ω-Transaminases are biocatalysts capable of asymmetrically synthesizing high-value chiral amines through the reductive amination of carbonyl compounds, and they are ubiquitously distributed across diverse microorganisms. Despite their broad natural occurrence, the industrial utility of naturally occurring ω-transaminases remains constrained by their limited catalytic efficiency toward sterically bulky substrates. Over recent decades, the use of structure-guided molecular modifications, leveraging three-dimensional structures, catalytic mechanisms, and machine learning-driven predictions, has emerged as a transformative strategy to address this limitation. Notably, these advancements have unlocked unprecedented progress in the asymmetric synthesis of bulky chiral amines, which is exemplified by the industrial-scale production of sitagliptin using engineered ω-transaminases. This review systematically explores the structural and mechanistic foundations of ω-transaminase engineering. We first delineate the substrate binding regions of these enzymes, focusing on their defining features such as substrate tunnels and dual pockets. These structural elements serve as critical targets for rational design to enhance substrate promiscuity. Next, we dissect the catalytic and substrate recognition mechanisms of (S)- and (R)-ω-transaminases. Drawing on these insights, we consolidate recent advances in engineering ω-transaminases to highlight their performance in synthesizing bulky chiral amines and aim to guide future research and the industrial implementation of tailored ω-transaminases.

Melt blending is an effective way to prepare new composite materials, but most polymers are incompatible. In order to reduce the interfacial tension and obtain fine and stable morphology with internal symmetric micro-textures, suitable compatibilizers should be added to the blend. The two immiscible polymers, low-density polyethylene (LDPE) and polystyrene (PS), were compatibilized by styrene/ethylene/butylene/styrene block copolymers grafted with maleic anhydride (SEBS-g-MAH) and organomontmorillonite (OMMT). The scanning electron microscope results indicated that the size of the PS phase decreased with increasing the content of SEBS-g-MAH. By introducing OMMT into LDPE/PS/SEBS-g-MAH composites, the compatibility of composites was further improved. The rheological analysis and Cole–Cole plot analysis indicated that the addition of SEBS-g-MAH and OMMT increased the interaction between the two phases. The tensile strength, elongation at break, and impact strength of the LDPE/PS/SEBS-g-MAH (70/30/7, wt%) composite increased by 64%, 255%, and 380%, respectively, compared with the LDPE/PS (70/30, wt%) composite. A small amount of OMMT could synergistically compatibilize the LDPE/PS composite with SEBS-g-MAH. After adding 0.3% OMMT into the LDPE/PS/SEBS-g-MAH system, the tensile strength, elongation at break, and impact strength of the composite were further increased to 18.57 MPa, 71.87%, and 33.28 kJ/m², respectively.

Acetohydroxyacid synthase (AHAS) is a key enzyme in the first step of the branched-chain amino acid synthesis pathway, and the production of acetohydroxybutyrate from one molecule of 2-ketobutyric acid and one molecule of pyruvate. AHAS is inhibited by feedback from L-valine, L-leucine, and L-isoleucine, and the expression of ilvBN, the gene encoding AHAS, is regulated by all three branched-chain amino acids. A change in amino acids 20–22 on the regulatory subunit (M13 mutation) removes the feedback inhibition by valine. We cloned the gene encoding AHAS (ilvBN) into a vector and then transfected it into Escherichia coli BL21 for expression with targeted changes in amino acids 20–22 on the regulatory subunit, and then determined the activity of the mutated AHAS and its inhibitory effects on valine, isoleucine, and leucine. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Previous studies have suggested that the binding sites for the three branched-chain amino acids may be at the same variable center. We investigated the enzymatic properties of wild-type and mutant AHAS, modeled their crystal structures, and resolved the mechanism of feedback inhibition induced by mutant M13, which will be useful for continuing the modification of AHAS and the design of broad-spectrum herbicides.

(S)-N-Boc-3-hydroxypiperidine is an important intermediate of the anticancer drug ibrutinib and is mainly synthesized by the asymmetric reduction catalyzed by ketoreductase coupled with glucose dehydrogenase at present. In this study, the coexpression recombinant strains E. coli/pET28-K-rbs-G with single promoter and E. coli/pETDuet-K-G with double promoters were first constructed for the coexpression of ketoreductase and glucose dehydrogenase in the same cell. Then, the catalytic efficiency of E. coli/pET28-K-rbs-G for synthesizing (S)-N-Boc-3-hydroxypiperidine was found to be higher than that of E. coli/pETDuet-K-G due to the more balanced activity ratio and higher catalytic activity. On this basis, the catalytic conditions of E. coli/pET28-K-rbs-G were further optimized, and finally both the conversion of the reaction and the optical purity of the product were higher than 99%. In the end, the cell-free extract was proved to be a better catalyst than the whole cell with the improved catalytic efficiency of different recombinant strains. This study developed a better coexpression strategy for ketoreductase and glucose dehydrogenase by investigating the effect of activity ratios and forms of the biocatalysts on the catalytic efficiency deeply, which provided a research basis for the efficient synthesis of chiral compounds.

Saccharomyces cerevisiae, widely used in industrial fermentation, still suffers from inherent problems in the efficient utilization of carbon sources. Here, a strategy for alleviating glucose effect and improving S-adenosyl-L-methionine (SAM) production by deleting Mig1 combined with introducing MetK1 from Leishmania infantum was applied in S. cerevisiae. The deletion of Mig1 improved glucose utilization by increasing the expression levels of genes related to glucose transport and glycolysis, thereby increasing the levels of glycolytic intermediates and increasing both the transcriptional levels of ACS1 and ALD6 and the activity of ADH2, which promotes the conversion of ethanol into acetyl-CoA. The deletion of Mig1 also upregulated the transcripts of genes involved in the metabolism of precursor amino acids of SAM and ultimately responsible for the improvement in SAM synthesis. Finally, MetK1 was introduced into yeast to redirect carbon flux toward SAM biosynthesis. As expected, the SAM production of the mutant YMig1ΔPMetK1 reached 8.91 g/L in a 10 L fermenter, which was 72.3% higher than that of the parent strain S. cerevisiae CGMCC 2842 (5.17 g/L) reported in our previous studies. This study revealed that the strategy of alleviating glucose effect and redirecting carbon flux to nonethanol products by Mig1 deletion combined with heterologous MetK1 introduction possesses great potential for improving SAM synthesis in yeast cells.