转录调控因子CsgD强化重组大肠埃希菌生物被膜催化转化桑树黄酮苷的研究  

Study on Enhancing Conversion of Mulberry Flavonoid Glycoside Catalyzed by Accelerating Recombinant Escherichia coli Biofilm Formation via Transcriptional Regulator CsgD

作　　者：陈欢 耿丽恬 黄婷 宫璐婵 吴福安 王俊[1,2] Chen Huan;Geng Litian;Huang Ting;Gong Luchan;Wu Fuan;Wang Jun(School of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang Jiangsu 212100,China;Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhenjiang Jiangsu 212100,China)

机构地区：[1]江苏科技大学生物技术学院,江苏镇江212100 [2]中国农业科学院蚕业研究所,江苏镇江212100

出　　处：《蚕业科学》2023年第6期533-543,共11页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金面上项目(22278196,21676130)。

摘　　要：生物被膜催化剂具有可再生、可持续和可扩展等优势,但其生长慢、产量低等问题限制了其在桑树黄酮苷生物转化中的应用。本文以产α-L-鼠李糖苷酶RhaB1的重组大肠埃希菌为研究对象,通过过表达其生物被膜转录调控因子基因csgD,考察CsgD对胞外聚合物分泌、细菌运动和群体感应等生物被膜形成过程关键因子及其催化性能的调控影响。结果表明,过表达菌株形成生物被膜在20 h达到成熟期,比原始菌株延长了25%,生物被膜产量提高了61.51%,说明过表达csgD对生物被膜生长具有促进作用。培养24 h后,过表达菌株分泌的紧密型胞外多聚物中多糖和蛋白质质量浓度为47.54 mg/L和54.72 mg/L,比原始菌株提高了18.24%和37.44%;过表达菌株的游动、群集和蹭行运动半径分别为3.41 mm、7.29 mm和10.61 mm,比原始菌株提高了7.23%、8.00%和7.06%。荧光定量PCR结果显示,过表达菌株中胞外聚合物基因(pgaA、bcsA)、运动性基因(fimH、motB)和群体感应基因(luxS、lsrB、aphA)的表达量显著上升,表明过表达csgD对胞外聚合物分泌、细菌运动和群体感应均具有促进作用。此外,过表达菌株的最适生物被膜催化条件为pH 6.0、温度35℃,此时芦丁转化为异槲皮苷的得率为69.29%,比原始菌株提高了56.74%。本研究表明,csgD基因对重组大肠埃希菌生物被膜形成及催化性能的强化作用,为生物转化桑树黄酮苷生产高附加值产品提供了新的思路。Biofilm catalyst has the advantages of renewability, sustainability, and extensibility, but its slow growth and low biomass limit its application in the bioconversion of mulberry flavonoid glycosides. In this study, the recombinant Escherichia coli producing α-L-rhamnosidase RhaB1 was used as the research object, and its biofilm transcriptional regulator gene csgD was overexpressed to investigate the effects of CsgD on key factors in biofilm formation processes such as extracellular polymer secretion, bacterial motility, and quorum sensing, as well as on the catalytic performance. The results showed that the biofilm formation of the overexpression strain reached maturity at 20 h, which was 25% longer than that of the original strain, and the biofilm biomass increased by 61.51%, indicating that the overexpression of csgD promoted the growth of biofilm. After 24 h of cultivation, the contents of polysaccharide and protein in the tight extracellular polymeric substances of the overexpression strain were 47.54 mg/L and 54.72 mg/L, which were 18.24% and 37.44% higher than those of the original strain, respectively. In addition, the motility radii of swimming, swarming, and twitching of the overexpression strain were 3.41 mm, 7.29 mm, and 10.61 m, which were 7.23%, 8.00%, and 7.06% higher than those of the original strain, respectively. The qPCR results showed that the expression levels of extracellular polymeric genes(pgaA, bcsA), motility genes(fimH, motB), and quorum sensing genes(luxS, lsrB, aphA) were significantly increased in overexpression strains, indicating that csgD overexpression could promote the secretion of extracellular polymers, the bacterial motility, and the quorum sensing. What's more, the optimal catalytic conditions for the biofilm of overexpression strains were pH 6.0 and 35 ℃, and the catalytic yield of rutin converted to isoquercitrin under this condition was 69.29%, which was 56.74% higher than that of the original strain. Our reseach reported the enhancive effect of csgD gene on the format

关 键 词：生物被膜 转录调控因子 桑树黄酮苷 异槲皮苷 

分 类 号：Q815[生物学—生物工程]