Please wait a minute...
 
 
食品与发酵工业  2021, Vol. 47 Issue (3): 10-17    DOI: 10.13995/j.cnki.11-1802/ts.025160
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
2'-岩藻糖基乳糖的生物合成菌株构建及发酵条件研究
李晨晨, 李梦丽, 江波, 张涛*
食品科学与技术国家重点实验室(江南大学),江苏 无锡,214122
Strain development and fermentation optimization for biosynthesis of 2'-fucosyllactose
LI Chenchen, LI Mengli, JIANG Bo, ZHANG Tao*
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
下载:  HTML   PDF (3719KB) 
输出:  BibTeX | EndNote (RIS)      
摘要 2'-岩藻糖基乳糖(2'-fucosyllactose,2'-FL)可作为益生元添加到婴幼儿配方奶粉中,对婴幼儿肠道菌群和免疫系统的发育至关重要。通过共表达L-岩藻糖激酶/GDP-L-岩藻糖焦磷酸化酶(L-fucokinase/GDP-L-fucose pyrophosphorylase,fkp)基因和α-1,2-岩藻糖基转移酶(α-1,2-fucosyltransferase,futC)基因,以大肠杆菌Escherichia coli BL21star(DE3)为出发菌株,异源表达脆弱拟杆菌(Bacteroides fragilis)和幽门螺杆菌(Helicobacter pylori)来源的fkpfutC基因,建立了2'-FL的合成途径。通过CRISPR/Cas9系统敲除β-半乳糖苷酶(β-galactosidase,LacZ)和UDP-葡萄糖脂质载体转移酶(UDP-glucose lipid carrier transferase,WcaJ)基因,阻断中间代谢产物的分解,探究该类基因对2'-FL合成的影响。实验结果显示:单敲除LacZ基因促进2'-FL的产量并提高1.88倍,对菌体生长影响不显著;叠加敲除基因LacZWcaJ后,2'-FL的产量提高4.89倍,且对菌体生长没有显著影响。通过摇瓶发酵优化,确定了发酵条件为:采用限定性培养基(DM培养基),异丙基-β-D-硫代半乳糖苷诱导浓度为0.2 mmol/L,诱导温度为25 ℃,接种量为5%。在此条件下摇瓶培养,2'-FL产量最高可达1.44 g/L。研究表明敲除β-半乳糖苷酶和UDP-葡萄糖脂质载体转移酶基因可显著提高2'-FL产量,为扩大其工业化生产提供参考依据。
服务
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章
李晨晨
李梦丽
江波
张涛
关键词:  2'-岩藻糖基乳糖  重组大肠杆菌  基因敲除  代谢工程  发酵优化    
Abstract: As a kind of prebiotics, 2'-fucosyllactose (2'-FL) can be added to infant formula, and is essential for the development of the intestinal flora and immune system of infants. The synthetic pathway of 2'-FL was established by heterologously co-expressing L-fucose kinase/GDP-L-fucose pyrophosphorylase gene fkp and α-1,2 fucosyltransferase gene futC in Escherichia coli BL21star (DE3), which were derived from Bacteroides fragilis and Helicobacter pylori respectively. Secondly, the β-galactosidase and UDP-glucose lipid carrier transferase genes lacZ and wcaJ were deleted through the CRISPR/Cas9 system to block the decomposition of intermediate metabolites and explore the influence of these genes on 2'-FL synthesis. The fermentation results showed that single deletion of lacZ gene promoted the yield of 2'-FL by 1.88 times, which had no significant effect on cell growth. After double mutation of genes lacZ and wcaJ, the yield of 2'-FL increased by 4.89 times, and the cell growth was not affected significantly. Under optimized shake flask fermentation, the fermentation conditions were determined as follows: a defined medium was used, the induction concentration of IPTG was 0.2 mmol/L, the induction temperature was 25 ℃, and the inoculation amount was 5%. Under these conditions, the yield of 2'-FL reached 1.44 g/L. This study confirmed that deletion of β-galactosidase and UDP-glucose lipid carrier transferase genes could significantly increase the yield of 2'-FL, which provides a reference for expanding its industrial production.
Key words:  2'-fucosyllactose    recombinant E. coli    gene knockout    metabolic engineering    fermentation optimization
收稿日期:  2020-07-24      修回日期:  2020-08-05           出版日期:  2021-02-15      发布日期:  2021-03-08      期的出版日期:  2021-02-15
基金资助: 十三五重点研发计划项目(2017YFD0400600)
作者简介:  硕士研究生(张涛教授为通讯作者,E-mail:zhangtao@jiangnan.edu.cn)
引用本文:    
李晨晨,李梦丽,江波,等. 2'-岩藻糖基乳糖的生物合成菌株构建及发酵条件研究[J]. 食品与发酵工业, 2021, 47(3): 10-17.
LI Chenchen,LI Mengli,JIANG Bo,et al. Strain development and fermentation optimization for biosynthesis of 2'-fucosyllactose[J]. Food and Fermentation Industries, 2021, 47(3): 10-17.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.025160  或          http://sf1970.cnif.cn/CN/Y2021/V47/I3/10
[1] ZIVKOVIC A M,GERMAN J B,LEBRILLA C B,et al.Human milk glycobiome and its impact on the infant gastrointestinal microbiota[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(Suppl.1):4 653-4 658.
[2] NINONUEVO M R,PARK Y,YIN H F,et al.A strategy for annotating the human milk glycome[J].Journal of Agricultural and Food Chemistry,2006,54(20):7 471-7 480.
[3] THONGARAM T,HOEFLINGER J L,CHOW J,et al.Human milk oligosaccharide consumption by probiotic and human-associated Bifidobacteria and Lactobacilli[J].Journal of Dairy Science,2017,100(10):7 825-7 833.
[4] BODE L.Human milk oligosaccharides:Every baby needs a sugar mama[J].Glycobiology,2012,22(9):1 147-1 162.
[5] 逯莹莹, 刘鹏,孙景珠,等.母乳低聚糖的研究进展[J].中国乳品工业,2018,46(12):23-33.
LU Y Y,LIU P,SUN J Z,et al.Research progress of human milk oligosaccharides[J].China Dairy Industry,2018,46(12):23-33.
[6] VANDENPLAS Y,BERGER B,CARNIELLI V P,etal.Human milk oligosaccharides:2'-fucosyllactose(2'-FL) and Lacto-N-Neotetraose (LNnT) in infant formula[J].Nutrients,2018,10(9):1 161-1 161.
[7] LEE W H,PATHANIBUL P,QUARTERMAN J,et al.Whole cell biosynthesis of a functional oligosaccharide,2'-fucosyllactose,using engineered Escherichia coli[J].Microbial Cell Factories,2012,11(1):48-48.
[8] HEGAR B,WIBOWO Y,BASROWI R W,et al.The role of two human milk oligosaccharides,2'-fucosyllactose and Lacto-N-Neotetraose,in Infant nutrition[J].Pediatric Gastroenterology Hepatology Nutrition,2019,22(4):330-340.
[9] 王永胜, 王硕,张慧林,等.L-岩藻糖对母乳寡糖(HMOs)合成的意义及其产业化研究进展[J].中国农学通报,2019,35(11):127-132.
WANG Y S,WANG S,ZHANG H L,et al.L-fucose:The significance to synthesis of human milk oligosaccharides (HMOs) and its research progress of industrialization[J].Chinese Agricultural Science Bulletin,2019,35(11):127-132.
[10] CHIN Y W,SEO N,KIM J H,et al.Metabolic engineering of Escherichia coli to produce 2'-fucosyllactose via salvage pathway of guanosine 5'-diphosphate (GDP)-L-fucose[J].Biotechnology and Bioengineering,2016,113(11):2 443-2 452.
[11] ZHAI Y F,HAN D L,PAN Y,et al.Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering[J].Enzyme and Microbial Technology,2015,69:38-45.
[12] 陈坚, 邓洁莹,李江华,等.母乳寡糖的生物合成研究进展[J].中国食品学报,2016,16(11):1-8.
CHEN J,DENG J Y,LI J H,et al.Advances in biosynthesis of breast milk oligosaccharides[J].Journal of Chinese Institute of Food Science and Technology,2016,16(11):1-8.
[13] CHIN Y W,KIM J Y,LEE W H,et al.Enhanced production of 2'-fucosyllactose in engineered Escherichia coli BL21 star(DE3) by modulation of lactose metabolism and fucosyltransferase[J].Joural of Biotechnology,2015(210):107-115.
[14] ENGELS L,ELLING L.WbgL:A novel bacterial α1,2-fucosyltransferase for the synthesis of 2'-fucosyllactose[J].Glycobiology,2014,24(2):170-178.
[15] 萨姆布鲁克J,拉塞尔著.分子克隆实验指南[M].(第三版).北京:科学出版社,2003:1 217-1 265.
SAMBROOK J,RUSSELL D W.Molecular cloning:A laboratory manual[M].3rd ed.Beijing:Science Press,2003:1 217-1 265.
[16] JIANG Y,CHEN B,DUAN C L,et al.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system[J].Applied and Environmental Microbiology,2015,81(7):2 506-2 514.
[17] TANG Q,LOU C B,LIU S J.Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit[J].Journal of Biological Engineering,2017,11(1):32-32.
[18] JIANG W Y,BIKARD D,COX D,et al.RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J].Nature Biotechnology,2013,31(3):233-241.
[19] WANG X Z,HE J H,LE K Y.Making point mutations in Escherichia coli BL21 genome using CRISPR-Cas9 system[J].FEMS Microbiology Letters,2018,365(14):1-6.
[20] WANG G,BOULTON P G,CHAN N W C,et al.Novel Helicobacter pylori alpha1,2-fucosyltransferase,a key enzyme in the synthesis of Lewis antigens[J].Microbiology,1999,145(11):3 245-3 253.
[21] KOTAKE T,HOJO S,TAJIMA N,et al.A bifunctional enzyme with L-fucokinase and GDP-L-fucose pyrophosphorylase activities salvages free L-fucose in Arabidopsis[J].Journal of Biological Chemistry,2008,283(13):8 125-8 135.
[22] 肖玲玲. IPTG对大肠杆菌内重组牙龈卟啉单胞菌GroEL表达的影响[D].南京:南京大学,2017.
XIAO L L.The effect of IPTG concentrations on recombinant Porphyromonas gingivalis GroEL expression in Escherichia coli[D].Nanjing:Nanjing University,2017.
[23] 叶姣, 陈长华,夏杰,等.温度对重组大肠杆菌生长及外源蛋白表达的影响[J].华东理工大学学报,2008,28(2):364-367.
YE J,CHEN C H,XIA J,et al.Effect of temperature on the growth of recombinant E.coli and on the expression of recombinant protein[J].Journal of East China University of Science and Technology,2008,28(2):364-367.
[1] 彭文坚, 张娟, 刘松. 采用组合策略提高灰色链霉菌胰蛋白酶在毕赤酵母中的表达[J]. 食品与发酵工业, 2021, 47(20): 15-21.
[2] 徐佳, 黄雪芹, 杨建飞, 易媛, 马倩, 胡琨, 左勇. 酿酒酵母中BAT2基因敲除对桑葚酒中高级醇的影响[J]. 食品与发酵工业, 2021, 47(19): 133-139.
[3] 张博, 史永吉, 杨辉, 吴梓丹, 陈开, 蔡雪, 柳志强, 郑裕国. 通过发酵优化提高大肠杆菌生产L-半胱氨酸产量[J]. 食品与发酵工业, 2021, 47(18): 175-180.
[4] 李旋, 王加初, 刘益宁, 蒋帅, 吴鹤云, 谢希贤. 代谢工程改造大肠杆菌生产L-丝氨酸[J]. 食品与发酵工业, 2021, 47(17): 1-7.
[5] 刘慧, 陈胜玲, 徐建中, 张伟国. α-法尼烯在巴斯德毕赤酵母中的生物合成[J]. 食品与发酵工业, 2021, 47(16): 9-14.
[6] 朱灵桓, 徐沙, 李由然, 张梁, 石贵阳. 微生物法从头合成2-苯乙醇的研究进展[J]. 食品与发酵工业, 2021, 47(16): 271-277.
[7] 马巍, 邹祥. 发酵法生产L-岩藻糖的研究进展[J]. 食品与发酵工业, 2021, 47(16): 308-312.
[8] 李俊毅, 王大红, 苏佳杰, 姬翔, 李阳. 敲除mig1基因对马克斯克鲁维酵母利用葡萄糖和木糖的影响[J]. 食品与发酵工业, 2021, 47(14): 51-56.
[9] 诸亚锋, 徐铮. 来源于Dictyoglomus sp.NZ13-RE01的纤维二糖差向异构酶酶学性质和乳果糖制备研究[J]. 食品与发酵工业, 2021, 47(13): 9-15.
[10] 李梦莹, 吕雪芹, 刘延峰, 李江华, 堵国成, 吴剑荣, 刘龙. 代谢工程改造大肠杆菌合成L-组氨酸[J]. 食品与发酵工业, 2021, 47(12): 1-9.
[11] 刘益宁, 秦臻, 李旋, 蒋帅, 吴鹤云, 谢希贤. 胞苷合成途径改造对大肠杆菌嘧啶核苷发酵的影响[J]. 食品与发酵工业, 2021, 47(12): 10-16.
[12] 段晓莉, 江波, 张涛. 产阿魏酸酯酶菌株的筛选与产酶条件优化[J]. 食品与发酵工业, 2021, 47(12): 154-160.
[13] 庞远祥, 谢远红, 金君华, 刘慧, 张红星. 低嘌呤、高纳豆激酶活性枯草芽孢杆菌SH21筛选及发酵条件优化[J]. 食品与发酵工业, 2021, 47(11): 194-199.
[14] 桑昆昆, 刘晓凤, 熊智强, 张汇, 王光强, 宋馨, 艾连中, 夏永军. 透明质酸分子质量调控进展[J]. 食品与发酵工业, 2021, 47(11): 272-278.
[15] 楼志华, 刘翔, 张劲楠. 嗜糖假单胞菌麦芽四糖酶基因在地衣芽孢杆菌中的异源表达[J]. 食品与发酵工业, 2021, 47(1): 50-54.
No Suggested Reading articles found!
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
版权所有 © 《食品与发酵工业》编辑部
地址:北京朝阳区酒仙桥中路24号院6号楼111室
本系统由北京玛格泰克科技发展有限公司设计开发  技术支持:support@magtech.com.cn