2′-岩藻糖基乳糖的微生物合成研究进展

摘要/Abstract

摘要： 摘要：2′-岩藻糖乳糖（2'-fucosyllactose, 2′-FL）是母乳寡糖（human milk oligosaccharides, HMOs）中含量最高的寡糖（约占31％），在婴幼儿的生长发育中起重要作用。2019年底，2′-FL已经在欧盟获批作为新型食品投入市场，因此，如何高效、安全的生产2′-FL成为当下的研究热点。与其他方法相比，利用微生物细胞工厂进行2′-FL的合成，具有成本低、安全、快捷等特点，是实现大规模生产 2′-FL的可行方法。本文主要针对微生物合成2′-FL过程中关键酶的活性、产物的分泌和积累、底物和中间代谢产物的分解代谢、以及辅因子再生等方面进行简要综述，并对未来的发展趋势进行展望。

关键词: 关键词：2′-岩藻糖乳糖（2′-FL）, 微生物合成, 途径改造, 发展趋势

Abstract: Abstract: 2 '- fucosyllactose (2' - FL) is the highest oligosaccharide in human milk oligosaccharides (HMOs) （about 31％）, which plays an important role in the growth and development of infants. By the end of 2019, 2' - FL has been approved as a new type of food put into the market in the EU,. Therefore, how to produce 2' - FL efficiently and safely has become a research hotspot. Compared with other methods, the synthesis of 2' - FL by using microbial cell factory has the advantages of low cost, safety and quickness, which is a feasible method to achieve mass production of 2' - FL. This paper mainly focuses on the activity of key enzymes, secretion and accumulation of products, metabolism of substrates and intermediate metabolites, and regeneration of cofactors in the process of microbial synthesis of 2' - FL, and the future development trend was prospected.

Key words: Key words:2 '- fucosyllactose (2' - FL), microbial synthesis, process modification, development trend

中图分类号:

中图分类号：Q939.97 文献标志码：A

瓮茹茹卫鑫慧李浩正路福平李玉. 2′-岩藻糖基乳糖的微生物合成研究进展[J]. 食品科学, 0, (): 0-0.

Ru-Ru WENG. Research progress in microbial synthesis of 2 '- fucosyllactose[J]. FOOD SCIENCE, 0, (): 0-0.

参考文献

参考文献：

[1] 陈坚, 邓洁莹, 李江华, et al. 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.DOI: 10.16429/j.1009-7848.2016.11.001.
[2] PETSCHACHER B, NIDETZKY B. Biotechnological production of fucosylated human milk oligosaccharides: Prokaryotic fucosyltransferases and their use in biocatalytic cascades or whole cell conversion systems [J]. Journal of Biotechnology, 2016, 235(61-83).DOI: 10.1016/j.jbiotec.2016.03.052.
[3] PUCCIO G, ALLIET P, CAJOZZO C, et al. Effects of Infant Formula With Human Milk Oligosaccharides on Growth and Morbidity: A Randomized Multicenter Trial [J]. Journal of Pediatric Gastroenterology and Nutrition, 2017, 64(4): 624-31. DOI: 10.1097/MPG.0000000000001520.
[4] MARRIAGE B J, BUCK R H, GOEHRING K C, et al. Infants Fed a Lower Calorie Formula With 2 ' FL Show Growth and 2 ' FL Uptake Like Breast-Fed Infants [J]. Journal of Pediatric Gastroenterology and Nutrition, 2015, 61(6): 649-58. DOI: 10.1097/mpg.0000000000000889.
[5] BYCH K, MIKS M H, JOHANSON T, et al. Production of HMOs using microbial hosts - from cell engineering to large scale production [J]. Current Opinion in Biotechnology, 2019, 56(130-7). DOI: 10.1016/j.copbio.2018.11.003.
[6] HUANG D, YANG K, LIU J, et al. Metabolic engineering of Escherichia coli for the production of 2'-fucosyllactose and 3-fucosyllactose through modular pathway enhancement [J]. Metab Eng, 2017, 41(23-38). DOI: 10.1016/j.ymben.2017.03.001.
[7] MORROW A L, RUIZ-PALACIOS G M, ALTAYE M, et al. Human milk oligosaccharides are associated with protection against diarrhea in breast-fed infants [J]. Journal of Pediatrics, 2004, 145(3): 0-303. DOI: 10.1016/j.jpeds.2004.04.054.
[8] HE Y, LIU S, KLING D E, et al. The human milk oligosaccharide 2\"-fucosyllactose modulates CD14 expression in human enterocytes, thereby attenuating LPS-induced inflammation [J]. Gut, 2014, gutjnl-2014-307544. DOI: 10.1136/gutjnl-2014-307544.
[9] RUIZ-PALACIOS G M, CERVANTES L E, RAMOS P, et al. Campylobacter jejuni Binds Intestinal H(O) Antigen (Fuc?1, 2Gal?1, 4GlcNAc), and Fucosyloligosaccharides of Human Milk Inhibit Its Binding and Infection [J]. Jbiolchem, 2003, 278(16): 14112-20. DOI: 10.1074/jbc.M207744200.
[10] MORROW A L, RUIZ-PALACIOS G M, JIANG X, et al. Human-Milk Glycans That Inhibit Pathogen Binding Protect Breast-feeding Infants against Infectious Diarrhea [J]. Journal of Nutrition, 2005, 135(5): 1304-7. DOI: 10.1093/jn/135.5.1304.
[11] BERGER P K, PLOWS J F, JONES R B, et al. Human milk oligosaccharide 2'-fucosyllactose links feedings at 1 month to cognitive development at 24 months in infants of normal and overweight mothers [J]. Plos One, 2020, 15(2):e0228323.DOI:10.1371/journal.pone.0228323.
[12] NEWBURG D S, RUIZ-PALACIOS G M, MORROW A L. HUMAN MILK GLYCANS PROTECT INFANTS AGAINST ENTERIC PATHOGENS [J]. Annual Review of Nutrition, 2005, 25(1): 37-58. DOI: 10.1146/annurev.nutr.25.050304.092553.
[13] MATTHIES H, STAAK S, KRUG M. Fucose and fucosyllactose enhance in-vitro hippocampal long-term potentiation [J]. Brain research, 1996, 725(2): 276-80. DOI: 10.1016/0006-8993(96)00406-4.
[14] Bienenstock J, Buck RH, Linke H, Forsythe P, Stanisz AM, Kunze W A. Fucosylated but not sialylated milk oligosaccharides diminish colon motor contractions. PLoS One 2013;8(10):e76236. DOI:10.1371/journal.pone.0076236.
[15] VAN BERLO D, WALLINGA A E, VAN ACKER F A, et al. Safety assessment of biotechnologically produced 2 '-Fucosyllactose, a novel food additive [J]. Food and Chemical Toxicology, 2018, 118(84-93. DOI: 10.1016/j.fct.2018.04.049.
[16] PRIEM B, GILBERT M, WAKARCHUK W W, et al. A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria [J]. Glycobiology, 2002, 12(4): 235-40. DOI: 10.1093/glycob/12.4.235.
[17] JUNG S-M, CHIN Y-W, LEE Y-G, et al. Enhanced production of 2'-fucosyllactose from fucose by elimination of rhamnose isomerase and arabinose isomerase in engineered Escherichia coli [J]. Biotechnology and Bioengineering, 2019, 116(9): 2412-7. DOI: 10.1002/bit.27019.
[18] ALBERMANN C, PIEPERSBERG W, WEHMEIER U F. Synthesis of the Milk Oligosaccharide 2′-Fucosyllactose Using Recombinant Bacterial Enzymes [J]. Carbohydrate Research, 2001, 334(2): 97-103. DOI: 10.1016/S0008-6215(01)00177-X.
[19] HAN N S, KIM T J, PARK Y C, et al. Biotechnological production of human milk oligosaccharides [J]. Biotechnology Advances, 2012, 30(6). DOI: 10.1016/j.biotechadv.2011.11.003.
[20] STEVENSON G, ANDRIANOPOULOS K, HOBBS M, et al. Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid [J]. Journal of Bacteriology, 1996, 178(16): 4885-93. DOI: 10.1128/jb.178.16.4885-4893.1996.
[21] CHRISTOPH A, JüRGEN D, WOLFGANG P. Preparative synthesis of GDP-β-L-fucose by recombinant enzymes from enterobacterial sources [J]. Glycobiology, 2000, (9): 9. DOI: 10.1093/glycob/10.9.875.
[22] BECKER D J, LOWE J B. Fucose: biosynthesis and biological function in mammals [J]. Glycobiology, 2003, 7): 7. DOI: 10.1093/glycob/cwg054.
[23] COYNE, M. J. Human Symbionts Use a Host-Like Pathway for Surface Fucosylation [J]. ence, 2005, 307(5716): p.1778-81. DOI: 10.1126/science.1106469.
[24] BODE L, CONTRACTOR N, BARILE D, et al. Overcoming the limited availability of human milk oligosaccharides: challenges and opportunities for research and application [J]. Nutrition Reviews, 2016, 74(10): 635-44. DOI: 10.1093/nutrit/nuw025.
[25] CHEMLER J A, FOWLER Z L, MCHUGH K P, et al. Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering [J]. Metabolic Engineering, 2010, 12(2): 96-104. DOI: 10.1016/j.ymben.2009.07.003.
[26] SAUER, U. The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli [J]. Journal of Biological Chemistry, 2003, 279(8): 6613-9. DOI: 10.1074/jbc.M311657200.
[27] LEE W H, CHIN Y W, HAN N S, et al. Enhanced production of GDP-l-fucose by overexpression of NADPH regenerator in recombinantEscherichia coli [J]. Applied Microbiology & Biotechnology, 2011, 91(4): 967-76. DOI: 10.1007/s00253-011-3271-x.
[28] BYUN S-G, KIM M-D, LEE W-H, et al. Production of GDP-L-fucose, L-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli [J]. Applied Microbiology and Biotechnology, 2007, 74(4): 768-75. DOI: 10.1007/s00253-006-0730-x.
[29] ZHAI Y, HAN D, PAN Y, et al. Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering [J]. Enzyme & Microbial Technology, 2015, 69(38-45). DOI: 10.1016/j.enzmictec.2014.12.001.
[30] LEE W H, SHIN S Y, KIM M D, et al. Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-l-fucose production in recombinant Escherichia coli [J]. Applied Microbiology & Biotechnology, 2012, 93(6): p.2327-34. DOI: 10.1007/s00253-011-3776-3.
[31] DENG J, GU L, CHEN T, et al. Engineering the Substrate Transport and Cofactor Regeneration Systems for Enhancing 2 '-Fucosyllactose Synthesis in Bacillus subtilis [J]. Acs Synthetic Biology, 2019, 8(10): 2418-27. DOI: 10.1021/acssynbio.9b00314.
[32] CHIN Y-W, KIM J-Y, LEE W-H, et al. Enhanced production of 2 '-fucosyllactose in engineered Escherichia coli BL21star(DE3) by modulation of lactose metabolism and fucosyltransferase [J]. Journal of Biotechnology, 2015, 210(107-15). DOI: 10.1016/j.jbiotec.2015.06.431.
[33] CHIN Y W, KIM J Y, KIM J H, et al. Improved production of 2′-fucosyllactose in engineered Escherichia coli by expressing putative α-1,2-fucosyltransferase, WcfB from Bacteroides fragilis [J]. Journal of Biotechnology, 2017,257(192-198). DOI: 10.1016/j.jbiotec.2016.11.033.
[34] EMINE, SEYDAMETOVA, JIWON, et al. Search for bacterial α1,2-fucosyltransferases for whole-cell biosynthesis of 2'-fucosyllactose in recombinant Escherichia coli [J]. Microbiological Research, 2019,222(35-42) DOI: 10.1016/j.micres.2019.02.009.
[35] BYCH K, MIK? M H, JOHANSON T, et al. Production of HMOs using microbial hosts — from cell engineering to large scale production [J]. Current Opinion in Biotechnology, 2019, 56(130-7). DOI: 10.1016/j.copbio.2018.11.003.
[36] JENNEWEIN S, HUEFNER E, PARKOT J. Method for the production of fucosyllactose in bacterial cells [M]. Google Patents. 2014.US008652808B2.2014-2-18.
[37] HOLLANDS K, BARON C M, GIBSON K J, et al. Engineering two species of yeast as cell factories for 2 '-fucosyllactose [J]. Metab Eng, 2019, 52(232-42). DOI: 10.1016/j.ymben.2018.12.005.
[38] 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, 11:48. DOI: 10.1186/1475-2859-11-48.
[39] BAUMGAERTNER F, SEITZ L, SPRENGER G A, et al. Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2 '-fucosyllactose [J]. Microbial Cell Factories, 2013, 12:40. DOI：10.2514/6.2012-2528.
[40] MASSIMO M, M. M J, IAN H M. Biosynthesis of human milk oligosaccharides in engineered bacteria, US10487346 [P/OL]. 2019-11-26.
[41] DUMON C, PRIEM B, MARTIN S L, et al. In vivo fucosylation of lacto-N-neotetraose and lacto-N-neohexaose by heterologous expression of Helicobacter pylori alpha-1,3 fucosyltransferase in engineered Escherichia coli [J]. Glycoconjugate Journal, 2001, 18(6): 465-74. DOI: 10.1023/A:1016086118274.
[42] GOTTESMAN S, STOUT V. Regulation of capsular polysaccharide synthesis in Escherichia coli K12 [J]. Molecular microbiology, 1991, 5(7): 1599-606. DOI: 10.1111/j.1365-2958.1991.tb01906.x.
[43] CHAKRABARTI T, CHEN Y M, LIN E C. Clustering of genes for L-fucose dissimilation by Escherichia coli [J]. Journal of bacteriology, 1984, 157(3): 984-6. DOI: 10.1128/JB.157.3.984-986.1984.
[44] CHEN Y M, TOBIN J F, ZHU Y, et al. Cross-induction of the L-fucose system by L-rhamnose in Escherichia coli [J]. Journal of bacteriology, 1987, 169(8): 3712-9. DOI: 10.1128/jb.169.8.3712-3719.1987.
[45] MALAMY M H. Frameshift Mutations in the Lactose Operon of E. coli [J]. Cold Spring Harb Symp Quant Biol, 1966, 31(189-201). DOI: 10.1101/SQB.1966.031.01.027.
[46] CHIN Y-W, SEO N, KIM J-H, et al. Metabolic Engineering of Escherichia coli to Produce 20-Fucosyllactose via Salvage Pathway of Guanosine 5 '-Diphosphate (GDP)-L-Fucose [J]. Biotechnology and Bioengineering, 2016, 113(11): 2443-52. DOI: 10.1002/bit.26015.
[47] 丁华建, 王金晶, 朱佳琪, et al. 酿酒酵母在传代过程中生理变化研究进展 [J]. 生物工程学报, 2018, 34(03): 311-9. DOI: 10.13345/j.cjb.170226.
[48] HASHIMOTO H, SAKAKIBARA A, YAMASAKI M, et al. Saccharomyces cerevisiae VIG9 Encodes GDP-mannose Pyrophosphorylase, Which Is Essential for Protein Glycosylation [J]. Journal of Biological Chemistry, 1997, 272(26): 16308-14. DOI: 10.1074/jbc.272.26.16308.
[49] MATTILA P, RABINA J, HORTLING S, et al. Functional expression of Escherichia coli enzymes synthesizing GDP-L-fucose from inherent GDP-D-mannose in Saccharomyces cerevisiae [J]. Glycobiology, 2000, 10(10): 1041-7. DOI: 10.1093/glycob/10.10.1041.
[50] NINA, J?RVINEN, MINNA, et al. Cloning and expression ofHelicobacter pyloriGDP-l-fucose synthesizing enzymes (GMD and GMER) inSaccharomyces cerevisiae [J]. European Journal of Biochemistry, 2001,268(6458-6464). DOI：10.1046/j.0014-2956.2001.02601.x.
[51] LIU T-W, ITO H, CHIBA Y, et al. Functional expression of L-fucokinase/guanosine 5 '-diphosphate-L-fucose pyrophosphorylase from Bacteroides fragilis in Saccharomyces cerevisiae for the production of nucleotide sugars from exogenous monosaccharides [J]. Glycobiology, 2011, 21(9): 1228-36. DOI: 10.1093/glycob/cwr057.
[52] LIU J-J, KWAK S, PATHANIBUL P, et al. Biosynthesis of a Functional Human Milk Oligosaccharide, 2 '-Fucosyllactose, and L-Fucose Using Engineered Saccharomyces cerevisiae [J]. Acs Synthetic Biology, 2018, 7(11): 2529-36. DOI: 10.1021/acssynbio.8b00134.
[53] YU S, LIU J-J, YUN E J, et al. Production of a human milk oligosaccharide 2 '-fucosyllactose by metabolically engineered Saccharomyces cerevisiae [J]. Microbial Cell Factories, 2018, 17(1):101. DOI: 10.1186/s12934-018-0947-2.
[54] PARK J, HONG S-K, CHANG Y K. Production of DagA and ethanol by sequential utilization of sugars in a mixed-sugar medium simulating microalgal hydrolysate [J]. Bioresource Technology, 2015, 191(414-9). DOI: 10.1016/j.biortech.2015.02.072.
[55] VAN DIJL J M, HECKER M. Bacillus subtilis: from soil bacterium to super-secreting cell factory [J]. Microbial Cell Factories, 2013, 12(1):1-6. DOI: 10.1186/1475-2859-12-3.
[56] LIU Y, LI J, DU G, et al. Metabolic engineering of Bacillus subtilis fueled by systems biology: Recent advances and future directions [J]. Biotechnology Advances, 2017,35(20-30). DOI:10.1016/j.biotechadv.2016.11.003.
[57] ADAM, WESTBROOK, XIANG, et al. Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis [J]. Biotechnology & Bioengineering, 2018;115:216-231.DOI: 10.1002/bit.26459.
[58] LIU Y, LIU L, LI J, et al. Synthetic Biology Toolbox and Chassis Development in Bacillus subtilis [J]. Trends in Biotechnology, 2019, 37(5): 548-62. DOI: 10.1016/j.tibtech.2018.10.005.
[59] DENG J, GU L, CHEN T, et al. Engineering the Substrate Transport and Cofactor Regeneration Systems for Enhancing 2′-Fucosyllactose Synthesis in Bacillus subtilis [J]. ACS synthetic biology, 2019, 8(10): 2418-27. DOI: 10.1021/acssynbio.9b00314.
[60] DOO E H, LEE W H, SEO H S, et al. Productivity of cyclohexanone oxidation of the recombinant Corynebacterium glutamicum expressing chnB of Acinetobacter calcoaceticus [J]. Journal of Biotechnology, 2009, 142(2): 164-9. DOI: 10.1016/j.jbiotec.2009.04.008.
[61] CHIN Y W, PARK J B, PARK Y C, et al. Metabolic engineering of Corynebacterium glutamicum to produce GDP-L-fucose from glucose and mannose [J]. Bioprocess & Biosystems Engineering, 2013, 36(6): 749-56. DOI: 10.1007/s00449-013-0900-z.
[62] SEO J-H, CHIN Y-W, JO H-Y. METHOD OF PRODUCING 2'-FUCOSYLLACTOSE USING CORYNEBACTERIUM GLUTAMICUM [J]. US20180298389A1.2018-10-18.