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 of γ-aminobutyrate-producing recombinant strains of Escherichia coli

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 ΔgabPΔpuuE/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。

Abstract:

[Objective] To construct the γ-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 producing γ-aminobutyrate. [Results] The target proteins were highly expressed in the recombinant strains harboring the constructed plasmids. The highest concentration of γ-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 of γ-aminobutyrate reached 8.4 g/L. The concentration of γ-aminobutyrate was slightly lower in the fermentation broth of the recombinant strain E. coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB-SNO1-SNZ1, probably due to the excessive consumption of energy. The highest concentration of γ-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 of γ-aminobutyrate produced by the recombinant strain. This finding lays a foundation for the industrial production of γ-aminobutyrate.