Engineering Escherichia coli for Highly Efficient Biosynthesis of Lacto-N-difucohexaose II through De Novo GDP-l-fucose Pathway

Journal of Agricultural and Food Chemistry, Journal Year: 2024, Volume and Issue: 72(18), P. 10469 - 10476

Published: April 25, 2024

Lacto-N-difucohexaose II (LNDFH II) is a typical fucosylated human milk oligosaccharide and can be enzymatically produced from lacto-N-tetraose (LNT) by specific α1,3/4-fucosyltransferase Helicobacter pylori DMS 6709, referred to as FucT14. Previously, we constructed an engineered Escherichia coli BL21(DE3) with single plasmid for highly efficient biosynthesis of LNT. In this study, two additional plasmids harboring the de novo GDP-L-fucose pathway module FucT14, respectively, were further introduced construct strain successful LNDFH II. FucT14 was actively expressed, major product, lacto-N-fucopentaose (LNFP) V minor trace amount LNFP 3-fucosyllactose very products. Additional expression α1,3-fucosyltransferase FutM1 Bacteroidaceae bacterium gut metagenome could obviously enhance biosynthesis. After optimization induction conditions, maximum titer reached 3.011 g/L shake-flask cultivation. During fed-batch cultivation, efficiently highest 18.062 productivity yield 0.301 g/L·h.

Language: Английский

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Highly Efficient In Vivo Production of Sialyllacto-N-tetraose C via Screening of Beneficial β1,4-galactosyltransferase and α2,6-sialyltransferase

Journal of Agricultural and Food Chemistry, Journal Year: 2025, Volume and Issue: 73(9), P. 5376 - 5384

Published: Feb. 18, 2025

Biological production of human milk oligosaccharides (HMOs) using metabolically engineered strains is a research hotspot in food biotechnology, but less effort has been made on the biological sialylated complex HMOs. Sialyllacto-N-tetraose c only monosialylated HMO top 15 In this study, metabolic pathway LST was constructed Escherichia coli BL21(DE3) by introducing three sequential glycosyltransferases: β1,3-N-acetylglucosaminyltransferase, β1,4-galactosyltransferase, and α2,6-sialyltransferase. The cytidine 5′-monophospho (CMP)-N-acetylneuraminic acid (Neu5Ac) enhanced to improve production. β1,4-galactosyltransferase from Helicobacter pylori J99 (HpGalT) α2,6-sialyltransferase Vespertiliibacter pulmonis (ED6ST) were screened as pair key glycosyltransferases for enhancing final strain could produce 1.718 9.745 g/L shake-flask fed-batch cultivation, respectively, indicating feasibility efficient biosynthesis

Language: Английский

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Efficient Biosynthesis of Sialyllacto-N-tetraose a by a Metabolically Engineered Escherichia coli BL21(DE3) Strain

Journal of Agricultural and Food Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: March 4, 2025

Recently, the construction of metabolically engineered strains for microbial synthesis human milk oligosaccharides (HMOs) has attracted increasing attention. However, fewer efforts were made in vivo biosynthesis complex HMOs, especially sialylated HMOs. In this study, we Escherichia coli BL21(DE3) to efficiently produce sialyllacto-N-tetraose a (LST-a) efficiently. Three sequential glycosylation steps introduced construct LST-a pathway, catalyzed by β1,3-N-acetylglucosaminylation, β1,3-galactosylation, and α2,3-sialylation. Pathway genes cytidine 5'-monophospho (CMP)-N-acetylneuraminic acid (Neu5Ac) support sialylation donor supply. Production was improved deleting competitive CMP-Neu5Ac synthesis, screening more efficient α2,3-sialyltransferase, combinatorial optimization pathway gene expression. finally produced with titer 1.235 4.85 g/L shake-flask fed-batch cultivation, respectively, demonstrating feasibility production

Language: Английский

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Comprehensive review on human Milk oligosaccharides: Biosynthesis, structure, intestinal health benefits, immune regulation, neuromodulation mechanisms, and applications

Food Research International, Journal Year: 2025, Volume and Issue: 209, P. 116328 - 116328

Published: April 6, 2025

Language: Английский

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Glycosyltransferases in human milk oligosaccharide synthesis: structural mechanisms and rational design

Current Opinion in Biotechnology, Journal Year: 2025, Volume and Issue: 93, P. 103315 - 103315

Published: May 9, 2025

Language: Английский

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