Systematic modular engineering of genome-integrated Escherichia coli MG1655 for high-level 2'-fucosyllactose production.

Plasmid- and Inducer-Free Biosynthesis of Difucosyllactose through Rational Fusion Enzyme Engineering and Systematic Pathway Optimization.

Difucosyllactose (DFL), a fucosylated human milk oligosaccharide, exhibits significant application potential in the food and pharmaceutical industries. However, the microbial production of DFL faces challenges such as the accumulation of the intermediate 2'-fucosyllactose (2'-FL) and the high costs associated with the use of antibiotics and inducers. In this study, we constructed a plasmid- and inducer-free Escherichia coli MG1655 strain to enhance the biosynthesis of DFL while minimizing 2'-FL accumulation. Initially, the de novo DFL biosynthetic pathway was established by introducing different α-1,3-fucosyltransferases (α-1,3-FucT) into a 2'-FL-producing strain. Combinatorial metabolic engineering strategies were then employed to improve DFL accumulation. Subsequently, guided by the in silico multienzyme assembling by reshaping space (iMARS) framework, a fusion enzyme, FucTaY218K-L60-FutC, was rationally designed to effectively alleviate 2'-FL accumulation. Furthermore, modulation of the hydrophobic microenvironment in the substrate-binding pocket of FucTaY218K yielded the beneficial mutant FucTaY218K/W31R. Finally, modifications to the lactose operon significantly improved strain growth while concurrently enhancing DFL production. The best-performing strain achieved a DFL titer of 74 g/L in a 5-L bioreactor, corresponding to a productivity of 0.96 g/L/h, representing the highest productivity reported to date. This study establishes a robust and environmentally friendly platform for the industrial-scale production of DFL and provides a strategy for the microbial synthesis of other high-value human milk oligosaccharides.

Systematic pathway engineering of Escherichia coli for enhanced 3’-sialyllactose production

3’-Sialyllactose (3’-SL), the simplest sialylated human milk oligosaccharide (HMO), is widely incorporated into infant formula due to its roles in shaping the gut microbiota, inhibiting pathogen adhesion, and supporting neurodevelopment. In this study, we developed an efficient microbial cell factory for 3’-SL production by enhancing carbon utilization and CTP regeneration. Following the identification of a highly active α-2,3-sialyltransferase, metabolic flux was redirected through pfkA deletion and fbaA overexpression. The precursor pool was strengthened by optimizing the expression of key enzymes involved in UDP-GlcNAc and CMP-Neu5Ac biosynthesis. To further improve pathway balance and stability, promoter engineering was applied to optimize the expression of four key genes (neuA, neuB, neuC and ST). The optimized multigene cassette was subsequently integrated into the chromosome using a one-step multicopy integration strategy, generating a plasmid-free and antibiotic marker-free production strain. The resulting strain achieved 3’-SL titers of 7.75 g L−1 in shake flasks and 102.18 g L−1 in a 5-L fed-batch bioreactor, representing, to the best of our knowledge, the highest reported level to date. This work demonstrates a robust strategy combining metabolic rewiring, adaptive gene expression tuning and multicopy genome integration, providing a versatile platform for the sustainable production of 3’-SL and other high-value HMOs.

Tailoring Precise Genomic Integration Toward Isolate-to-Industry Strain Development for Scalable High-Titer Production of Polyhydroxyalkanoate

An expression framework for production of recombinant methionine aminopeptidase in Escherichia coli: a case study of upstream process.

Incomplete removal of the initiator methionine is a frequent bottleneck in Escherichia coli-based production of recombinant proteins, causing heterogeneity and increased immunogenicity of biopharmaceuticals. Methionine aminopeptidase (MAP) is the key enzyme responsible for this post-translational modification, yet its endogenous activity is rapidly saturated under high-level expression conditions. Here, we report a case-study evaluation of a pBR322-derived expression system, previously applied to therapeutic peptides and insulin analogs, in combination with fed-batch cultivation for recombinant production of methionine aminopeptidase in E. coli. The map gene from E. coli BL21(DE3) was cloned into the pF644 vector to generate pF1492. During fed-batch cultivation under the tested conditions, specific productivity reached 127.03 ± 8.66 mg·g-1 and volumetric productivity of total cell-associated MAP reached 2.71 ± 0.18 g·L-1 by the final hour of induction. MAP accumulated predominantly as insoluble inclusion bodies, which is a common outcome for recombinant protein expression in E. coli at high rates. This study reports upstream production and inclusion body formation only; functional recovery and enzymatic activity were not assessed. Acetate remained moderate (35-60 mM) and biomass was stable, indicating balanced metabolism. The present study evaluates the performance of this system for methionine aminopeptidase as a stress-sensitive model protein under the tested conditions, without a side-by-side comparison with alternative expression systems.

Systematic engineering of Pichia pastoris for high-level production of recombinant human granulocyte-macrophage colony-stimulating factor.

Human granulocyte-macrophage colony-stimulating factor (hCSF2) is a clinically important therapeutic glycoprotein with growing market demand, underscoring the need for efficient recombinant production platforms. To enable high-level production of recombinant hCSF2 (rhCSF2), the Pichia pastoris GS115 strain was systematically engineered through multi-copy integration of the expression cassette, screening for suitable secretory signal peptides, and enhancement of protein folding and transport pathways. The combination of multi-copy integration at multiple genomic loci with the combinatorial use of endogenous and exogenous signal peptides proved critical for improving rhCSF2 titers, while reinforcement of the folding and transport machinery further alleviated the bottleneck of protein throughput. Following high-density fermentation in a 3‑L bioreactor, the optimally engineered strain achieved an rhCSF2 titer of 3.4 g/L-markedly exceeding the highest titer previously reported in yeast hosts by an order of magnitude. Importantly, this engineering strategy was successfully extended to a glycoengineered strain SuperMan5, where it enabled production of rhCSF2 with more homogeneous and humanized N‑glycans and a titer of 4.3 g/L under high‑density fermentation condition. Our work demonstrates that the high-yield production of recombinant human therapeutic glycoproteins bearing homogeneous, humanized N-glycans in engineered yeast offers promising prospects.

Systems metabolic engineering of Corynebacterium glutamicum for efficient production of L-isoleucine

L-Isoleucine is an essential branched-chain amino acid for livestock and poultry, supporting protein accretion and regulating energy metabolism, immune function, and stress resilience. A genetically stable L-isoleucine producer, Corynebacterium glutamicum cgl-Ile0, was obtained via biosensor-assisted ARTP mutagenesis, and produced 11.52 g/L L-isoleucine in a 5-L fermenter, with a yield of 0.11 g/g and a productivity of 0.24 g/L/h. Whole-genome resequencing revealed four mutations associated with the phenotype, including aspBV346I, asdP27E, brnEL87S, and brnFR28P. Structure-guided protein engineering of two rate-limiting enzymes—threonine dehydratase (TD) and acetohydroxyacid synthase (AHAS)—generated strain cgl-Ile1, increasing titer, yield, and productivity by 39.50%, 27.27%, and 37.50%, respectively, relative to strain cgl-Ile0. Subsequent modular optimization of L-isoleucine biosynthesis, oxaloacetate supply module, cofactor-supply module, and transport/export module yielded the final strain cgl-Ile8. Through optimization pH and dissolved oxygen, the titer, yield and productivity of L-isoleucine produced by strain cgl-Ile8 in a 5-L fermenter were 48.49 g/L, 0.31 g/g and 1.01 g/L/h, which were 4.21-, 2.82-, and 4.21-fold those of strain cgl-Ile0, respectively. Scale-up to a 50-L fermenter further increased the titer, yield and productivity to 50.12 g/L, 0.32 g/g and 1.04 g/L/h, respectively, representing the highest reported L-isoleucine titer in C. glutamicum to date. We developed an industrial L-isoleucine-producing C. glutamicum strain by integrating biosensor-guided ARTP mutagenesis, structure-guided protein engineering, and modular pathway rewiring, providing a practical and transferable framework for constructing GRAS amino-acid producers for animal nutrition.