代谢工程改造大肠杆菌底物利用途径促进L-赖氨酸生产

Metabolic engineering of the substrate utilization pathway in Escherichia coli increases L-lysine production

摘要:

L-赖氨酸作为一种必需氨基酸，广泛应用于饲料、食品、医药等领域。针对大肠杆菌(Escherichia coli)发酵生产L-赖氨酸存在底物利用效率差、糖酸转化率低等问题，本研究通过敲除全局调控因子基因mlc，异源表达来源于麦芽糖磷酸转移酶基因malAP，提高菌株对二糖、三糖的利用效率，得到菌株E. coli XC3，其L-赖氨酸产量、产率和生产强度分别提高到160.00 g/L、63.78%和4.44 g/(L·h)；在此基础上，在菌株E. coli XC3中过表达谷氨酸脱氢酶基因gdhA、来源于枯草芽孢杆菌(Bacillus subtilis)硝酸盐还原酶基因BsnasBC和来源于E. coli的亚硝酸盐还原酶基因EcnirBD，构建硝酸盐同化路径，获得工程菌E. coli XC4，其L-赖氨酸产量、产率和生产强度分别提高到188.00 g/L、69.44%和5.22 g/(L·h)，进一步通过优化残糖浓度和碳氮比，在5 L发酵罐中将L-赖氨酸产量、产率和生产强度分别提高到204.00 g/L、72.32%、5.67 g/(L·h)，比出发菌株XC1分别提高了40.69%、20.03%、40.69%。本研究通过强化菌株的底物利用途径，构建了L-赖氨酸高产菌株，为L-赖氨酸的工业化生产奠定了坚实基础。

关键词:大肠杆菌;L-赖氨酸;代谢工程;底物利用

Abstract:

L-lysine is an essential amino acid with broad applications in the animal feed, human food, and pharmaceutical industries. The fermentation production of L-lysine by Escherichia coli has limitations such as poor substrate utilization efficiency and low saccharide conversion rates. We deleted the global regulatory factor gene mlc and introduced heterologous genes, including the maltose phosphotransferase genes (malAP) from Bacillus subtilis, to enhance the use efficiency of disaccharides and trisaccharides. The engineered strain E. coli XC3 demonstrated improved L-lysine production, yield, and productivity, which reached 160.00 g/L, 63.78%, and 4.44 g/(L‧h), respectively. Furthermore, we overexpressed the glutamate dehydrogenase gene (gdhA) and assimilated nitrate reductase genes (BsnasBC) from B. subtilis, along with nitrite reductase genes (EcnirBD) from E. coli, in strain E. coli XC3. This allowed the construction of E. coli XC4 with a nitrate assimilation pathway. The L-lysine production, yield, and productivity of E. coli XC4 were elevated to 188.00 g/L, 69.44%, and 5.22 g/(L‧h), respectively. After optimization of the residual sugar concentration and carbon to nitrogen ratio, the L-lysine production, yield, and productivity were increased to 204.00 g/L, 72.32%, and 5.67 g/(L‧h), respectively, in a 5 L fermenter. These values represented the increases of 40.69%, 20.03%, and 40.69%, respectively, compared with those of the starting strain XC1. By engineering the substrate utilization pathway, we successfully constructed a high-yield L-lysine producing strain, laying a solid foundation for the industrial production of L-lysine.

Key words:Escherichia coli;L-lysine;metabolic engineering;substrate utilization