乙酰氨基葡萄糖合成关键酶氨基葡萄糖-6-磷酸乙酰转移酶的筛选及酶学性质分析

Screening of Glucosamine-6-phosphate N-acetyltransferase for N-acetylglucosamine Production and Enzyme Kinetics Analysis

摘要:

N-乙酰氨基葡萄糖（N-acetylglucosamine，GlcNAc)是氨基葡萄糖（Glucosamine，GlcN)的衍生物，在保健食品和医药领域具有广泛应用。氨基葡萄糖-6-磷酸乙酰转移酶（glucosamine-6-phosphate N-acetyltransferase，GNA）是N-乙酰氨基葡萄糖合成途径中的关键酶。本研究在前期构建的产GlcNAc枯草芽孢杆菌（Bacillus. subtilis，B. subtilis）工程菌的基础上，通过过量表达不同来源的GNA，筛选出来源于秀丽隐杆线虫（Caenorhabditis elegans-）的氨基葡萄糖-6-磷酸乙酰基转移酶（Cegna1）最适于加强GlcNAc合成途径。在B. subtilis工程菌中表达Cegna1，摇瓶水平发酵GlcNAc产量达到7.31 g/L，与过量表达来源于酿酒酵母（Saccharomyces cerevisiae，S. cerevisiae）的GNA的对照菌株相比，GlcNAc的产量提高了24.51%。在3 L发酵罐中进行分批发酵，GlcNAc产量达到24.23 g/L，相较于对照菌株GlcNAc的产量提高了24.69%。进一步研究发现重组Cegna1对GlcN-6P和乙酰辅酶A（Ac-CoA）有着较高的亲和力和催化效率，最后对重组Cegna1的基本酶学性质进行了考察，发现底物GlcN-6P对重组Cegna1的抑制作用明显，下一步可采用定向进化策略改造Cegna1，解除底物GlcN-6P对重组Cegna1的抑制作用。本研究结果为应用定向进化策略改造GNA的实现提供了理论依据。

Abstract:

N-acetylglucosamine（GlcNAc），derivative of glucosamine（glucosamine，GlcN），is widely used in the field of health food and pharmaceutical. Glucosamine-6-phosphate N-acetyltransferase（GNA） is one of the crucial enzymes in the GlcNAc synthesis pathway. On the basis of recombinant GlcNAc-producing Bacillus subtilis，we overexpressed glucosamine-6-phosphate N-acetyltransferase，originates from Caenorhabditis elegans（Cegna1），in the engineered B. subtilis，which enhanced GlcNAc titer to 7.31 g/L. Compared with the engineered B. subtilis with overexpression of glucosamine-6-phosphate N-acetyltransferase derived from Saccharomyces cerevisiae，the GlcNAc yield of the recombinant B. subtilis with Cegna1 increased by 24.51%. By fed-batch fermentation in 3 L bioreactor，GlcNAc titer further increased to 24.23 g/L，which is 24.69% higher than the reference strain. Further investigation results showed that the recombinant Cegna1 has higher affinity and catalytic efficiency for GlcN-6P and Ac-CoA. Finally，it is found that the substrate GlcN-6P has obvious inhibition on the the recombinant Cegna1. Identified substrate inhibition of Cegna1 provides future directions for improvement of GlcNAc production by directed evolution of Cegna1 for alleviating substrate inhibition.