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

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

2'-Fucosyllactose (2'-FL), the most abundant human milk oligosaccharide (HMO), has attracted considerable interest for its prebiotic and immunomodulatory functions, with broad applications in infant nutrition. In this study, we report the development of a high-yield, genome-integrated 2'-FL-producing strain based on Escherichia coli MG1655 through systematic modular optimization. Starting from a single-copy BKHT strain (MGC06), we first optimized the copy number of the α-1,2-fucosyltransferase (α-1,2-FT) gene BKHT. Subsequently, the GDP-L-fucose supply was enhanced through coordinated genomic integration of the gene clusters cpsG-cpsB and gmd-fcl, while the multidrug efflux transporter gene mdfA was integrated to improve product export and strain robustness. BKHT copy number was then re-evaluated in the optimized background, with four copies yielding the highest production. The final engineered strain, harboring all genetic modifications stably integrated into the chromosome, produced 17.18 g/L 2'-FL in shake-flask culture. In fed-batch fermentation using a 5-L bioreactor, this strain achieved a titer of 154.12 g/L after 60 h, with a productivity of 2.57 g/L/h. Notably, throughout the entire fermentation process, no antibiotics or inducers were supplemented, underscoring the genetic stability and regulatory compliance of this plasmid-free system. To our knowledge, this represents the highest 2'-FL titer reported to date, positioning our engineered strain as a promising candidate for commercial 2'-FL production.

Construction and systematic engineering of Saccharomyces cerevisiae for efficient de novo biosynthesis of pentostatin.

Pentostatin is a potent adenosine deaminase inhibitor, yet its industrial application is hindered by low extraction yields and complex chemical synthesis. Here, we report an efficient de novo biosynthesis platform for pentostatin in Saccharomyces cerevisiae. Starting with the heterologous expression of cns3 from Cordyceps militaris, we optimized the cell factory via promoter engineering, multicopy integration, and AAH1 knockout. This integration strain achieved a maximum pentostatin titer of 16.28mg/L in shake-flask cultivation, representing a 19.38-fold improvement over our initial production. Separately, to alleviate severe product toxicity, we implemented flux balance analysis (FBA)-guided transporter engineering; the engineered strain expressing the episomal efflux pump Cns4 yielded a titer of 8.27mg/L while significantly accelerating the production process. Molecular docking revealed a distinct binding cavity where key residues (e.g., Asp296, Ala292) capture pentostatin via specific hydrogen bonds and hydrophobic interactions. Furthermore, transcriptomics demonstrated that Cns4 globally reprograms carbon and energy metabolism to boost precursor supply and cellular robustness. This work integrates structural insights with systems metabolic engineering, providing a generalizable paradigm for biosynthesizing toxic nucleoside natural products.

A Pichia pastoris-based platform for large-scale production of functional recombinant human serum albumin with therapeutic efficacy

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.

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, evaluating 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.

Advances in Key Genetic Elements and Strategies for High-Yield Heterologous Protein Expression in Komagataella phaffii

This review summarizes recent advances in engineering key expression elements underlying heterologous protein production in K. phaffii, with particular emphasis on promoter architecture redesign, signal peptide replacement and sequence engineering, molecular chaperone co-expression, and quantitative regulation of the unfolded protein response.