As one of the main components of human milk oligosaccharides (HMOs), 3-fucosyllactose (3-FL) constitutes approximately 5% of HMOs.It plays important roles in modulating gut microbiota, providing immune support, and promoting brain development.At present, the research on the synthesis of 3-FL mainly focuses on Escherichia coli.This study introduced lac12 from Kluyveromyces lactis, gmd and gmer from Mortierella alpina, and screened six α-1,3-fucosyltransferases (α-1,3-Fut).Among them, fut3Bc from Neobacillus cucumis was selected, enabling the synthesis of 3-FL in Yarrowia lipolytica for the first time, with a yield of 0.66 g/L.Further overexpression of related genes identified the metabolic step catalyzed by α-1,3-Fut as the rate-limiting step in 3-FL synthesis.Subsequently, by adding a small ubiquitin-related modifier (SUMO) tag to Fut3Bc and multi-copy expressing SUMO-fut3Bc, the 3-FL yield increased to 2.28 g/L.Finally, enhancing the supply of NADPH further improved the 3-FL yield to 2.84 g/L.After fed-batch fermentation in 5 L fermentor, the 3-FL yield reached 14.70 g/L.This study explores the feasibility of producing 3-FL in Yarrowia lipolytica and provides valuable insights for constructing microbial cell factories for the production of HMOs.
HUANG Yong
,
ZHANG Yiran
,
XIN Yu
,
GU Zhenghua
,
LI Moying
,
ZHU Rui
,
GUO Zhongpeng
,
ZHANG Liang
. Metabolic engineering of Yarrowia lipolytica for synthesis of 3-fucosyllactose[J]. Food and Fermentation Industries, 2026
, 52(4)
: 10
-18
.
DOI: 10.13995/j.cnki.11-1802/ts.042052
[1] ZHOU Y L, SUN H, LI K F, et al.Dynamic changes in human milk oligosaccharides in Chinese population:A systematic review and meta-analysis[J].Nutrients, 2021, 13(9):2912.
[2] BYCH K, MIKŠ 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, 56:130-137.
[3] DU Z H, LI Z Y, GUANG C E, et al.Recent advances of 3-fucosyllactose in health effects and production[J].Archives of Microbiology, 2024, 206(9):378.
[4] LI Z Y, ZHU Y Y, NI D W, et al.Occurrence, functional properties, and preparation of 3-fucosyllactose, one of the smallest human milk oligosaccharides[J].Critical Reviews in Food Science and Nutrition, 2023, 63(28):9364-9378.
[5] CHEN G, WU H, ZHU Y Y, et al.Glycosyltransferase from Bacteroides gallinaceum is a novel a-1,3-fucosyltransferase that can be used for 3-fucosyllactose production in vivo by metabolically engineered Escherichia coli[J].Journal of Agricultural and Food Chemistry, 2022, 70(6):1934-1942.
[6] DU Z H, ZHU Y Y, LU Z, et al.Combinatorial optimization strategies for 3-fucosyllactose hyperproduction in Escherichia coli[J].Journal of Agricultural and Food Chemistry, 2024, 72(25):14191-14198.
[7] LEE J W, KWAK S, LIU J J, et al.Enhanced 2′-fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate[J].Metabolic Engineering, 2020, 62:322-329.
[8] 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):2529-2536.
[9] 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.
[10] XU M Y, SUN M T, MENG X F, et al.Engineering pheromone-mediated quorum sensing with enhanced response output increases fucosyllactose production in Saccharomyces cerevisiae[J].ACS Synthetic Biology, 2023, 12(1):238-248.
[11] LIU H H, JI X J, HUANG H.Biotechnological applications of Yarrowia lipolytica:Past, present and future[J].Biotechnology Advances, 2015, 33(8):1522-1546.
[12] BLANK L M, LEHMBECK F, SAUER U.Metabolic-flux and network analysis in fourteen hemiascomycetous yeasts[J].FEMS Yeast Research, 2005, 5(6-7):545-558.
[13] HOLLANDS K, BARON C M, GIBSON K J, et al.Engineering two species of yeast as cell factories for 2′-fucosyllactose[J].Metabolic Engineering, 2019, 52:232-242.
[14] FAIJES M, CASTEJÓN-VILATERSANA M, VAL-CID C, et al.Enzymatic and cell factory approaches to the production of human milk oligosaccharides[J].Biotechnology Advances, 2019, 37(5):667-697.
[15] XIE Y K, WU X Y, FU C, et al.Rational design of an a-1,3-fucosyltransferase for the biosynthesis of 3-fucosyllactose in Bacillus subtilis ATCC 6051a via de novo GDP-l-fucose pathway[J].Journal of Agricultural and Food Chemistry, 2024, 72(2):1178-1189.
[16] HUANG D, YANG K X, LIU J, et al.Metabolic engineering of Escherichia coli for the production of 2′-fucosyllactose and 3-fucosyllactose through modular pathway enhancement[J].Metabolic Engineering, 2017, 41:23-38.
[17] LI M L, LI C C, LUO Y J, et al.Multi-level metabolic engineering of Escherichia coli for high-titer biosynthesis of 2′-fucosyllactose and 3-fucosyllactose[J].Microbial Biotechnology, 2022, 15(12):2970-2981.
[18] NI Z J, WU J Y LI Z K, et al.Enhanced bioproduction of fucosylated oligosaccharide 3-fucosyllactose in engineered Escherichia coli with an improved de novo pathway[J].Bioscience, Biotechnology, and Biochemistry, 2021, 85(7):1772-1781.
[19] CHOI Y H, KIM J H, PARK B S, et al.Solubilization and iterative saturation mutagenesis of a1,3-fucosyltransferase from Helicobacter pylori to enhance its catalytic efficiency[J].Biotechnology and Bioengineering, 2016, 113(8):1666-1675.
[20] XIE X, WU P, HUANG X C, et al.Retro-protein XXA is a remarkable solubilizing fusion tag for inclusion bodies[J].Microbial Cell Factories, 2022, 21(1):51.
[21] LIN L, GONG M Y, LIU Y F, et al.Combinatorial metabolic engineering of Escherichia coli for de novo production of 2′-fucosyllactose[J].Bioresource Technology, 2022, 351:126949.
[22] ZHANG Y, ZHANG X J, LIU H Y, et al.Efficient production of 2′-fucosyllactose in unconventional yeast Yarrowia lipolytica[J].Synthetic and Systems Biotechnology, 2023, 8(4):716-723.
[23] HUANG H Y, YU W W, XU X H, et al.Combinatorial engineering of Escherichia coli for enhancing 3-fucosyllactose production[J].American Chemical Society Synthetic Biology, 2024, 13(6):1866-1878.
[24] CHENG H L, WANG S Q, BILAL M, et al.Identification, characterization of two NADPH-dependent erythrose reductases in the yeast Yarrowia lipolytica and improvement of erythritol productivity using metabolic engineering[J].Microbial Cell Factories, 2018, 17(1):133.
