基于CRISPR/Cas9技术的植物乳植杆菌YZH81胞外多糖合成基因簇功能鉴定  

Functional Characterization of the Exopolysaccharide Gene Clusters of Lactiplantibacillus plantarum YZH81 Using CRISPR/Cas9 Technology

作　　者：黄淑婷 孙娅西 宦海琳[2] 贾思思 赵芳[1] 吕广萍 杨瑶[1] HUANG Shuting;SUN Yaxi;HUAN Hailin;JIA Sisi;ZHAO Fang;LÜGuangping;YANG Yao(School of Food Science and Pharmaceutical Engineering,Nanjing Normal University,Nanjing 210023,China;Institute of Animal Science,Jiangsu Academy of Agricultural Sciences,Nangjing 210014,China)

机构地区：[1]南京师范大学食品与制药工程学院,江苏南京210023 [2]江苏省农业科学院畜牧研究所,江苏南京210014

出　　处：《食品科学》2025年第6期133-141,共9页Food Science

基　　金：江苏省自然科学基金面上项目(BK20231280);江苏省农业科技自主创新资金项目(CX(22)2019)。

摘　　要：胞外多糖(exopolysaccharides,EPS)合成基因簇是揭示乳酸菌EPS构效关系的基础。本研究前期发现一株高产EPS的植物乳植杆菌Lactiplantibacillus plantarum YZH81菌株含有可能的2个EPS合成基因簇cps1和cps2,构建的YZH81?cps1菌株EPS产量下降。本研究首先利用CRISPR/Cas9技术敲除了YZH81菌株cps2基因簇。与YZH81相比,YZH81?cps1和YZH81?cps2的EPS产量分别下降了22.88%和35.42%,化学组成改变也较大,突变株均未检出鼠李糖单糖组分,而甘露糖和葡萄糖占比大幅提高。进一步利用蛋白质组学分析了不同发酵时间(10、32 h)两个不同cps簇的缺失相比于野生型YZH81菌株的差异蛋白29个和22个,京都基因与基因组百科全书和基因本体分析结果显示差异蛋白参与YZH81菌株的生物学过程、细胞组成的代谢与加工过程,但敲除cps2簇引起的差异蛋白功能更为丰富,包括脂质代谢、核苷酸代谢和肽聚糖的生物合成与代谢等。综上,本研究明确了YZH81菌株具有两个独立的不同cps簇,并分别承担了不同功能,为后期开展YZH81菌株的EPS益生功能构效关系的研究提供了理论支撑。The exopolysaccharide(EPS)gene clusters are the basis for understanding the structure-function relationship of EPS in lactic acid bacteria.In our earlier work,we had constructed the cps1 deletion strain(YZH81Δcps1),with a decreased capacity to produce EPS,from Lactiplantibacillus plantarum YZH81,a strain with high EPS production ability,which had been found to possibly contain two capsular polysaccharide syntheis(cps)gene clusters,cps1 and cps2.In this study,the cps2 gene cluster of strain YZH81 was knocked out using CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9)technology.Compared with YZH81,the EPS production of YZH81Δcps1 and YZH81Δcps2 decreased by 22.88%and 35.42%,respectively,and the monosaccharide compositions of EPS from the two cps strains were altered considerably.The monosaccharide rhamnose was not detected in any of the mutant strains,and the percentages of mannose and glucose increased dramatically.Proteomics identified a total of 29 and 22 differential proteins in YZH81Δcps1 and YZH81Δcps2 versus YZH81 after fermentation for 10 and 32 h,respectively,and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)functional analyses showed that the differential proteins were involved in the biological processes,the metabolism and processing of cellular components in YZH81,but those resulting from knockout of the cps2 cluster had richer functions such as lipid metabolism,nucleotide metabolism,and glycan biosynthesis and metabolism. In conclusion, the present study provides theoretical support for future research on thestructure-probiotic function relationship of EPS from L. plantarum YZH81.

关 键 词：植物乳植杆菌 胞外多糖 cps基因簇 蛋白功能 

分 类 号：Q71[生物学—分子生物学]