摘要
2’-岩藻糖基乳糖(2’-Fucosyllactose,2’-FL)是一种岩藻糖化人乳低聚糖,具有预防肠道疾病、提高免疫力、促进大脑发育等重要生理功能。基于基因组水平,通过敲除大肠杆菌Escherichia coli BL21(DE3)中2’-FL合成前体物质相关代谢基因,如β-半乳糖苷酶基因(β-galactosidase,lac Z)、UDP-葡萄糖脂质载体转移酶基因(undecaprenyl-phosphate glucose-1-phosphate transferase,wca J)和GDP-甘露糖基水解酶基因(GDP-mannose mannosyl hydrolase,nud D),构建2’-FL合成底盘细胞;在此基础上,将外源α-1,2-岩藻糖基转移酶基因(α-1,2-Fucosyltransferase,wbg L)和磷酸甘露糖变位酶基因(phosphomannomutase,man B)、甘露糖-1-磷酸鸟苷转移酶基因(annose-1-phosphate guanylyltransferas,man C)、GDP-D-甘露糖-4,6-脱水酶基因(GDP-mannose 4,6-dehydratase,gmd)和GDP-岩藻糖合酶基因(GDP-L-fucose synthase,wca G)引入到大肠杆菌基因组中,探究在基因组水平过表达2’-FL合成基因对2’-FL产量的影响。结果表明,2’-FL在所构建菌株B-05的摇瓶含量达到了1.99 g·L^(-1)。进一步通过摇瓶发酵优化确定了最优发酵条件:IPTG诱导浓度为0.4 mmol·L^(-1)、诱导时OD600为2.4,诱导温度为28℃。在此条件下,摇瓶中2’-FL合成量达到了3.92 g·L^(-1)。最后在5 L发酵罐中,2’-FL含量达到了43.48 g·L^(-1)。研究表明通过将外源2’-FL相关合成基因导入基因组中进行过表达,实现了2’-FL高效合成,为构建整合型2’-FL菌株提供了数据支撑。
2'-Fucosyllactose(2'-FL)is a fucosylated human milk oligosaccharides,which has many im-portant physiological functions such as preventing intestinal diseases,improving immunity and promoting brain development.Based on the genome level,this study constructed 2'-FL synthetic chassis cells by knocking out of 2'-FL precursor synthesis related metabolic genes in Escherichia coli BL21(DE3),such asβ-galactosidase(lacZ),undecaprenyl-phosphate glucose-1-phosphate transferase(wcaJ)and GDP-mannose mannosyl hydrolase(nudD);on this basis,the exogenous geneα-1,2-Fucosyltransferase(wbgL)and phosphomannomutase(manB),man-nose-1-phosphate guanylyltransferas(manC),GDP-mannose 4,6-dehydratase(gmd)and GDP-L-fucose syn-thase(wcaG)were introduced into the genome of E.coli to explore the effect of overexpression of 2'-FL synthesis gene on 2'-FL yield at the genomic level.The results showed that the accumulation of 2'-FL reached 1.99 g·L^(-1)in shake-flask fermentation with B-05 strain.The optimal fermentation conditions were determined by shake-flask fermentation optimization:IPTG induction concentration was 0.4 mmol·L^(-1),OD600 was 2.4,and induction tem-perature was 28℃.Under these conditions,the synthesis amount of 2'-FL in shake-flask fermentation reached 3.92 g·L^(-1).In 5 L fed-batch fermentation,the content of 2'-FL reached 43.48 g·L^(-1).The results showed that the efficient synthesis of 2'-FL was achieved by introducing exogenous 2'-FL-related synthetic genes into the ge-nome for overexpression,which provides data support for the construction of integrated 2'-FL strains.
作者
劳才文
吴金勇
李忠奎
廖迎雪
王纪
袁丽霞
陈祥松
姚建铭
LAO Caiwen;WU Jinyong;LI Zhongkui;LIAO Yingxue;WANG Ji;YUAN Lixia;CHEN Xiangsong;YAO Jianming(Institutes of Physical Sciences and Information Technology,Anhui University,Hefei 230601;Institute of Plasma Physics,Hefei Institutes of Physical Science,Chinese Academy of Sciences,Hefei 230031;Huainan New Energy Research Center,Huainan 232000;Wuhan Zhongke Optics Valley Green Biotechnology Co.Ltd.,Wuhan 430200)
出处
《安徽农业大学学报》
CAS
CSCD
2024年第1期124-131,共8页
Journal of Anhui Agricultural University
基金
湖北省中国科学院科技合作专项项目(2021-01-09)
武汉市科技计划项目企业技术创新专项(2020020602012119)共同资助。
关键词
2’-岩藻糖基乳糖
重组大肠杆菌
基因敲除
发酵优化
2'-Fucosyllactose
recombinant Escherichia coli
gene knockout
fermentation optimization
