-batch fermentation was studied.The key enzyme in the acetyl-CoA transportation system, carnitine acetyltransferase (CAT) was engineered. One copy of CAT gene was knocked out, and recombinant strain CZ-15 was constructed. Both the features of morphology and growth of CZ-15 were familiar with those of F10-1, the original strain. Compared with F10-1, DCA13 concentration and alkane conversion of CZ-15 were increased by 18.3 % and 12.0 % respectively, and the characteristics of CZ-15 could be inherited stably.Based on the metabolic network models of Candida tropicalis, the fermentation data were analyzed. The results indicated that, compared with F10-1, in CZ-15, the acetyl-CoA transported into TCA by CAT was 50% lower, and theβ-oxidation of DCA13 was also decreased, showing that the reduction of CAT activity could decrease the excessive consumption of DCA13.Sequentially, both of the two copies of CAT genes were knocked out, and the recombinant strain CKC-11 was obtained, in which no CAT activity was detected. CKC-11 could not grow on alkane and produce DCA13, which indicated that the total更多还原