Human milk oligosaccharides (HMOs) play an important role in regulating intestinal flora and protecting infant health.Lacto-N-triose Ⅱ (LNT Ⅱ) is the core structural unit of HMOs.Hitherto, the developing methods for LNT Ⅱ synthesis are expensive and complicated.In this work, lactose and N-acetylglucosamine were used as substrate to synthesize LNT Ⅱ by the three-strain coupling fermentation strategy.In order to synthesize N-acetylglucosamine-1-phosphate (GlcNAc-1-P), the engineering bacteria Escherichia coli JM109(DE3)/pET28a-nahK was constructed, the highest yield of GlcNAc-1-P was 16.88 g/L and the conversion rate was 70.05% using optimizing the fermentation conditions.Then three engineering strains containing agx1, agx2, and pmglmU genes were constructed, respectively.The selected E.coli JM109(DE3)/pET28a-agx1 had the highest ability to synthesize uridine diphosphate acetylglucosamine (UDP-GlcNAc), the maximum yield of UDP-GlcNAc was 24.78 g/L with the optimized fermentation system.The engineering strain E.coli JM109(DE3)/pET28a-lgtA was constructed and coupled with E.coli JM109(DE3)/pET28a-nahK and E.coli JM109(DE3)/pET28a-agx1 for producing LNT Ⅱ.The final optimized yield of LNT Ⅱ reached to 3.03 g/L, which was nearly 4 times higher than that before optimization.Based on the three-strain coupling fermentation strategy, this project realized the synthesis of LNT Ⅱ This strategy has the advantages of strong versatility, simplicity and low cost, which will provide a new method for the large-scale production of HMOs.
LI Yu
,
LI Zhongxia
,
DENG Mingchao
,
WANG Zhijie
,
ZHOU Wen
,
Wengang CHAI
,
ZHANG Hongtao
. Modular synthesis of lacto-N-triose Ⅱ based on coupling fermentation strategy[J]. Food and Fermentation Industries, 2024
, 50(15)
: 16
-24
.
DOI: 10.13995/j.cnki.11-1802/ts.035263
[1] BODE L.Human milk oligosaccharides:Prebiotics and beyond[J].Nutrition Reviews, 2009, 67(suppl.2):S183-S191.
[2] BODE L.Human milk oligosaccharides:Every baby needs a sugar Mama[J].Glycobiology, 2012, 22(9):1147-1162.
[3] CHENG L H, KIEWIET M B G, GROENEVELD A, et al.Human milk oligosaccharides and its acid hydrolysate LNT2 show immunomodulatory effects via TLRs in a dose and structure-dependent way[J].Journal of Functional Foods, 2019, 59:174-184.
[4] CHENG L H, KONG C L, WANG W J, et al.The human milk oligosaccharides 3-FL, lacto-N-neotetraose, and LDFT attenuate tumor necrosis factor-α induced inflammation in fetal intestinal epithelial cells in vitro through shedding or interacting with tumor necrosis factor receptor 1[J].Molecular Nutrition & Food Research, 2021, 65(7):e2000425.
[5] 尹晓娟. 化学酶法合成LNT系列人乳寡糖[D].济南:山东大学, 2012.
YIN X J.Chemoenzymatic synthesis LNT series HMOs[D].Jinan:Shandong University, 2012.
[6] BANDARA M D, STINE K J, DEMCHENKO A V.The chemical synthesis of human milk oligosaccharides:Lacto-N-neotetraose (Galβ1→4GlcNAcβ1→3Galβ1→4Glc)[J].Carbohydrate Research, 2019, 483:107743.
[7] BLIXT O, VAN DIE I, NORBERG T, et al.High-level expression of the Neisseria meningitidis lgtA gene in Escherichia coli and characterization of the encoded N-acetylglucosaminyltransferase as a useful catalyst in the synthesis of GlcNAc β 1:→3Gal and GalNAc β 1:→3Gal linkages[J].Glycobiology, 1999, 9(10):1061-1071.
[8] LI Y H, XUE M Y, SHENG X, et al.Donor substrate promiscuity of bacterial β1-3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1-4-galactosyltransferases[J].Bioorganic & Medicinal Chemistry, 2016, 24(8):1696-1705.
[9] DONG X M, LI N, LIU Z M, et al.CRISPRi-guided multiplexed fine-tuning of metabolic flux for enhanced lacto-N-neotetraose production in Bacillus subtilis[J].Journal of Agricultural and Food Chemistry, 2020, 68(8):2477-2484.
[10] 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):235-240.
[11] COYNE M J, REINAP B, LEE M M, et al.Human symbionts use a host-like pathway for surface fucosylation[J].Science, 2005, 307(5716):1778-1781.
[12] LEE Y C, CHIEN H C R, HSU W H.Production of N-acetyl-D-neuraminic acid by recombinant whole cells expressing Anabaena sp.CH1 N-acetyl-D-glucosamine 2-epimerase and Escherichia coli N-acetyl-D-neuraminic acid lyase[J].Journal of Biotechnology, 2007, 129(3):453-460.
[13] 曹海萍. 酿酒酵母全细胞催化合成尿苷二磷酸-N-乙酰葡糖胺的机理研究[D].南京:南京工业大学, 2008.
CAO H P.Study on the mechanism of synthesis of uridine diphosphate-N-acetylglucosamine catalyzed by Saccharomyces cerevisiae[D].Nanjing:Nanjing University of Technology, 2008.
[14] LIU Y H, MA J W, SHI R, et al.Biochemical characterization of a β-N-acetylhexosaminidase from Catenibacterium mitsuokai suitable for the synthesis of lacto-N-triose Ⅱ[J].Process Biochemistry, 2021, 102:360-368.
[15] LI Y H, YU H, CHEN Y, et al.Substrate promiscuity of N-acetylhexosamine 1-kinases[J].Molecules, 2011, 16(8):6396-6407.
[16] BOURGEAUX V, PILLER F, PILLER V.Two-step enzymatic synthesis of UDP-N-acetylgalactosamine[J].Bioorganic & Medicinal Chemistry Letters, 2005, 15(24):5459-5462.
[17] WANG-GILLAM A, PASTUSZAK I, ELBEIN A D.A 17-amino acid insert changes UDP-N-acetylgalactosamine pyrophosphory lase specificity from UDP-GalNAc to UDP-GlcNAc[J].Journal of Biological Chemistry, 1998, 273(42):27055-27057.
[18] CHEN Y, THON V, LI Y H, et al.One-pot three-enzyme synthesis of UDP-GlcNAc derivatives[J].Chemical Communications, 2011, 47(38):10815-10817.
[19] 薛梦阳. 氨基糖核苷酸的酶法合成研究及其在糖缀合物制备中的应用[D].济南:山东大学, 2014.
XUE M Y.Study of enzymatic synthesis of amino sugar nucleotides and their applications in preparation of glycoconjugates[D].Jinan:Shandong University, 2014.
[20] NISHIMOTO M, KITAOKA M.Identification of N-acetylhexosamine 1-kinase in the complete lacto-N-biose I/galacto-N-biose metabolic pathway in Bifidobacterium longum[J].Applied and Environmental Microbiology, 2007, 73(20):6444-6449.
