强化厌氧表达dld基因以提高大肠杆菌工程菌发酵产L-乳酸的光学纯度

Increase of optical purtiy of L-lactic produced by engineered Escherichia coli by enhancing anaerobic expression of dld gene

为去除廉价发酵原料中的D-乳酸从而提高发酵最终产物L-乳酸的光学纯度,该研究通过构建带有不同厌氧诱导启动子(pflBp6、pnirB、pflBp6-pnirB)的D-乳酸脱氢酶基因(dld)的表达质粒,并将其分别转入大肠杆菌(Escherichia coli)HBUT-L,加强其在发酵产L-乳酸的同时消除外源D-乳酸的能力。通过装液量与转速的单因素试验筛选最优的质粒表达条件及菌株,并在无机盐培养基与玉米浆培养基中进行发酵验证。结果表明,在装液量为200 mL/250 mL、转速为150 r/min的条件下,含有启动子pflBp6-pnirB的表达质粒的工程菌株HBUT-L4发酵18 h时D-乳酸脱氢酶活力最高,为81.1 U/g。在此条件下进行无机盐培养基和玉米浆培养基发酵时,工程菌株HBUT-L4相对于出发菌株HBUT-L,D-乳酸消耗速率分别提高250%、217%,L-乳酸的光学纯度分别从96.32%、98.48%提升至99.95%、99.98%。在大肠杆菌工程菌发酵L-乳酸的过程中,带有厌氧诱导启动子pflBp6-pnirB的表达质粒可提高D-乳酸脱氢酶活力,强化菌株消除外源D-乳酸,进而提高L-乳酸的光学纯度的能力,具有重要的工业化应用价值。

In order to eliminate D-lactic acid in cheap fermentation raw materials and improve the optical purity of L-lactic acid in fermentation final product,the expression plasmids of D-lactate dehydrogenase gene(dld)with different anaerobic inducible promoters(pflBp6,pnirB,pflBp6-pnirB)were constructed.They were transferred into the Escherichia coli HBUT-L to enhance its ability to eliminate exogenous D-lactic acid while producing L-lactic acid by fermentation.The optimal plasmid expression conditions and strains were screened by single factor experiments of liquid loading volume and rotation speed,and the fermentation was verified in inorganic salt and corn pulp media.The results showed that the D-lactate dehydrogenase activity of engineered strain HBUT-L4 containing expression plasmid with promoter pflBp6-pnirB was the highest(81.1 U/g)fermentation for 18 h under the conditions of liquid loading volume 200 ml/250 ml and rotation speed 150 r/min.When fermentation with inorganic salt and corn pulp media under these conditions,compared to original strain HBUT-L,the D-lactic acid consumption rate of engineered strain HBUT-L4 increased by 250%and 217%,respectively,and the optical purity of L-lactic acid increased from 96.32%,98.48%to 99.95%,99.98%,respectively.In the process of L-lactic acid fermentation by engineered E.coli,the expression plasmid with anaerobic inducible promoter pflBp6-pnirB could increase the D-lactate dehydrogenase activity,and strengthen the ability of strain to eliminate exogenous D-lactic acid,and improve the optical purity of L-lactic acid,which had important industrial application value.