大肠杆菌色氨酸转运系统多基因敲除对色氨酸生产的影响

Effect of mufti-gene knockout of L-tryptophan transport system on L-tryptophan production in Escherichia coli

摘要:

大肠杆菌中色氨酸向胞内的转运主要是由mtr、tnaB和aroP 3个基因编码的通透酶进行调控。利用Red重组技术，在mtr单基因敲除菌的基础上，成功构建了mtr. tnaB和mtr. aroP双基因敲除菌以及mtr. tnaB. aroP三基因敲除菌，并通过发酵实验首次考察了色氨酸转运系统多基因缺失对大肠杆菌合成色氨酸的影响。发酵结果表明，mtr. tnaB和mtr. aroP双基因缺失后，色氨酸产量分别达到1.38 g/L和1.27 g/L，与出发菌株相比分别提高了17％和9％，而mtr. tnaB. aroP三基因缺失后，菌体生长受到了明显抑制，发酵后色氨酸产量仅为0.63 g/L。在补料分批发酵实验中，mtr. tnaB双基因敲除菌的色氨酸产量进一步提高至12.2 g/L，与出发菌株相比色氨酸产量提高了27％。

Abstract:

In Escherichia coli, uptake of L-tryptophan is done by three distinct permeases, encoded by mtr, tnaB, and aroP. Based on the mtr single-gene knockout, we constructed the mtr.tnaB and mtr.aroP double-gene knockout mutants and the mtr.tnaB.aroP triple-gene knockout mutant. The fermentation results showed that the mtr.tnaB and mtr.aroP knockout mutants produced 1.38 g/L and 1.27 g/L L-tryptophan, respectively, which was 17% and 9% higher than that of the mtr knockout mutant. However, the mtr.tnaB.aroP knockout mutant was significantly affected on cell growth and only produced 0.63 g/L L-tryptophan. During the fed-batch fermentation in a 3-L fermentor, the mtr.tnaB knockout mutant produced 12.2 g/L L-tryptophan, which was 27% higher than that of the mtr knockout mutant. This study demonstrates the effect of multi-gene knockouts of L-tryptophan transport system of Escherichia coli on the biosynthesis of L-tryptophan.