Lacto-N-biose(LNB) is a major component of human milk oligosaccharides (HMOs) and one of the candidates for bifidus factors.However, there is a lack of economical large-scale synthesis technology.This study took advantage of the non-dependent energy-charge regeneration of sucrose phosphorylase and LNB phosphorylase, and established a dual-strain co-culture method for efficient LNB synthesis using Saccharomyces cerevisiae as the energy supply cell.Firstly, the engineered strains E.coli JM109(DE3)/pET28a-GalT1, E.coli JM109(DE3)/pET28a-GalT2 and E.coli JM109(DE3)/pET28a-BLUSP were constructed, and the optimal strain E.coli JM109(DE3)/ET28a-GalT1 with a yield up to 10.5 g/L of UDP-Glc was screened.Then it was coupled fermentation with the constructed engineered strains E.coli JM109(DE3)/pET28a-SPase, E.coli JM109(DE3)/pET28a-GalE and E.coli JM109(DE3)/pET28a-BiGalHexNAcP, and the LNB yield was 6.78 g/L.Finally, the co-expression strain E.coli JM109(DE3) (containing plasmids pRSFDuet1-SPase-GalT1 and pETDuet1-GalE-BiGalHexNAcP) was constructed with exogenous addition of cofactor UMP (at a concentration of 20 mmol/L) and coupled with yeast to fermentation, and the highest LNB yield was 28.66 g/L, which increased the yield by 323% compared to the four-strain coupled fermentation.This is the highest reported yield of LNB to date.The results of the study provide a new technological route for the low-cost production of LNB.
LI Zhuying
,
ZHANG Hongtao
,
LI Jiayi
,
ZHANG Zimeng
,
HAN Cheng
,
XU Chun
. Production of lacto-N-biose by coupled high-density fermentation with engineered strain-yeast[J]. Food and Fermentation Industries, 2025
, 51(6)
: 120
-126
.
DOI: 10.13995/j.cnki.11-1802/ts.039337
[1] URASHIMA T, ASAKUMA S, LEO F, et al.The predominance of type I oligosaccharides is a feature specific to human breast milk[J].Advances in Nutrition, 2012, 3(3):473S-482S.
[2] ZHU Y Y, WAN L, LI W, et al.Recent advances on 2′-fucosyllactose:Physiological properties, applications, and production approaches[J].Critical Reviews in Food Science and Nutrition, 2022, 62(8):2083-2092.
[3] MOORE R E, XU L L, TOWNSEND S D.Prospecting human milk oligosaccharides as a defense against viral infections[J].ACS Infectious Diseases, 2021, 7(2):254-263.
[4] CHEN X.Chapter four human milk oligosaccharides (HMOS):Structure, function, and enzyme-catalyzed synthesis[J].Advances in Carbohydrate Chemistry and Biochemistry, 2015, 72:113-190.
[5] KIYOHARA M, TACHIZAWA A, NISHIMOTO M, et al.Prebiotic effect of lacto-N-biose I on bifidobacterial growth[J].Bioscience, Biotechnology, and Biochemistry, 2009, 73(5):1175-1179.
[6] BODE L.Human milk oligosaccharides:Every baby needs a sugar mama[J].Glycobiology, 2012, 22(9):1147-1162.
[7] SAKURAMA H, KIYOHARA M, JUN W D, et al.Lacto-N-biosidase encoded by a novel gene of Bifidobacterium longum subspecies longum shows unique substrate specificity and requires a designated chaperone for its active expression[J].Journal of Biological Chemistry, 2013, 288(35):25194-25206.
[8] DU Z Q, LIU Z Y, TAN Y S, et al.Lacto-N-biose synthesis via a modular enzymatic cascade with ATP regeneration[J].iScience, 2021, 24(3):102236.
[9] VETERE A, MILETICH M, BOSCO M, et al.Regiospecific glycosidase-assisted synthesis of lacto-N-biose I (Galbeta1-3GlcNAc) and 3′-sialyl-lacto-N-biose I (NeuAcalpha2-3Galbeta1-3GlcNAc)[J].European Journal of Biochemistry, 2000, 267(4):942-949.
[10] D’ALMEIDA A, IONATA M, TRAN V, et al.An expeditious and efficient synthesis of β-D-galactopyranosyl-(1→3)-D-N-acetylglucosamine (lacto-N-biose) using a glycosynthase from Thermus thermophilus as a catalyst[J].Tetrahedron:Asymmetry, 2009, 20(11):1243-1246.
[11] KITAOKA M.Bifidobacterial enzymes involved in the metabolism of human milk oligosaccharides[J].Advances in Nutrition, 2012, 3(3):422S-429S.
[12] JUN W D, ANDO T, KIYOHARA M, et al.Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure[J].Applied and Environmental Microbiology, 2008, 74(13):3996-4004.
[13] 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.
[14] NISHIMOTO M.Large scale production of lacto- N-biose I, a building block of type I human milk oligosaccharides, using sugar phosphorylases[J].Bioscience, Biotechnology, and Biochemistry, 2020, 84(1):17-24.
[15] HAN N S, KIM T J, PARK Y C, et al.Biotechnological production of human milk oligosaccharides[J].Biotechnology Advances, 2012, 30(6):1268-1278.
[16] LI F Y, MA W, LIU L L, et al.Reprogramming the metabolic network in Kluyveromyces lactis with a transcriptional switch for de novo lacto-N-biose synthesis[J].Journal of Agricultural and Food Chemistry, 2023, 71(23):9031-9039.
[17] 周文, 游星, 张洪涛, 等.多细胞耦合转化N-乙酰氨基葡萄糖和乳糖生产唾液酸乳糖[J].生物工程学报, 2023, 39(11):4621-4634.
ZHOU W, YOU X, ZHANG H T, et al.Multicellular coupling fermentation for 3′-sialyllactose conversion using N-acetyl-glucosamine and lactose[J].Chinese Journal of Biotechnology, 2023, 39(11):4621-4634.
[18] 陈德康. 酵母酶系合成三磷酸核苷的研究[D].南京:南京工业大学, 2004.
CHEN D K.Study on synthesis of nucleoside triphosphate by yeast enzyme system[D].Nanjing:Nanjing University of Technology, 2004.
[19] LEE Y C, CHIEN H 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.
[20] CHEN L J, QIN Y M, MA L, et al.Biosynthesis of lacto-N-biose I from starch and N-acetylglucosamine via an in vitro synthetic enzymatic biosystem[J].Synthetic and Systems Biotechnology, 2023, 8(3):555-562.
