Production of L-phenylalanine from trans-cinnamic acids by high-level expression of phenylalanine ammonia lyase gene from Rhodosporidium toruloides in Escherichia coli

摘要

A combined promoter expression vector pBV-PAL for high-level expression of phenylalanine ammonia lyase gene of Rhodosporidium toruloides was constructed.Pal gene was cloned and inserted into the region between Sail and Pstl restriction sites of expression vector pBV220(containing P_LP_R promoter)to obtain recombinant expression vector pBV220-PAL The tac promoter obtained from the plasmid pKtac was inserted into the expression vector pBV220-PAL to construct expression vector pBV-PAL.The recombinant plasmid pBV220-PAL and pBV-PAL were introduced into Escherichia coli JM109 by transformation.The result showed that the transformant E.coli JM109(pBV-PAL)gave a much higher PAL activity than that transformant E.coli JM109(pBV220-PAL).Recombinant PAL expression level of the transformant JM109(pBV-PAL)was about 9.6% of total cellular protein,specific enzyme activity was 2.3-fold higher than that of the transformant JM109(pBV220-PAL),reached 35 U/g(dry cells weight,DON).PAL specific activity of 123 U/g(DCW)could be achieved in a 5-1 fermentor.80.5% conversion rate of trans-cinnamic acid to L-phenylalanine and 5.12 g/1 L-phenylalanine were obtained after 3 h bioconversion using the transformant JM109(pBV-PAL).The recombinant strain JM109 containing the combined promoter expression vector pBV-PAL was shown to be effective and practical to product L-phenylalanine.