产γ-氨基丁酸基因工程重组大肠杆菌的构建及其发酵特性  

Construction and fermentation characterization ofγ-aminobutyrate-producing recombinant strains of Escherichia coli

作　　者：管付瑶 陆佳杰 朱志文 王配泽 鄢楚洋 于平[1] GUAN Fuyao;LU Jiajie;ZHU Zhiwen;WANG Peize;YAN Chuyang;YU Ping(School of Food Science and Biotechnology,Zhejiang Gongshang University,Hangzhou 310018,Zhejiang,China)

机构地区：[1]浙江工商大学食品与生物工程学院,浙江杭州310018

出　　处：《微生物学报》2024年第2期489-501,共13页Acta Microbiologica Sinica

基　　金：浙江省自然科学基金(LY21C200006)。

摘　　要：【目的】构建高产γ-氨基丁酸的基因工程重组大肠杆菌(E.coli),并研究其发酵特性。【方法】首先通过分子生物学方法构建重组质粒pTrc99a-gadB和pTrc99a-gadB-SNO1-SNZ1,然后分别将它们转入基因敲除菌株E.coli K12/ΔgabTΔgabPΔpuuE。通过对重组菌进行培养,研究其产γ-氨基丁酸的发酵过程并进行优化。【结果】构建的重组质粒转入大肠杆菌后,筛选的重组菌经聚丙烯酰胺凝胶电泳分析表明目的蛋白均得到高效表达。重组菌E.coliK12ΔgabTΔgabPΔpuuE/pTrc99a-gadB在含10g/L底物L-谷氨酸钠的发酵液中产γ-氨基丁酸的最高浓度为4.6 g/L。与原始菌相比,提高了21.9倍。该菌株在含不同底物浓度(5、10、20、30、40 g/L)的发酵液中的发酵过程表明,在20g/L的底物浓度下,底物的转化率达到最高值,此时γ-氨基丁酸的产量为8.4 g/L。当2个外源基因SNO1和SNZ1同时引入重组菌E.coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB时,由于能量的过度消耗,重组菌E.coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB-SNO1-SNZ1产γ-氨基丁酸的能力略有下降。重组菌E.coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB在含20 g/L底物L-谷氨酸钠的1 L发酵液中的扩大培养结果表明,重组菌培养24 h时,其发酵液中γ-氨基丁酸的含量达到最高值9.4 g/L。【结论】经基因工程构建的重组大肠杆菌产γ-氨基丁酸的能力明显提高,该研究结果为γ-氨基丁酸的产业化生产提供了良好基础。[Objective]To construct theγ-aminobutyrate-producing recombinant strains of Escherichia coli and investigate their fermentation characteristics.[Methods]We constructed two recombinant plasmids pTrc99a-gadB and pTrc99a-gadB-SNO1-SNZ1 and then respectively transformed them into the gene-knockout strain E.coli K12/ΔgabTΔgabPΔpuuE.We investigated and optimized the fermentation process of the recombinant strains for producingγ-aminobutyrate.[Results]The target proteins were highly expressed in the recombinant strains harboring the constructed plasmids.The highest concentration ofγ-aminobutyrate was 4.6 g/L in the fermentation broth of E.coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB cultured in the medium containing 10 g/L L-monosodium glutamate and was 21.9 times higher than that in the fermentation broth of the wild type strain.At the L-monosodium glutamate concentration of 20 g/L,the conversion rate of substrate was the highest and the concentration ofγ-aminobutyrate reached 8.4 g/L.The concentration ofγ-aminobutyrate was slightly lower in the fermentation broth of the recombinant strain E.coli K12ΔgabTΔgabPΔpuuE/pTrc99agadB-SNO1-SNZ1,probably due to the excessive consumption of energy.The highest concentration ofγ-aminobutyrate was 9.4 g/L when E.coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB was cultured in 1 L fermentation medium containing 20 g/L L-monosodium glutamate.[Conclusion]We obviously increased the yield ofγ-aminobutyrate produced by the recombinant strain.This finding lays a foundation for the industrial production ofγ-aminobutyrate.

关 键 词：重组大肠杆菌 Γ-氨基丁酸 生物转化 发酵特性 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]