茯苓多糖合成途径磷酸葡萄糖变位酶和UDP-葡萄糖焦磷酸化酶基因鉴定

Identification of Phosphoglucomutase and UDP-Glucose Pyrophosphorylase Involved in Biosynthesis of Polysaccharide from Wolfiporia cocos

采用已报道的灵芝磷酸葡萄糖变位酶(PGM)和UDP-葡萄糖焦磷酸化酶(UGPP)基因序列对茯苓基因组进行搜索比对,获得茯苓WcPGM和WcUGPP候选基因序列;以茯苓的cDNA为模板,成功克隆获得WcPGM和WcUGPP基因;随后利用pPIC9K构建2个基因的表达载体,并转化至毕赤酵母进行异源表达。序列分析表明WcPGM和WcUGPP基因全长分别为2021 bp和2144 bp,其中WcPGM基因含有5个外显子和4个内含子,编码564个氨基酸;WcUGPP基因含有11个外显子和10个内含子,编码502个氨基酸。氨基酸序列比对表明,WcPGM和WcUGPP与来源于绣球菌的ScPGM和ScUGPP的同源性分别为87%和91%。酶活测定表明,重组酶WcPGM可转化葡萄糖-1-磷酸(G1P)为葡萄糖-6-磷酸(G6P),酶活达1540 U·mL^(-1);重组酶WcUGPP可催化G1P和尿苷三磷酸(UTP)合成UDP-葡萄糖(UDP-Glc),酶活达660 U·mL^(-1),表明从茯苓中筛选到的2个酶WcPGM和WcUGPP具有PGM和UGPP活性。分子模拟表明,WcPGM在催化可逆反应过程中S114为磷酸供体和受体,R24和K379作为催化酸和碱实现底物的磷酸化和去磷酸化,而E366和S368可实现底物的2种朝向,保障了中间产物葡萄糖-1,6-二磷酸的翻转,确保了WcPGM的可逆反应;而WcUGPP活性中心的核苷酸结合Loop和糖结合Loop保障了底物的正确朝向,K390作为催化碱实现了底物UTP/G1P与UDP-Glc的可逆反应。该研究为茯苓多糖合成途径的解析和代谢工程提升茯苓多糖产量奠定了基础。

We obtained candidate gene sequences of WcPGM and WcUGPP in Wolfiporia cocos(W.cocos)by search and comparison of the genome of W.cocos bu using gene sequences between the reported Ganoderma lucidum phosphoglucomutase(PGM)and UDP-glucose pyrophosphorylase(UGPP),and successfully cloned the genes of WcPGM and WcUGPP by using the cDNA of W.cocos as a template.Then,we used pPIC9K to construct the expression vector of the two genes and transformed them into Pichia pastoris for heterologous expression.Gene sequence analysis shows that the total length of WcPGM and WcUGPP genes are 2021 bp and 2144 bp,respectively.The WcPGM gene contains 5 exons and 4 introns,encoding 564 amino acids.The WcUGPP gene contains 11 exons and 10 introns,encoding 502 amino acids.Amino acid sequence comparison shows that the homology of WcPGM enzyme and WcUGPP enzyme in W.cocos with ScPGM enzyme and ScUGPP enzyme in Sparasis crispa are 87%and 91%,respectively.Enzyme activity assay shows that the recombinant enzyme WcPGM can convert glucose-1-phosphate(G1P)into glucose-6-phosphate(G6P)with enzyme activity of 1540 U·mL^(-1),recombinant enzyme WcUGPP can catalyze the synthesis of UDP-glucose(UDP-Glc)from G1P and UTP with enzyme activity of 660 U·mL^(-1),indicating that the screened two enzymes in W.cocos exhibit PGM and UGPP activities.Molecular simulation shows that in the reversible reaction process by WcPGM enzyme,S114 acts as phosphate donor and acceptor,R24 and K379 act as catalytic acid and base respectively to achieve phosphorylation and dephosphorylation of substrate,while E366 and S368 can realize two orientation of substrates,ensuring the inversion of the intermediate product glucose-1,6-diphosphate and ensuring the reversible reaction of WcPGM enzyme.The nucleotide-binding Loop and sugar-binding Loop in the active center of WcUGPP enzyme ensure the correct orientation of the substrate,and K390 serves as catalytic base to realize the reversible reaction of substrate UTP/G1P with UDP-Glc.This study lays the foundation for elucidating the synthetic pathway of W.cocos polysaccharide and improving the yield of W.cocos polysaccharide by metabolic engineering.