利用DNA重组技术生产L-苯丙氨酸的研究

Study on production of L-phenylalanine by DNA recombination technology

L-苯丙氨酸(L-phenylalanine,L-Phe)是人体八大必须氨基酸之一,在人类的新陈代谢中起着重要的作用。而现在生产L-Phe的技术还远远满足不了市场的需求,本研究目的是利用DNA重组技术以及微生物发酵的方法提高L-Phe产量。研究方法包括利用易错PCR技术对rpoA基因进行点突变建立一个基因库,通过突变作用于rpoA的正常转录从而调控工程菌株产生L-Phe的量;利用质粒ptrc99a作为载体转化到感受态细胞中,并从中筛选出高产菌株;通过质粒的构建、筛选、验证、发酵等一系列试验,结果表明工程菌株构建成功,为L-Phe规模化生产提供科学依据。

L-phenylalanine is one of the eight essential amino acids and plays an important role in human metabolism.Now the production of L-Phe technology cannot satisfy the demand of the market.Therefore,the goal of this study is to improve the yield of L-phe by using reombinant DNA technology and the method of microorganism fermentation.In the process of the study,a gene pool was established by using error-prone PCR to carry out point mutation of rpoA gene.The amount of L-phe produced by the engineered strain was regulated by the mutation acting on the normal transcription of rpoA.Plasmid ptrc99a was used as a vector to transfer into competent cells,and high-yield strains were screened from it.The plasmid was constructed,screened,validated,and verified and a series of tests were used to state the feasibility of this method in this process.The experimental results showed that the engineered strain was successfully constructed and this study provides scientific basis for L-Phe large-scale production.