Sheng wu gong cheng xue bao = Chinese journal of biotechnology最新文献

{"title":"[Directed evolution of amine dehydrogenase for the catalytic resolution of (1<i>R</i>,5<i>S</i>)-3-ethylbicyclo [3.2.0] hept-3-en-6-one, a chiral intermediate of mirogabalin].","authors":"Qingxia Luo, Xiaohan Zhang, Jinhui Feng, Qinggang Li, Zixiang Xu, Xi Chen, Qiaqing Wu, Dunming Zhu","doi":"10.13345/j.cjb.260241","DOIUrl":"https://doi.org/10.13345/j.cjb.260241","url":null,"abstract":"<p><p>(1<i>R</i>,5<i>S</i>)-3-ethylbicyclo[3.2.0]hept-3-en-6-one[(1<i>R</i>,5<i>S</i>)-<b>1a</b>] is the key chiral starting material for the synthesis of mirogabalin. Existing chemical and biocatalytic resolution processes both suffer from cumbersome post-processing procedures and high production costs. To achieve the efficient and green resolution of (1<i>R</i>,5<i>S</i>)-<b>1a</b>, the present study employed racemic ketone <b>1a</b> as the substrate and identified <i>Lf</i>AmDH(M0) as the template for directed evolution through enzyme library screening. The substrate binding site and the key amino acid residues around the substrate access channel were selected, and site-directed mutagenesis and iterative mutagenesis were performed in turn to construct a multi-round mutant library. Following library screening, the specific activities of the mutants were elevated to 1.3- to 5.9-fold that of the starting enzyme, demonstrating significantly optimized catalytic performance. The substrate spectrum characteristics of the starting enzyme M0 and the dominant mutants were investigated, revealing that the mutants exhibited appreciable catalytic activity and stereoselectivity toward aliphatic ketones, cyclic ketones, arylalkyl ketones and aromatic ketones. In a 50 mL reaction system, the optimal mutant L40T/L125R/V291A(M3) converted 150 mmol/L of substrate. After the reaction was completed, the product was subjected to extraction and acid washing, affording the chiral ketone with an <i>ee</i> value of 97.1% and a yield of 45.4%. This study successfully developed a novel amine dehydrogenase-catalyzed resolution approach for (1<i>R</i>,5<i>S</i>)-<b>1a</b>, which simplifies the post-processing workflow of the product and reduces the consumption of organic reagents, thereby providing robust technical support for the efficient industrial-scale biomanufacturing of (1<i>R</i>,5<i>S</i>)-<b>1a</b>.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"42 9","pages":"4154-4167"},"PeriodicalIF":0.0,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149025994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[An integrated system for transient expression and stable transformation in <i>Nicotiana benthamiana</i> suspension cells].","authors":"Yuxi Zhang, Jing Yu, Qiuxian Wang, Jiahong Liu, Yue Zhao, Rong Fan, Zhichao Li","doi":"10.13345/j.cjb.260144","DOIUrl":"https://doi.org/10.13345/j.cjb.260144","url":null,"abstract":"<p><p><i>Nicotiana benthamiana</i> suspension cells, exhibiting advantages such as short growth cycles, facile genetic manipulation, ease of product purification, and suitability for large-scale cultivation, are emerging as a novel industrial biosynthesis platform for plant-derived natural products. Transient expression and stable transformation are core technologies for validating gene function and constructing plant cell factories. However, current methods have been developed primarily for whole-plant systems, systematic investigations in plant suspension cells are lacking, let alone a technological framework for integrating both approaches. Plant suspension cells, by virtue of cultivating characteristics, are suitable to develop integrated systems for transient expression and stable transformation. This study constructed an integrated system for transient expression and stable transformation (ISTS) by incorporating an efficient expression module, a transient expression enhancement module, and a stable integration screening module. ISTS enabled higher transient transformation efficiency and expression intensity in <i>N</i>. <i>benthamiana</i> suspension cells than classical pCAMBIA1300. Moreover, upon selection and cultivation of transiently expressing cells, stably transformed cell lines with efficient target gene integration and high expression levels can be obtained readily. In this study, <i>tHMGR</i>, <i>tGGPPS</i>, and <i>tTS</i> were cloned into ISTS vector individually and co-transformed into <i>N</i>. <i>benthamiana</i> suspension cells, which enabled the one-step multigene transient expression and stable integration. This approach successfully yielded taxadiene-producing tobacco cell lines. ISTS offers advantages such as simplified vector construction, fewer procedural steps, and shorter operational cycle. This work provides key technical support for establishing efficient, stable plant suspension cell factories and facilitating sustainable biosynthesis of complex natural products.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"42 9","pages":"4305-4317"},"PeriodicalIF":0.0,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149026038","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[High-level expression of PET depolymerases in <i>Pichia pastoris</i> and construction of a crude enzyme stabilization system].","authors":"Jiaxin Zhou, Haohong Lin, Xiujuan Qian, Anming Xu, Min Jiang","doi":"10.13345/j.cjb.260313","DOIUrl":"https://doi.org/10.13345/j.cjb.260313","url":null,"abstract":"<p><p>Improving the production of polyethylene terephthalate (PET) depolymerases is essential for reducing the cost of enzymatic PET recycling and facilitating its industrial application. To enhance the production of PET hydrolases, the fungal-derived hydrolase HiC and the bacterial-derived hydrolase LCC^ICCG were employed as model enzymes. Strategies including recombinant strain construction, gene copy number optimization, molecular chaperone-assisted expression, fermentation scale-up, and crude enzyme stabilization were systematically investigated. The strains with secretory expression were generated by chromosomal integration, and multicopy transformants were obtained through high-stringency zeocin selection. To further improve protein production, we introduced PDI, ERO1, SEC53, SEC1, HAC1, and GCN4 as helper factors. GCN4 showed the strongest enhancement effect on HiC production, whereas ERO1 and HAC1 were more effective for LCC^ICCG. No significant synergistic effect was observed when ERO1 and HAC1 were co-expressed. In 5-L bioreactors, the optimized strains achieved the titers of 2.36 g/L for HiC and 1.72 g/L for LCC^ICCG. Furthermore, in a 50 L fermenter, the maximum extracellular protein concentration of HiC reached 8.5 g/L. A crude enzyme stabilization system composed of 0.1% Kathon, 10% glycerol, and 0.5% phenoxyethanol was subsequently established. After storage at room temperature for 30 days, HiC and LCC^ICCG retained 56.43% and 78.11% of their initial activities, respectively. The results demonstrate that coordinated optimization of expression and formulation can improve both the production and storage stability of PET depolymerases, thereby supporting their practical application in enzymatic PET recycling.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"42 9","pages":"4289-4304"},"PeriodicalIF":0.0,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149026055","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Metabolic engineering of <i>Yarrowia lipolytica</i> for astaxanthin production].","authors":"Shuo Sun, Qun Yang, Ziyue Zhou, Yutong Fan, Yunying Zhao, Yu Deng","doi":"10.13345/j.cjb.260148","DOIUrl":"https://doi.org/10.13345/j.cjb.260148","url":null,"abstract":"<p><p>Astaxanthin, a high-value terpenoid secondary metabolite that exhibits excellent antioxidant activity, has broad application potential in the pharmaceutical and nutraceutical industries. However, its sophisticated biosynthetic pathway poses a great challenge for efficient production and industrial-scale application of astaxanthin. In this study, the unconventional oleaginous yeast <i>Yarrowia lipolytica</i> was employed as the chassis organism, and a multidimensional metabolic engineering strategy combined with fermentation process optimization was used to construct a microbial cell factory for astaxanthin production. Firstly, a heterologous β-carotene biosynthetic pathway was reconstituted, the carbon flux supply of the mevalonate (MVA) pathway was systematically enhanced, and endogenous branch metabolism was blocked, leading to the initial construction of an astaxanthin-producing strain. Subsequently, a RIDD-RIAD protein interaction tag was introduced to optimize the expression patterns of the key rate-limiting enzymes of β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ), thereby effectively improving the efficiency of astaxanthin biosynthesis. Following this, six rounds of iterative targeted metabolic modification combined with shake-flask fermentation significantly enhanced the biosynthetic potential of the engineered strains. The results showed that the engineered strain YAX11 achieved an astaxanthin titer of 107.4 mg/L in shake-flask fermentation, and the titer was increased to 1 148.4 mg/L in a 5 L bioreactor. Through the deep integration of metabolic network remodeling and fermentation process optimization, this study successfully constructed a <i>Y</i>. <i>lipolytica</i> cell factory for astaxanthin production, providing a theoretical reference for the green and large-scale manufacturing of astaxanthin, as well as metabolic engineering strategies for the heterologous synthesis of other terpenoid compounds.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"42 9","pages":"4067-4088"},"PeriodicalIF":0.0,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149026069","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Rational design for improving the thermostability of β-glucosidase from <i>Bacillus thermoamylovorans</i>].","authors":"Panpan Dong, Tong Liu, Keyi Xue, Ruili Wang, Yandie Wang, Modan Qi, Chunsheng Zuo, Guosi Li, Nailiang Zhu","doi":"10.13345/j.cjb.260227","DOIUrl":"https://doi.org/10.13345/j.cjb.260227","url":null,"abstract":"<p><p>The β-glucosidase from <i>Bacillus thermoamylovorans</i> (Bgl52) has been identified as a thermophilic and acidophilic β-glucosidase, which constitutes the most critical component in the cellulolytic enzyme system for cellulose hydrolysis. Although Bgl52 is a thermophilic enzyme, its poor thermal stability at high temperatures restricts its long-term industrial application under elevated-temperature conditions. In this study, rational design strategies, including two free energy calculation programs (Fireprot and FoldX) and homologous sequence alignment, were integrated to perform single-point and multi-point combinatorial mutagenesis on Bgl52. The enzymatic kinetic stability assay and molecular dynamics simulation were conducted to further investigate the catalytic performance and structural variations of positive mutants. After screening, the optimal stacked mutant M5 exhibited increases in <i>T</i>_m, optimal temperature, and <i>T</i>₅₀ by 8.7, 10 and 8℃, respectively, compared with Bgl52 (wild type, WT), and its half-life <i>t</i>_1/2 at 80℃ was enhanced by 32.5 folds. The catalytic efficiency (<i>K</i>_cat/<i>K</i>_m) of M5 showed a slight decrease compared with that of the wild-type enzyme. Furthermore, M5 hydrolyzed native cellulose into glucose more efficiently at the high temperature of 85℃. Structural analysis and molecular dynamics simulation revealed that the improved thermostability was attributed to increased hydrogen bonds, the proline effect, additional salt bridges, and enhanced hydrophobic interactions. The results indicate that the multi-strategy integrated rational design approach demonstrates outstanding efficiency and reliability in enzyme engineering modification, enhancing the industrial application potential of β-glucosidase.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"42 9","pages":"4195-4209"},"PeriodicalIF":0.0,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149025712","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Research progress in synthesis of cordycepin by microorganisms].","authors":"Ying Tian, Lishuangfei Chen, Yirui Liang, Qiqi Zhu, Chuanbo Zhang, Wenyu Lu","doi":"10.13345/j.cjb.260120","DOIUrl":"https://doi.org/10.13345/j.cjb.260120","url":null,"abstract":"<p><p>Cordycepin is a natural nucleoside antibiotic isolated from the fruiting bodies of <i>Cordyceps militaris</i>. It possesses diverse health-beneficial and medicinal values. However, the conventional fermentation processes for cordycepin production suffer from inherent drawbacks, including low yields, prolonged production cycles, and batch-to-batch inconsistency. These problems result in the exorbitant market price of cordycepin and severely restrict the potential for market application. The advent of synthetic biology has offered a revolutionary approach to address this challenge. This article systematically reviews the development trajectory of cordycepin production, spanning from conventional fermentation to modern synthetic biology-based manufacturing. It focuses on analyzing the breakthrough advancements in the construction of microbial cell factories such as <i>Saccharomyces cerevisiae</i> and cordycepin synthesis. Finally, it provides perspectives on the future development of more efficient, eco-friendly, and intelligent cordycepin biomanufacturing technologies by leveraging enzyme engineering and synthetic biology. And this study offers theoretical support and technical references for constructing high-cordycepin-producing strains, which is critical to facilitate the industrialization of cordycepin.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"42 9","pages":"3867-3883"},"PeriodicalIF":0.0,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149025805","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Research progress and prospects of cyanobacterial cell factories for ethylene production].","authors":"Yanwei Gao, Hongxiu Xu, Chunjiang Ye, Tao Zhu, Xuefeng Lü","doi":"10.13345/j.cjb.260210","DOIUrl":"https://doi.org/10.13345/j.cjb.260210","url":null,"abstract":"<p><p>Ethylene, a fundamental feedstock in the petrochemical industry, is conventionally produced <i>via</i> energy-intensive steam cracking of fossil fuels, a process associated with high energy consumption and carbon emissions. Constructing photosynthetic cyanobacterial cell factories for the direct, solar-driven conversion of CO₂ to ethylene offers a promising route for the green production of ethylene. This review systematically summarizes recent advances in this field, highlighting the core catalytic mechanism of the ethylene-forming enzyme (EFE), the screening and optimization of key genetic elements, metabolic engineering strategies (including gene copy number amplification, precursor supply enhancement, rewiring of carbon flux, and alleviation of by-product inhibition), and the optimization of cultivation processes. Furthermore, we identify the key challenges currently impeding cyanobacterial production of ethylene, such as low yields and efficiencies, insufficient genetic stability, and bottlenecks in large-scale cultivation. Finally, we outline future development directions-advancing synthetic biology tools, enabling precise regulation of metabolic networks, designing efficient photobioreactors, and integrating industrial technologies, with the aim of providing a theoretical foundation for establishing robust and high-performance cyanobacterial cell factories for ethylene production and facilitating their practical application.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"42 9","pages":"3829-3845"},"PeriodicalIF":0.0,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149025813","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Coenzyme-free <i>in vitro</i> biotransformation for the synthesis of galacto-<i>N</i>-biose].","authors":"Hao Fang, Yuan Li, Haotian Cheng, Fuping Lu, Yi-Heng P J Zhang","doi":"10.13345/j.cjb.260330","DOIUrl":"https://doi.org/10.13345/j.cjb.260330","url":null,"abstract":"<p><p>Galacto-<i>N</i>-biose (GNB), a core structural unit of O-glycan and a key component of gastrointestinal mucins, plays important physiological roles in regulating intestinal microecology and maintaining the mucosal barrier. Traditional enzymatic routes for the synthesis of GNB rely on the supplies of ATP and UDP-sugars. To address this disadvantage, we designed an <i>in vitro</i> ATP-free and UDP-sugar-free enzymatic pathway to synthesize GNB directly from lactose and <i>N</i>-acetylgalactosamine (GalNAc). This pathway consisted of two core enzymes, lactose phosphorylase (LacP) and lacto-<i>N</i>-biose phosphorylase (LnbP), as well as one auxiliary enzyme, polyphosphate glucokinase (PPGK). By incorporating PPGK to remove the byproduct glucose, this pathway not only pushed the reaction equilibrium toward the biosynthesis of GNB, but also achieved the regeneration of inorganic phosphate. Through the systematic optimization of key parameters such as reaction temperature, pH, and enzyme dosage, a molar yield of 76% was achieved with 10 mmol/L GalNAc. This <i>in vitro</i> biotransformation pathway provides a technical basis for the low-cost, large-scale green production of GNB and other high-value functional oligosaccharides.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"42 9","pages":"4141-4153"},"PeriodicalIF":0.0,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149025982","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Construction of an integrative recombinant <i>Escherichia coli</i> strain for efficient biosynthesis of chondroitin].","authors":"Lingxin Huang, Weiwei Meng, Hejia Qi, Xiangting Song, Linhao Zhang, Liming Liu, Jing Wu, Xiaomin Li","doi":"10.13345/j.cjb.260132","DOIUrl":"https://doi.org/10.13345/j.cjb.260132","url":null,"abstract":"<p><p>Chondroitin and its derivatives are essential glycosaminoglycans in articular cartilage and connective tissue, with broad applications in the nutraceutical and pharmaceutical industries. Conventional chemical and enzymatic synthesis approaches are often limited by process complexity, low efficiency, and high substrate costs. In contrast, microbial fermentation offers a promising alternative for the sustainable production of chondroitin due to its shorter production cycle, lower cost, and scalability. To obtain a genetically stable chondroitin-producing strain, in this study, an efficient genome-integrated <i>Escherichia coli</i> strain for chondroitin production was developed based on the strain GZ17 previously constructed in our laboratory. First, a chondroitin synthase from <i>Moraxella canis</i> (<i>Mc</i>CS) was identified through phylogenetic analysis and stably expressed under a strong promoter <i>via</i> genomic integration. Subsequently, key amino acid residues were identified through structural modeling and molecular dynamics simulations, followed by iterative mutagenesis to obtain a highly active mutant, <i>Mc</i>CS^{S602A/N632S/K637L}. Finally, two copies of the mutant <i>Mc</i>CS^{S602A/N632S/K637L} gene were integrated into the genome of GZ17, which significantly enhanced chondroitin biosynthesis. The resulting strain, GZ-2ASLM, achieved a chondroitin titer of 7.41 g/L, a glycerol yield of 0.03 g/g, and a productivity of 0.10 g/(L·h) by fed-batch fermentation in a 5-L fermenter. Notably, this engineered strain utilizes glycerol as the sole carbon source for chondroitin production without depending on plasmids. This study provides a robust strategy for developing efficient microbial cell factories for the production of glycosaminoglycans.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"42 9","pages":"4018-4037"},"PeriodicalIF":0.0,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149025990","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Cofactor preference engineering of <i>meso</i>-diaminopimelate dehydrogenase and its application in d-phenylalanine synthesis].","authors":"Hong Xu, Yaxin Liao, Fangyuan Niu, Siqi Li, Meijuan Xu","doi":"10.13345/j.cjb.260259","DOIUrl":"https://doi.org/10.13345/j.cjb.260259","url":null,"abstract":"<p><p>d-phenylalanine is an important d-amino acid with a wide range of applications in the pharmaceutical and chemical industries. <i>meso</i>-diaminopimelate dehydrogenase (DAPDH) catalyzes the asymmetric reductive amination of α-keto acids to produce d-amino acids with high optical purity, serving as a key enzymatic module for constructing multi-enzyme cascade systems. However, the strict dependence of wild-type DAPDH on NADPH results in high cofactor costs, limiting its industrial application. To address this issue, this study focused on a previously engineered yet still NADPH-preferring type I DAPDH from <i>Corynebacterium glutamicum</i> (<i>Cg</i>DAPDH). Based on structural analysis of the protein, a three-pronged engineering strategy involving steric hindrance reduction, local electrostatic potential modulation, and conformational flexibility enhancement was proposed. Through site-directed and combinatorial mutagenesis of residues at the cofactor-binding pocket entrance (Ser35, Arg36, Arg37) and conserved regions (Ser68, Thr88, Asp120), a mutant S35E/R36E/R37A/S68T/T88A was obtained. This mutant exhibited a catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) of 274.87 mmol/(L·s) toward NADH, representing a 16.9-fold improvement over the starting enzyme, while achieving comparable catalytic competence with both NADH and NADPH. This mutant was coupled with formate dehydrogenase (FDH) to construct an NADH self-regeneration system for whole-cell catalytic reductive amination using phenylpyruvate as the substrate. After 6 h of reaction, the titer of d -phenylalanine reached 36.22 g/L with a conversion rate of 90.12%. When this system was applied to three-enzyme cascade employing l-phenylalanine as the substrate, the d-phenylalanine titer reached 16.25 g/L, which was comparable to that obtained with a glucose dehydrogenase/NADPH regeneration system (17.21 g/L). This study demonstrates that cofactor engineering can alleviate the NADPH dependence of DAPDH and achieve efficient conversion of phenylpyruvate to d-phenylalanine, thereby providing a new enzymatic module and a technical route for cost-effective biomanufacturing of d-amino acids.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"42 9","pages":"4124-4140"},"PeriodicalIF":0.0,"publicationDate":"2026-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149026073","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}