大肠杆菌转运蛋白SstT和RhtC的改造对L-苏氨酸产量的影响

Effect of Transport Proteins SstT and RhtC Modification on L-threonine Production in Escherichia coli

摘要:

L-苏氨酸是一种必需氨基酸，广泛应用于食品、医药和饲料等领域。有报道表明降低胞内L-苏氨酸浓度，解除其对合成途径酶的反馈作用，有利于提高L-苏氨酸产量，因此，本实验利用Red重组技术和基因过表达技术，以L-苏氨酸生产菌Escherichia coli TRFC为出发菌株，成功构建了TRFCΔsstT和TRFCΔsstT+pSTV28-rhtC。摇瓶发酵结果显示，与原菌相比，TRFCΔsstT的L-苏氨酸产量较原菌提高了4.00%，TRFCΔsstT+pSTV28-rhtC的L-苏氨酸产量较TRFCΔsstT+pSTV28提高了18.16%。此外，经检测发现重组菌株胞内L-苏氨酸浓度均低于原菌。最后进行了TRFCΔsstT+pSTV28-rhtC的5 L分批补料发酵实验，其L-苏氨酸产量及糖酸转化率分别较原菌提高了15.33%和16.14%。综上所述，通过改造L-苏氨酸转运系统的SstT和RhtC蛋白，能有效地增强细胞内L-苏氨酸外排能力，降低细胞内L-苏氨酸的浓度，进而有利于L-苏氨酸产量的提高。

Abstract:

L-threonine is an essential amino acid and has wide application in food, pharmaceutical and feed fields. It has been reported that the reduction of the intracellular L-threonine concentration could remove the feedbacks on the enzymes in L-threonine synthetic pathway, which was benefit for L-threonine production. Hence, in this work, gene sstT was deleted in E. coli TRFC, an L-threonine producer, by Red recombination technology and gene rhtC was over expressed in TRFCΔsstT. Compared to the control strain TRFC, TRFCΔsstT was found to produce 4% more L-threonine. And compared to TRFCΔsstT+pSTV28，TRFCΔsstT+pSTV28-rhtC produced 18.16% more L-threonine in shake flask fed-batch fermentation. Besides, the intracellular L-threonine concentrations of all mutants were lower than the control strains. Finally, in the 5 L fed-batch fermentation, L-threonine production and yield on glucose of TRFCΔsstT+pSTV28-rhtC were 15.33% and 16.14% higher than those of TRFC, respectively. The results suggested that modification of transport system could effectively decrease the intracellular L-threonine concentration and enhance the excretion of internal synthesized L-threonine, and could be benefit for L-threonine production.