草酸脱羧酶的进化和异源表达对罗伊氏乳杆菌草酸盐降解能力的影响  

Effects of Evolution and Heterologous Expression of Oxalate Decarboxylase on Oxalate Degradation Ability of Limosilactobacillus reuteri

作　　者：牛冬玉 孔令慧 刘向勇[1] 秦加阳[1] NIU Dongyu;KONG Linghui;LIU Xiangyong;QIN Jiayang(School of Pharmacy,Binzhou Medical University,Yantai 264003,Shandong,China)

机构地区：[1]滨州医学院药学院,山东烟台264003

出　　处：《济南大学学报(自然科学版)》2025年第2期306-312,共7页Journal of University of Jinan(Science and Technology)

基　　金：国家自然科学基金项目(32302050);山东省自然科学基金项目(ZR2022QC004)。

摘　　要：为了开发能够治疗或辅助治疗高草酸尿症的益生菌,采用基于深度学习的方法对枯草芽孢杆菌的草酸脱羧酶(OXDC)进行蛋白质进化,并在罗伊氏乳杆菌Limosilactobacillus reuteri Q35中异源表达野生型OXDC和2个预测催化常数更大的突变型OXDC-M5和OXDC-M10,比较基因工程菌株在生长体系和全细胞转化体系中降解草酸盐的能力。结果表明:突变型、野生型OXDC的表达均能显著提高罗伊氏乳杆菌的草酸铵降解能力,且OXDC-M10表达菌株L_(3)的降解能力最强;生长体系中培养24 h,菌株L_(3)的草酸盐降解率为45%,比对照菌株的高52.03%,比野生型OXDC表达菌株L_(1)的高12.78%;转化体系中培养4 h,菌株L_(3)的草酸盐降解率为46.56%,比对照菌株的高23.24%,比野生型OXDC表达菌株L_(1)的高21.25%,利用基因工程和蛋白质工程相结合的方法可显著提高罗伊氏乳杆菌Q35的草酸盐降解能力。To develop probiotics for the treatment or assistance of hyperoxaluria,a deep learning-based approach was employed to perform protein evolution on Bacillus subtilis’oxalate decarboxylase(OXDC).Limosilactobacillus reuteri(L.reuteri)Q35 was used for heterologous expression of wild-type OXDC and two mutants,OXDC-M5 and OXDC-M10,which exhibited higher predictive catalytic constant,and the ability of these genetically engineered strains to degrade oxalate in both the growth system and whole cell transformation system was compared.The results show that the expression of both mutant and wild-type OXDC significantly enhance the ability of L.reuteri to degrade ammonium oxalate.Among the tested strains,strain L_(3) expressing OXDC-M10 exhibits superior degradation capability,achieving a 45%oxalate degradation rate after 24 h of cultivation,which is 52.03%higher than that observed in control strains and 12.78%higher than strain L_(1) expressing wild-type OXDC.During a 4 h period in the transformation system,strain L_(3) demonstrates a 46.56%oxalate degradation rate,surpassing control strains by 23.24%and outperforming strain L_(1) expressing wild-type OXDC by 21.25%.The oxalate degradation ability of L.reuteri Q35 can be significantly improved by the combination of genetic engineering and protein engineering.

关 键 词：蛋白质进化 草酸盐降解 异源表达 罗伊氏乳杆菌 草酸脱羧酶 

分 类 号：Q812[生物学—生物工程]