丙酮丁醇梭菌代谢工程菌的构建及其发酵性能

Metabolic engineering and fermentation properties of Clostridium acetobutylicum

丙酮丁醇梭菌Clostridium acetobutylicum可以利用葡萄糖、木糖、阿拉伯糖、纤维二糖等多种底物,发酵糖获得丙酮、丁醇、乙醇等产物,是一种优良的木质纤维素同步糖化发酵菌种。为获得具有更优良发酵性能的木质纤维素发酵菌株,使用代谢工程技术对丙酮丁醇梭菌进行改造。将乙酰乙酰CoA硫解酶基因(thl)的启动子和末端两个同源片段以及醛/醇脱氢酶基因(adhE)的开放阅读框连接到pUC18上,构建成整合型质粒pTAEE,电转化丙酮丁醇梭菌后在红霉素抗性平板筛选转化子。通过PCR扩增及产物序列分析表明,质粒pTAEE中的adhE基因以单交换的方式整合到转化子基因组中,增强adhE的表达。重组菌T4的乙醇得率为2.3%,比野生菌提高了15%,乙醇浓度为0.39 g/L,与野生菌相当;丁醇得率为41.6%,比野生菌提高了69%,丁醇浓度为6.9g/L,比野生菌提高了41%,获得了发酵性能更高的丙酮丁醇梭菌代谢工程菌株。

Clostridium acetobutylicum is a good strain for simultaneous saccharification and fermentation of ligno- cellulosic. It can produce acetone, butanol, ethanol and other products from a wide variety of carbohydrates such as glucose, xylose, arabinose and cellobiose. The metabolic flux of C. acetobutylicum was redirected through metabolic engineering technology to obtain strains for simultaneous saccharification and fermentation of lignocellulose with better fermentation properties. An integrational plasmid pTAEE containing two homologous fragments derived from the pro- moter and the terminal fragments of acetyl coenzyme A acetyltransferase gene （thl） and the open reading frame （ORF） of aldehyde/alcohol dehydrogenase E gene （adhE） as well as the erythromycin resistance gene, was con- structed and introduced into C. acetobutylicum ATCC 824 by electroporation. The recombinant transformants grown on plates containing erythromycin were validated by PCR amplification and sequencing of the amplified fragments. It indi- cated that adhE of plasmid pTAEE was integrated into the chromosome through single crossover recombination, how- ever thl gene remained undestroyed. The mutant＇s yield of ethanol was 2.3% , which was increased by 15% com- pared with wild type. The ethanol concentration was 0.39 g/L, which was equal to wild type. And the yield of buta- nol was 41.6% , which was increased by 69% compared with wild type. The butanol concentration was 6.9 g/L, which was increased by 41% compared with wild type. It provided novel technology basis for surveying of strain for lignocellulose fermentation with better properties.