Xylitol, a widely used sugar substitute in food and medicine, can be produced through microbial bioconversion using glucose as the primary substrate. In this process, a critical factor limiting xylitol production is the relatively low activity of xylitol dehydrogenase (XDH) during the biotransformation of D-arabitol to xylitol by resting cells of Gluconobacter oxydans. To improve the catalytic performance, GoXDH was engineered by site-saturation mutagenesis combined with a high-throughput screening method, and a variant GoXDH_M3 (S77C/S106N/A110N) with high activity was obtained, showing a 2.49-fold increase in catalytic activity. The structural analysis revealed that the S77C/S106N/A110N mutations enhanced proton and electron transfer rates while stabilizing the hydrophilic substrate-binding pocket and the tetrameric structure. Additionally, by optimizing the coenzyme regeneration system and enhancing the oxygen transfer efficiency, we developed an efficient biotransformation of D-arabitol to xylitol in G. oxydans. Using resting cells of G. oxydans/XDH_M3-GDH-VHb, a xylitol titer of 29.02 g/L were achieved from 40 g/L D-arabitol within 30 h. The findings suggest that boosting XDH activity through semi-rational engineering markedly improves xylitol productivity in G. oxydans.

Komagataella phaffii has emerged as an important eukaryotic microbial cell factory for the production of industrial enzymes, biopharmaceuticals, and food-related proteins owing to its rapid growth, capacity for eukaryotic post-translational modifications, and suitability for high-cell-density fermentation. However, efficient heterologous protein production remains constrained by limitations in transcriptional regulation, protein secretion, and endoplasmic reticulum (ER) protein-folding capacity. This review summarizes recent advances in engineering key expression elements underlying heterologous protein production in K. phaffii, with particular emphasis on promoter architecture redesign (e.g., upstream activating sequence (UAS) duplication and core promoter mutagenesis), signal peptide replacement and sequence engineering, molecular chaperone co-expression, and quantitative regulation of the unfolded protein response (UPR). Rather than focusing on individual expression modules, this review highlights a systems-level engineering perspective that integrates regulatory elements, intracellular processing, and secretory pathway optimization, illustrating the ongoing transition from empirical modification of individual expression elements to systems-level engineering of the secretory pathway. Recent advances in synthetic biology and artificial intelligence (AI)-assisted approaches are further accelerating the development of predictive and precision engineering approaches for K. phaffii. Together, these advances provide a foundation for the rational design of next-generation K. phaffii cell factories for the sustainable production of structurally complex and high-value recombinant proteins.

Ergothioneine (EGT), a naturally occurring amino acid derivative with potent antioxidant and cytoprotective properties, is widely applied in the food, cosmetic, and medical industries. Traditional production methods are limited by high costs, low efficiency, and environmental concerns, so microbial fermentation serves as a sustainable alternative for EGT production. In this study, Escherichia coli BL21 (DE3) was employed as the chassis strain. First, a basic EGT-producing engineered strain was constructed by heterologously expressing the egtB gene from Methylobacterium pseudosasicola along with the egtD and egtE genes from Mycobacterium smegmatis. This initial strain achieved a yield of 84.84 ± 1.64 mg/L of EGT in shake-flask cultures. To enhance production, solubility-enhancing tags were introduced to improve the soluble expression of the key enzymes, and metabolic pathways were rationally engineered to strengthen the supply of essential precursor amino acids. These modifications led to the development of a high-yield EGT strain. After optimizing the fermentation process, the best results were achieved using a medium with glycerol as the carbon source, 0.5 g/L of histidine, 1.5 g/L of methionine, and 1.0 g/L of cysteine, along with induction at 25 °C using 0.2 mM IPTG for 120 h. Under these conditions, the final EGT yield reached 385.70 ± 4.86 mg/L. The engineered strain for EGT synthesis and optimized fermentation strategy developed in this study offer a useful basis for further process development.

Recombinant collagen is an attractive alternative to animal-derived collagen for applications in tissue engineering, wound repair, regenerative medicine, and advanced aesthetic biomaterials. However, many microbial production hosts lack sufficient endogenous prolyl 4-hydroxylase (P4H) activity, resulting in inadequate proline hydroxylation, reduced triple-helix stability, and potentially impaired material performance. Engineering P4H therefore offers a promising strategy for enhancing proline hydroxylation and improving the triple-helix stability of recombinant collagen. This review summarizes recent advances in the structural characterization, catalytic mechanisms, and engineering of P4H systems from animal, plant, viral, and microbial sources. Particular attention is given to the structural and functional differences between animal-derived α₂β₂ tetrameric P4Hs and structurally simpler monomeric P4Hs, including their distinct requirements for subunit assembly, folding, substrate recognition, and heterologous expression. Structure-guided engineering strategies, including active-site remodeling, substrate-binding-pocket expansion, flexible-loop engineering, collagen-anchoring module fusion, and computationally guided protein design, are evaluated for their potential to improve hydroxylation efficiency toward long-chain collagen substrates and reduce nonproductive uncoupling. At the host–cell level, synthetic biology strategies for coordinating P4H expression, subunit assembly, chaperone-assisted folding, secretion, and cofactor metabolism are discussed. The review also highlights the importance of engineering α-ketoglutarate (α-KG), iron, and L-ascorbate metabolism to match cofactor availability with catalytic demand while minimizing cellular stress. In addition, the spatial and temporal coordination of collagen synthesis, P4H-mediated hydroxylation, intracellular folding, and secretion is discussed as a means of improving hydroxylation efficiency and product uniformity. A comprehensive quality-assessment framework is further proposed, integrating site-specific mass spectrometry for hydroxylation profiling, spectroscopic and thermal analyses for structural characterization, and cell-based assays for evaluating biological activity. Finally, the review addresses challenges associated with industrial scale-up, including oxygen-transfer limitations, dynamic cofactor management, process consistency, and regulatory compliance. Emerging approaches involving artificial intelligence, cell-free protein synthesis, and integrated computational–experimental workflows are also considered for accelerating the development of clinically and industrially viable recombinant collagen products.