新型高丝氨酸脱氢酶的挖掘与改造研究  

Research on mining and evolution of a novel homoserine dehydrogenase

作　　者：吴硕 黄新燕 李梦雅 徐宁[1] 魏亮 刘君[1] WU Shuo;HUANG Xinyan;LI Mengya;XU Ning;WEI Liang;LIU Jun(Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;College of Life Sciences,Hebei Normal University,Shijiazhuang 050024,China;College of Life Sciences,Nanjing Agricultural University,Nanjing 210000,China)

机构地区：[1]中国科学院天津工业生物技术研究所,天津300308 [2]河北师范大学生命科学学院,河北石家庄050024 [3]南京农业大学生命科学学院,江苏南京210000

出　　处：《食品与发酵工业》2024年第12期9-16,共8页Food and Fermentation Industries

基　　金：国家重点研发计划(2021YFC2100700);国家自然科学基金(32001671,31972061)。

摘　　要：高丝氨酸脱氢酶(homoserine dehydrogenase,HSD)是L-高丝氨酸和L-苏氨酸等天冬氨酸家族氨基酸生物合成的关键酶,然而由于其活性较低且受到L-苏氨酸等的反馈抑制作用,严重制约了L-高丝氨酸和L-苏氨酸等氨基酸的生物合成水平。该研究通过数据库检索,挖掘了8个不同来源的高丝氨酸脱氢酶。通过酶活测定分析法发现,来源于二穗短柄草(Brachypodium distachyon)的高丝氨酸脱氢酶BdHSD具有最高的催化活性,达到7.6 U/mg,且不受L-苏氨酸的反馈抑制,其催化最适pH值为10.5,最适催化温度为38℃。随后该研究进一步对BdHSD进行定向进化,提高BdHSD的催化活性。通过多轮筛选获得了3个具有更高催化活性的BdHSD突变体T186A、N283K、A137T/I188V,其中突变体T186A酶活性达到10.3 U/mg,比野生型提高了35.6%。通过L-高丝氨酸发酵分析发现,BdHSD突变体能有效提升L-高丝氨酸的合成水平。综上所述,该研究挖掘和改造了一个具有高效催化特性的高丝氨酸脱氢酶BdHSD,为L-高丝氨酸、L-苏氨酸和L-蛋氨酸等天冬氨酸家族氨基酸的高效生物合成提供了有力的催化元件。Homoserine dehydrogenase(HSD)is a key enzyme in the biosynthesis of aspartate-family amino acids such as L-homoserine and L-threonine.But HSD exhibited low activity and is feedback inhibited by L-threonine,which severely restricts the biosynthesis level of L-homoserine and L-threonine.In this study,eight HSDs from different species were mined through database search.Among of them,BdHSD derived from Brachypodium distachyon had the highest catalytic activity with 7.6 U/mg,and was not feedback-inhibited by L-threonine.The optimal catalytic pH of BdHSD was 10.5,and the optimal catalytic temperature was 38℃.To improve the catalytic activity of BdHSD,this study further performed the directed evolution of BdHSD,and three BdHSD mutants T186A,N283K,and A137T/I188V with higher catalytic activity were obtained through multiple rounds of screening.The enzyme activity of the mutant T186A reached 10.3 U/mg,which was 35.6%higher than that of the wild type.The L-homoserine fermentation analysis suggested that the BdHSD mutant could effectively enhance the synthesis level of L-homoserine.In summary,this study had mined and evolved the BdHSD with high efficient catalytic,which provided a powerful catalytic element for the efficient biosynthesis of L-homoserine,L-threonine,L-methionine,and other aspartate-family amino acids.

关 键 词：高丝氨酸脱氢酶 酶进化 二穗短柄草 L-高丝氨酸 抗苏氨酸 

分 类 号：TQ922[轻工技术与工程—发酵工程]