Metabolic engineering of Priestia megaterium for 2’-fucosyllactose production

Microbial Cell Factories, Journal Year: 2025, Volume and Issue: 24(1)

Published: Jan. 4, 2025

Abstract Background 2′-Fucosyllactose (2′-FL) is a predominant human milk oligosaccharide that significantly enhances infant nutrition and immune health. This study addresses the need for safe economical production of 2’-FL by employing Generally Recognized As Safe (GRAS) microbial strain, Priestia megaterium ATCC 14581. strain was chosen its robust growth established safety profile attributing suitable industrial-scale production. Results The engineering targets included deletion lacZ gene to prevent lactose metabolism interference, introduction α-1,2-fucosyltransferase derived from non-pathogenic optimization GDP-L-fucose biosynthesis pathway through overexpression manA manC . These changes, coupled with improvements in uptake utilization random mutagenesis, led high yield 28.6 g/L fed-batch fermentation, highlighting potential our metabolic strategies on P. Conclusions GRAS 14581 successfully engineered overproduce 2’-FL, valuable oligosaccharide, series genetic modifications optimizations. work underscores feasibility using strains oligosaccharides, paving way safer more efficient methods biotechnological applications. Future studies could explore additional fermentation conditions further enhance scalability.

Language: Английский

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Engineering Pichia pastoris for Efficient De Novo Synthesis of 2′-Fucosyllactose

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

Published: March 28, 2025

2′-Fucosyllactose (2′-FL), the most abundant in human milk oligosaccharides (HMOs), is a nutrient of great importance. As safe organism widely used industries, Pichia pastoris was tested here for 2′-FL production. The de novo biosynthesis pathway constructed using genome-editing technology based on CRISPR-Cas9 with an initial titer 1.01 g/L. Introducing N-terminal SUMO or Ub tag to FucT2 and transporter CDT2 from Neurospora crassa into P. found improve Then, modular metabolic engineering conducted production, enhancing GTP supply module, NADPH regeneration precursor module. Subsequently, key enzyme semirationally designed further increase Finally, production by engineered scaled up 3 L fermenter fed-batch mode, resulting 22.35 g/L that highest pastoris. results prove effectiveness strategies demonstrate could be potential chassis produce HMOs.

Language: Английский

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Microbial Synthesis of Lacto-N-fucopentaose I with High Titer and Purity by Screening of Specific Glycosyltransferase and Elimination of Residual Lacto-N-triose II and Lacto-N-tetraose

Journal of Agricultural and Food Chemistry, Journal Year: 2024, Volume and Issue: 72(8), P. 4317 - 4324

Published: Feb. 16, 2024

Lacto-N-fucopentaose I (LNFP I) has recently been approved as generally recognized safe, demonstrating its great commercial potential in the food industry. Microbial synthesis through metabolic engineering strategies is an effective approach for large-scale production of LNFP I. Biosynthesis requires consideration two key points: high titer with low byproduct 2′-fucosyllactose (2′-FL) generation and purity lacto-N-triose II (LNTri II) lacto-N-tetraose (LNT) residues. Herein, α1,2-fucosyltransferase from Thermoanaerobacterium sp. RBIITD was screened 16 selected I-producing glycosyltransferase candidates, showing highest vivo productivity. Chromosomal integration wbgO enhanced by improving precursor conversion LNTri to LNT. The best engineered strain produced 4.42 35.1 g/L shake-flask fed-batch cultivation, respectively. residual LNT were eliminated further cultivation a recombinant coexpressing Bifidobacterium bifidum β-N-acetylhexosaminidase lacto-N-biosidase. A strategy biosynthesis yield finally realized, providing support practical application production.

Language: Английский

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Metabolic Engineering of Escherichia coli BL21(DE3) for 2′-Fucosyllactose Synthesis in a Higher Productivity

ACS Synthetic Biology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 15, 2025

2′-Fucosyllactose (2′-FL) is the most abundant human milk oligosaccharides (HMOs). 2′-FL exhibits great benefits for infant health, such as preventing infantile diarrhea and promoting growth of intestinal probiotics. The microbial cell factory technique has shown promise massive production 2′-FL. Here, we aimed to construct a recombinant E. coli BL21(DE3) strain hyperproduction Initially, multicopy genomic integration expression lactose permease gene lacY reduced formation byproducts. Furthermore, more efficient Shine–Dalgarno sequence was used replace wild-type in manC-manB gmd-wcaG clusters, which significantly increased titer. Based on these results, overexpressed sugar efflux transporter SetA knocked out pgi gene. This further improved synthesis when glycerol sole carbon source. Finally, new α-1,2-fucosyltransferase identified Neisseria sp., exhibited higher capacity production. Fed-batch fermentation produced 141.27 g/L 45 h with productivity 3.14 × h. rate achieved highest recorded levels, indicating potential engineered BL21 (DE3) strains use industrial

Language: Английский

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Construction and Optimization of a Bgar-Based Lactose Biosensor for High-Throughput Selection of Human Milk Oligosaccharides Producers

Published: Jan. 1, 2025

Language: Английский

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Multidimensional Engineering of Escherichia coli MG1655 for the Efficient Biosynthesis of Difucosyllactose

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

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Biosynthesis and metabolic engineering of natural sweeteners

AMB Express, Journal Year: 2025, Volume and Issue: 15(1)

Published: March 18, 2025

Natural sweeteners have attracted widespread attention because they are eco-friendly, healthy, low in calories, and tasty. The demand for natural is increasing together with the popularity of green, low-carbon, sustainable development. With development synthetic biology, microbial cell factories emerged as an effective method to produce large amounts sweeteners. This technology has significantly progressed recent years. review summarizes pathways enzymes related biosynthesis sweeteners, such mogrosides, steviol glycosides, glycyrrhizin, glycyrrhetinic acid, phlorizin, trilobatin, erythritol, sorbitol, mannitol, thaumatin, monellin, brazzein. Moreover, it focuses on research about production these using biology methods, aiming provide a reference future

Language: Английский

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Efficient production of 2′-fucosyllactose in Pichia pastoris through metabolic engineering and constructing an orthogonal energy supply system

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

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Metabolic engineering Corynebacterium glutamicum ATCC13032 for 2′-fucosyllactose production

Systems Microbiology and Biomanufacturing, Journal Year: 2025, Volume and Issue: unknown

Published: April 25, 2025

Language: Английский

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De Novo Synthesis of Lacto-N-Neotetraose in Escherichia coli through Metabolic Engineering with Glucose as the Sole Carbon Source

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

Published: May 26, 2025

Lacto-N-neotetraose (LNnT), the predominant nonfucosylated human milk oligosaccharide (HMO), significantly enhances immune function of intestinal epithelial cells and prevents necrotizing enterocolitis. However, current LNnT synthesis processes depend on exogenous lactose supplementation, leading to increased production costs operational complexity. In this study, we established de novo by engineering an endogenous biosynthesis pathway with glucose as sole carbon source. First, pathway. Second, optimized metabolic network balance reduced flux branching modulating phosphorylation efficiency fine-tuning expression levels key modules. Following this, was through targeted enhancement transport UDP-Gal optimization. As a result, developed high-yielding E. coli engineered strain for production. The SC16-14 achieved titer 25.4 g/L during 79 h fermentation in 3-L bioreactor. This study not only demonstrates feasibility but also provides novel strategy reduce simplify manufacturing process.

Language: Английский

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