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Jian Chen

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Jul 2026

Precursor supply and translational machinery engineering of Saccharomyces cerevisiae for improving cellular protein content and biomass-based microbial protein bioproduction.

The sustainable production of biomass-based microbial protein (MP) requires efficient microbial cell factories for accumulating cellular protein with high content, which is beneficial both for improving protein production and downstream cellular protein isolation and purification. To overcome the limited protein content of the Saccharomyces cerevisiae, we designed a systematic multilevel metabolic engineering strategy. Initially, single-gene edits based on predictions using the genome-scale model Yeast 9.0.2 and the OptForce algorithm failed to increase protein content due to precursor supply limitations. Enhancing genes in nitrogen metabolic (GDH1, GDH2, GLN1, GLT1) and central carbon (CIT1, IDH1) pathways were implemented to synergistically enhance ammonium assimilation. Subsequently, overexpression of valyl-tRNA synthetase (VAS1) alleviated the translational bottleneck, increasing cellular protein content to 52.3 g/100 g dry cell weight (DCW). The ribosomal synthesis pathway was further enhanced via ribosomal regulator IFH1 and ribosomal protein gene overexpression, with cellular protein content reaching 57.3 g/100 g DCW. Finally, diploidization and global transcriptional regulator SUT1 integration in strain D3 achieved a protein content of 66.5 g/100 g DCW in shake flask culture. Under controlled 5 L bioreactor conditions, its protein content further increased to a peak of 75.2 g/100 g DCW, representing a 50.3% increase over the parental strain Y1. This study developed a multilevel engineering strategy to enhance yeast protein production by optimizing precursor supply, translation machinery, and diploid construction. Using marker-free editing and endogenous gene regulation, it provides both improved protein content and key targets for breeding high-protein microbial strains.

Yang Liu, Caiyin Jin, Wentao Shen et al. · 1 citation
Aug 2026

Mechanism-Guided Synergistic Engineering of Substrate Access and Catalytic Microenvironment in Oxidosqualene Cyclase for Enhanced Amyrin Biosynthesis.

Oxidosqualene cyclases (OSCs) catalyze the cyclization of 2,3-oxidosqualene into diverse triterpenoids, yet their intrinsically low catalytic efficiency restricts biosynthetic productivity. Here, we establish a mechanism-guided synergistic engineering strategy that extends beyond conventional active-site engineering by integrating distal substrate access regulation with catalytic microenvironment optimization to enhance the catalytic performance of CrAS from Catharanthus roseus. Structural modeling and mechanistic analyses revealed a conserved catalytic framework involving carbocation-mediated polycyclization and identified a surface-exposed constriction region that regulates substrate access. Guided by these insights, distal surface engineering of the constriction region was synergistically combined with active pocket optimization. The resulting combinatorial mutant, M3 (L323A/T327K/N565I), exhibited a 95.2% increase in catalytic efficiency and enhanced α-amyrin and β-amyrin by 53.2% and 49.7%, reaching 158 mg/L and 63 mg/L, respectively. Multi-scale analyses combining molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations revealed that the enhanced catalytic performance is attributable to increased flexibility of the substrate access pathway, reinforced electrostatic and cation-π interactions, and reduced reaction energy barriers. Notably, distal mutation T327K improved substrate ingress through dynamic modulation of the protein surface, while N565I optimized the catalytic microenvironment by enhancing hydrophobic packing and stabilizing key intermediates. Overall, our findings establish a generalizable framework for engineering complex cyclases and provide a foundation for the sustainable microbial production of high-value triterpenoids.

Yangyang Li, K. Jin, Jiangong Lu et al. · 0 citations