Regulation of Metabolic Pathways to Enhance Difucosyllactose Biosynthesis in Escherichia coli DOI

Shanquan Liang,

Qi Quan,

Dan Liu

et al.

Journal of Agricultural and Food Chemistry, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 19, 2024

Difucosyllactose (DFL), an important kind of fucosylated human milk oligosaccharides (HMOs), has garnered considerable attention due to its excellent physiological activities in infants. Previously, we achieved de novo biosynthesis DFL; however, substantial residual intermediates fucosyllactoses (FL) were detected. In this study, DFL was optimized, and FL reduced by regulating metabolic pathways. Different plasmid combinations used regulate gene expression, achieving optimal flux balance between 2′-FL DFL. The expression level key enzyme α-1,3-fucosyltransferase (α-1,3-FT, FucTa) then enhanced increasing copy number integrating fucTa into the chromosome. Exocytosis deleting sugar efflux transporter setA gene, thereby minimizing FL. Finally, strain BSF41 produced 55.3 g/L with only 2.59 a 5 L fermentor, representing highest reported titer date. This study provides foundation for advancing HMOs.

Language: Английский

De Novo Biosynthesis of Difucosyllactose by Artificial Pathway Construction and α1,3/4-Fucosyltransferase Rational Design in Escherichia coli DOI
Yingying Zhu, Roulin Chen, Hao Wang

et al.

Journal of Agricultural and Food Chemistry, Journal Year: 2024, Volume and Issue: unknown

Published: April 10, 2024

Difucosyllactose (DFL) is a significant and plentiful oligosaccharide found in human breast milk. In this study, an artificial metabolic pathway of DFL was designed, focusing on the de novo biosynthesis GDP-fucose from only glycerol. This achieved by engineering

Language: Английский

Citations

5
Efficient fermentative production of lactodifucotetraose by controlling sequential glycosyltransferase reactions in Escherichia coli DOI Open Access
Shu Moriyama,

Tomotoshi Sugita,

Makoto Yamashita

et al.

Biotechnology Progress, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 6, 2025

Abstract Lactodifucotetraose (LDFT) is a human milk oligosaccharide (HMO) that might reduce inflammation in infants. In this study, we established useful production process of LDFT by engineering two key enzymes, α1,2‐fucosyltransferase (α1,2‐FucT) and α1,3‐fucosyltransferase (α1,3‐FucT). First, verified which 2′‐fucosyllactose (2′‐FL) or 3‐fucosyllactose (3‐FL) (mostly unverified) was more useful. We searched for FucTs functioned efficiently vivo against the raw material lactose intermediates 2′‐FL 3‐FL external substrate addition to culture medium. found α1,2‐ FucT (HMFT) from Helicobacter mustelae N‐terminal truncated form α1,3‐FucT Bacteroides fragilis (BfFucTΔN10) had high potential. not converted LDFT, be attributed low reactivity HMFT as well uptake efficiency LacY, revealed growth test with exogenously added FL sole carbon source heterologously expressed intracellular fucosidase. Furthermore, because accumulation negative impact on cell growth, avoided route passing through 3‐FL. By adjusting copy numbers BffucTΔN10 , produced predominantly via 2′‐FL. Finally, 17.5 g/L (with 6.8 no residual lactose) accumulated 3‐L fed‐batch after 77 h. This study reports detailed analysis multiple pathways shows control glycosyltransferases can improve complex HMOs.

Language: Английский

Citations

0
Multidimensional Engineering of Escherichia coli MG1655 for the Efficient Biosynthesis of Difucosyllactose DOI
Yihan Chen, Chunhua Zhao,

Ruiyan Wang

et al.

Journal of Agricultural and Food Chemistry, Journal Year: 2025, Volume and Issue: 73(9), P. 5405 - 5413

Published: Feb. 24, 2025

Difucosyllactose (DFL), a representative bisfucosylated oligosaccharide found in human milk, has garnered significant attention due to its immense health benefits. To date, several plasmid-based engineered strains have been established for DFL synthesis. However, these face challenges such as antibiotic dependence and plasmid instability, which limit their commercial application the food industry. For this, plasmid-free Escherichia coli MG1655 were by integrating multicopy numbers of SAMT fut3Bc into genome overexpressing key genes involved GDP-l-fucose pathway. enhance catalytic efficiency, Fut3Bc was mutated based on AlphaFold 3, obtaining beneficial mutant (F24Y). The optimized strain, MGA2S-5, containing 2 copies 4 (F24Y) obtained. Eventually, strain MGA2S-5 synthesized 35.04 g/L 7 L bioreactor fed-batch cultivation, with no intermediate products remaining medium.

Language: Английский

Citations

0
Efficient production of 2′-fucosyllactose in Pichia pastoris through metabolic engineering and constructing an orthogonal energy supply system DOI Creative Commons
Yi Li, Xiang Wang,

Kaidi Chen

et al.

Synthetic and Systems Biotechnology, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

2'-fucosyllactose (2'-FL) holds significant role in the infants' nutrition. While microbial production of 2'-FL has predominantly utilized Escherichia coli and Saccharomyces cerevisiae, potential Pichia pastoris, renowned for its robust NADPH regeneration capability, remains underexplored. Herein, we systematically engineered metabolism P. pastoris to develop an efficient 2'-FL-producing cell factory. We first constructed de novo biosynthesis pathway achieving initial titer 0.143 g/L. By optimizing enzyme selection solubility α-1,2-fucosyltransferase (FutC), was enhanced by nearly ten folds. Subsequently, engineering supply further increased 170 %. Furthermore, energy incorporating orthogonal module based on methanol dissimilation increasing GTP availability, resulting a 32 % improvement production. Finally, through optimization fermentation condition, realized 3.50 g/L shake-flask, representing highest pastoris. These findings highlight as chassis produce chemicals providing abundant utilizing co-substrate sufficient energy.

Language: Английский

Citations

0
Structure, Function, Synthesis and Improved Strategies of Fucosylated Human Milk Oligosaccharides and Their Future Perspectives: A Review DOI

Ya-Ya Yang,

Shunli Jing,

Le Zhang

et al.

Food Bioscience, Journal Year: 2025, Volume and Issue: unknown, P. 106584 - 106584

Published: April 1, 2025

Language: Английский

Citations

0
Developing a Metabolic Flux Optimized Escherichia coli for Efficient Production of Difucosyllactose DOI
Yihan Chen, Yingying Zhu, Hao Wang

et al.

ACS Sustainable Chemistry & Engineering, Journal Year: 2024, Volume and Issue: 12(38), P. 14249 - 14257

Published: Sept. 9, 2024

Language: Английский

Citations

2
Iterative multimodule reprogramming and protein engineering for efficient lactodifucotetraose bioproduction DOI
Mengli Li, Tao Zhang, Chenchen Li

et al.

Food Bioscience, Journal Year: 2024, Volume and Issue: unknown, P. 105364 - 105364

Published: Oct. 1, 2024

Language: Английский

Citations

1
Regulation of Metabolic Pathways to Enhance Difucosyllactose Biosynthesis in Escherichia coli DOI

Shanquan Liang,

Qi Quan,

Dan Liu

et al.

Journal of Agricultural and Food Chemistry, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 19, 2024

Difucosyllactose (DFL), an important kind of fucosylated human milk oligosaccharides (HMOs), has garnered considerable attention due to its excellent physiological activities in infants. Previously, we achieved de novo biosynthesis DFL; however, substantial residual intermediates fucosyllactoses (FL) were detected. In this study, DFL was optimized, and FL reduced by regulating metabolic pathways. Different plasmid combinations used regulate gene expression, achieving optimal flux balance between 2′-FL DFL. The expression level key enzyme α-1,3-fucosyltransferase (α-1,3-FT, FucTa) then enhanced increasing copy number integrating fucTa into the chromosome. Exocytosis deleting sugar efflux transporter setA gene, thereby minimizing FL. Finally, strain BSF41 produced 55.3 g/L with only 2.59 a 5 L fermentor, representing highest reported titer date. This study provides foundation for advancing HMOs.

Language: Английский

Citations

1