Chun-Yue Weng

A robust and reusable Rhizomucor miehei lipase (RML) biocatalyst was prepared by integrating bio-imprinting with resin immobilization. RML was co-imprinted with lauric acid and polyethylene glycol (PEG600) followed by immobilization on an amine-functionalized epoxy resin (ES-103B), resulting in AE@RML-LA+PEG600. After immobilization process optimization, the AE@RML-LA + PEG 600 was verified by CD, SEM, FTIR, and XRD analysis. AE@RML-LA+PEG600 exhibited improved thermal, pH, and organic solvent stability compared with free RML. Under optimized conditions (10 h, 62 °C, palmitic-acid-enriched palm stearin/oleic acid = 1:10 mol/mol, 10 wt% enzyme), AE@RML-LA+PEG600 achieved a maximum OPO content of 45.80%, and the proportion of palmitic acid at the sn-2 position relative to total palmitic acid ((sn-2, P)/P) was 54.59%, which was higher than that of the unimprinted enzyme (35.52% OPO). After five cycles, AE@RML-LA+PEG600 maintained 69.52% of its original catalytic activity, indicating its excellent operational stability. This work provides a promising imprinting-immobilization strategy for preparing durable lipases for industrial OPO production.

S-Adenosyl-L-methionine (SAM), an important biochemical substance, is experiencing increased demand due to its versatile applications in the medical field. Although Saccharomyces cerevisiae has been adapted as a promising platform for SAM production, the issue of SAM productivity is still very interesting. In this study, a systematic approach that consists of genome shuffling, genetic engineering, and fermentation process optimization strategies was established. Firstly, haploids of mutant T11-1 and P15-33 were prepared and treated with ultraviolet irradiation (UV) and atmospheric and room temperature plasma (ARTP) compound mutagenesis, which aids to increase SAM production to 1.25, 1.34 g/L, respectively. Genome shuffling was performed with cycloheximide-resistant protoplast of mutant T5-8-1 and hygromycin B-resistant protoplasts of mutant P4-2-4. The resulting fusant TRP60, with a SAM titer of 1.36 g/L, was subjected to transcriptome analysis. Subsequently, hemoglobin from vitreoscilla has been introduced into fusant TRP60 to increase ATP supply and then strengthening the synthesis pathway of SAM, which significantly elevated SAM titer to 2.06 g/L. Finally, the SAM titer of fusant TRP60-vgb-SAM2 reached 14.22 g/L in a 5 L bioreactor with an optimized fed-batch fermentation. Therefore, fusant TRP60-vgb-SAM2 may serve as a candidate for industrial production of SAM.

Gibberellic acid 3 (GA ₃ ), a diterpenoid phytohormone industrially biosynthesized by Fusarium fujikuroi , serves as a pivotal plant growth regulator with extensive agricultural applications. Currently, industrial GA ₃ production predominantly relies on prolonged submerged microbial fermentation with F. fujikuroi as the main production strain, valued for its native biosynthetic capacity. Nevertheless, large‐scale industrialization of GA ₃ remains constrained by low production yields. In this study, a systematic multimodular metabolic engineering framework was implemented to enhance GA₃ biosynthesis in F. fujikuroi . The engineering strategy encompassed four synergistic modules: reinforcement of fatty acid biosynthesis, augmentation of acetyl‐CoA metabolic flux, optimization of redox cofactor homeostasis, and overexpression of the positive transcriptional regulator. This integrated approach yielded the engineered strain OE: Lae1‐AGP3 demonstrating a 2.58 g/L GA₃ titer in shake‐flask fermentation. Subsequent bioprocess optimization through exogenous fatty acid supplementation further elevated GA ₃ production to 2.86 g/L, representing a 10.9% increase. This study demonstrates the feasibility of coordinated metabolic modifications for improving GA ₃ biosynthesis in F. fujikuroi , offering practical insights for overcoming productivity limitations in fungal secondary metabolite fermentation processes.

High stereoselectivity and activity are the keys for the application of enzymes in the industrial context. Rational design of enzymes with improved fitness is essential in developing and optimizing enzymes for applications in multiple scenarios. However, the current in silico prediction tools show limited performance in the analysis of the effects of single or multiple amino acid substitutions. Here, we developed UniESA, a unified framework based on the AAindex or protein language model encoding, signal processing refinement and machine learning regression. Enzyme sequences were numerically represented as the input to build sequence-stereoselectivity/activity models. UniESA demonstrated satisfactory performance on six public datasets and successfully revealed two mutants with strict stereoselectivity (de_p > 99.5%) and one mutant with activity improved by 2.8-fold in the carbonyl reductase evolution task, requiring only one-tenth or even one-thousandth of the experimental workload necessary for traditional directed evolution. In essence, UniESA is a valuable tool that enables novel insights into enzyme engineering, dramatic experimental workload reduction, and the discovery of high-fitness enzymes in green industrial applications.

Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer‐assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCR_M4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)‐2‐chloro‐1‐(3,4‐difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I‐Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCR_M4‐V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCR_M4. Our computer‐assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production.

Carbonyl reductase (CR)‐catalyzed bioreduction in the organic phase and the neat substrate reaction system is a lasting challenge, placing higher requirements on the performance of enzymes. Protein engineering is an effective method to enhance the properties of enzymes for industrial applications. In the present work, a single point mutation E145A on our previously constructed CR mutant LsCR_M3, coevolved thermostability, and activity. Compared with LsCR_M3, the catalytic efficiency k_cat/K_M of LsCR_M3‐E145A (LsCR_M4) was increased from 6.6 to 21.9 s⁻¹ mM⁻¹. Moreover, E145A prolonged the half‐life t_1/2 at 40°C from 4.1 to 117 h, was increased by 5°C, was increased by 14.6°C, and T_opt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing LsCR_M4 completely reduced up to 600 g/L 2‐chloro‐1‐(3,4‐difluorophenyl)ethanone (CFPO) within 13 h at 45°C, yielding the corresponding (1S)‐2‐chloro‐1‐(3,4‐difluorophenyl)ethanol ((S)‐CFPL) in 99.5% ee_P, with a space‐time yield of 1.0 kg/L d, the substrate to catalyst ratios (S/C) of 600 g/g. Compared with LsCR_M3, the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCR_WT, the substrate loading was increased by 6.5 times. In contrast, LsCR_M4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system.

To improve S-Adenosyl-L-methionine (a compound with important physiological functions, SAM) production, atmospheric and room temperature plasma and ultraviolet-LiCl mutagenesis were carried out with Saccharomyces cerevisiae strain ZY 1–5. The mutants were screened with ethionine, L-methionine, nystatin and cordycepin as screening agents. Adaptive evolution of a positive mutant UV6-69 was further performed by droplet microfluidics cultivation with ethionine as screening pressure. After adaptation, mutant T11-1 was obtained. Its SAM titer in shake flask fermentation reached 1.31 g/L, which was 191% higher than that of strain ZY 1–5. Under optimal conditions, the SAM titer and biomass of mutant T11-1 in 5 L bioreactor reached 10.72 g/L and 105.9 g dcw/L (142.86% and 34.22% higher than those of strain ZY 1–5), respectively. Comparative transcriptome analysis between strain ZY 1–5 and mutant T11-1 revealed the enhancements in TCA cycle and gluconeogenesis/glycolysis pathways as well as the inhibitions in serine and ergosterol synthesis of mutant T11-1. The elevated SAM synthesis of mutant T11-1 may attribute to the above changes. Taken together, this study is helpful for industrial production of SAM.