Journal of biotechnology最新文献

{"title":"CRISPR-based precise methylation of specific FUT8 promoter regions allows isolation of CHO cells with a fine-tuned glycoprofile","authors":"Víctor Jiménez Lancho , Klaus Leitner , Kitty Agarwal , Arathi Krishnakumar , Anurag Khetan , Nicole Borth , Nicolas Marx","doi":"10.1016/j.jbiotec.2026.01.001","DOIUrl":"10.1016/j.jbiotec.2026.01.001","url":null,"abstract":"<div><div>A major advantage of producing therapeutic proteins in mammalian cells is their ability to tailor proteins with human-like posttranslational modifications such as glycosylation, which ultimately defines aspects like stability, protein folding or immunogenicity. However, producing therapeutic proteins with a consistent and reproducible glycoprofile remains a major challenge for the biopharmaceutical industry, especially with biosimilar production. While the enzymes responsible for glycosylation of proteins have been the subject of various cell engineering approaches, tuning their gene expression to precise levels is still difficult to achieve. While CRISPR/Cas9 enabled the genetic engineering of cells to drastically overexpress or remove a target gene, CRISPR/dCas9-based epigenetic editing by targeted DNA methylation promises to stably change the expression pattern of target genes after transient transfection of the CRISPR-tool. Application of targeted DNA methylation so far has mostly been used to completely silence gene expression by fully methylating the corresponding promoter regions. Here, we aim to tune expression of the associated gene by DNA methylation of confined promoter regions and to apply this technique as a new glycoengineering approach. By coupling CRISPR-based targeted DNA methylation with lectin-FACS assisted sorting we obtained CHO cell lines with a fine-tuned phenotype. First, dCas9-DNMT3A3L in combination with one single gRNA is targeted to the FUT8 promoter to induce confined DNA methylation, resulting in a phenotypically diversified population. Next, a window sorting strategy based on lectin-stained cells using five different sorting gates spanning from low to high FUT8 expression was applied to isolate single clones with a defined phenotype. Isolated clones were phenotypically assessed and re-sorted to obtain a homogenous expression profile. The resulting clonal cell lines showed either tuned or knock-down phenotypes with varying gene expression levels. Two out of seven clones that showed tuned FUT8 gene expression were phenotypically stable over 60 days. Gene expression levels, on the other hand, showed a steady decline over time that in part, however, can be explained by the general variation of FUT8 expression in different growth phases. Importantly, glycan analysis of recombinant EpoFc produced in the tuned clonal cell lines showed ranges of 35–70 % fucosylation, demonstrating that isolated clones can produce recombinant proteins with a distinct glycosylation profile. To understand why some clones showed tuned FUT8 gene expression levels while others were completely knocked-down, we analyzed the DNA methylation status of their respective FUT8 promoter. Critical areas within the FUT8 promoter were identified, with some associated with general repression and others with the tuning of FUT8 gene expression when affected by DNA methylation. Additionally, a combination of histone marks associated with active and rep","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 341-352"},"PeriodicalIF":3.9,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145933379","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structure–function analysis and exonuclease deletion yield an improved strand-displacing DNA polymerase from Aeribacillus pallidus for efficient recombinase polymerase amplification","authors":"Koki Nishi ,&nbsp;Eisuke Inoue ,&nbsp;Yuto Murakami ,&nbsp;Yuri Ishii ,&nbsp;Yukiko Nakura ,&nbsp;Itaru Yanagihara ,&nbsp;Kiyoshi Yasukawa ,&nbsp;Shinsuke Fujiwara","doi":"10.1016/j.jbiotec.2025.12.020","DOIUrl":"10.1016/j.jbiotec.2025.12.020","url":null,"abstract":"<div><div>Isothermal nucleic-acid amplification relies on strand-displacing DNA polymerases that synthesize DNA while unwinding duplex templates. We previously identified two thermostable family-A polymerases, C1-Pol from <em>Geobacillus zalihae</em> and H1-Pol from <em>Aeribacillus pallidus</em>, which show a characteristic and reproducible trade-off between stability and activity. C1-Pol is more thermostable, whereas H1-Pol exhibits stronger strand-displacement activity and higher recombinase polymerase amplification (RPA) efficiency. Domain-swapping analysis indicated that a chimeric construct (C1-Pol#4), in which the C1-Pol 5′→3′ exonuclease region was replaced with that of H1-Pol, enhanced strand-displacement and nucleotide-incorporation activities while maintaining parental-level stability, suggesting that this region modulates these important biochemical properties. However, C1-Pol#4 did not improve RPA efficiency. To clarify the functional contribution of this domain, we constructed Klenow-type deletion mutants lacking the 5′→3′ exonuclease region. The H1-Pol large fragment (H1-PolLF) exhibited reduced thermostability but markedly enhanced polymerase and strand-displacement activities at 40 °C, the optimal temperature for RPA reactions. At this temperature, H1-PolLF showed ∼10-fold higher strand-displacement activity than Bst DNA polymerase 2.0 and 4.4-fold higher than full-length H1-Pol. H1-PolLF also accelerated RPA, producing equivalent amplicons in half the reaction time and improving detection sensitivity 100-fold (6 × 10³ → 6 × 10¹ copies) than parental H1-Pol. Coupling H1-PolLF with branched-chain polyamine (BCPA)–conjugated magnetic beads enabled reliable detection of ∼10² target molecules from 10 mL saline. These findings demonstrate that removing the 5′→3′ exonuclease domain fine-tunes polymerase and strand-displacing functions, yielding an enzyme highly suited for rapid, ultrasensitive isothermal nucleic-acid detection when used in combination with BCPA beads.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 321-330"},"PeriodicalIF":3.9,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145900250","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Systematic engineering of cell wall for improving single cell protein (SCP) production","authors":"Linhai Xie ,&nbsp;Shuo Tian ,&nbsp;Zhehao Jin ,&nbsp;Tiantian Zou ,&nbsp;Mingxin Chang ,&nbsp;Hongting Tang ,&nbsp;Tao Yu ,&nbsp;Zhoukang Zhuang","doi":"10.1016/j.jbiotec.2025.12.011","DOIUrl":"10.1016/j.jbiotec.2025.12.011","url":null,"abstract":"<div><div>As an emerging alternative protein, Single cell protein (SCP) has gained considerable traction in livestock feed applications. Furthermore, it presents substantial potential for replacing traditional dietary proteins for human consumption. Yet current research remains focused on strain screening with limited advances in cellular metabolic engineering. Due to the complexity of protein synthesis and processing, enhancing the SCP production efficiency in microbial chassis remains a major challenge. Here, we present a metabolic engineering approach to systematic engineer the cell wall of <em>Saccharomyces cerevisiae</em> for improving SCP production. In this study, we constructed a strain by gene modifications of genes related to cell wall biogenesis and associated signaling pathways, achieving a 32.6 % increase in cellular protein content. Additionally, comparative transcriptomics analysis uncovered the regulatory mechanism whereby remodeling of carbon and nitrogen metabolism governs cellular protein biosynthesis, providing new insight for the rational design of microbial cell factories with enhanced protein content.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 173-183"},"PeriodicalIF":3.9,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145781399","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Antibacterial effects and mechanisms of fisetin-β-cyclodextrin complex under UVA light against Escherichia coli O157:H7","authors":"Tao Liu ,&nbsp;Yuzhang Zhu ,&nbsp;Jiahui Wang ,&nbsp;Kun Lin ,&nbsp;Xuan Zhang ,&nbsp;Huawei Ma ,&nbsp;Chaonan Kong ,&nbsp;Wei Cai ,&nbsp;Lifang Yang ,&nbsp;Mingguo Jiang","doi":"10.1016/j.jbiotec.2025.12.003","DOIUrl":"10.1016/j.jbiotec.2025.12.003","url":null,"abstract":"<div><div>The development of natural product photosensitizers was a critical cornerstone for applying photodynamic antimicrobial technology in the food industry. The objective of this study was to fabricate complex of fisetin-β-cyclodextrin (FIT-β-CD), explore their photodynamic antimicrobial efficacy and mechanisms, and characterize it through infrared spectroscopy and X-ray diffraction. The results indicatde that this method can produce good dispersibility and water solubility of FIT-β-CD complex.The photodynamic impact of FIT-β-CD was assessed by examining alterations in reactive oxygen species (ROS) production.The results demonstrated that FIT-β-CD can rapidly generate ROS under UVA, with hydrogen peroxide production being 3.73 times higher than in dark conditions. The antimicrobial efficacy of FIT-β-CD against <em>Escherichia coli</em> O157:H7 (<em>E.coli</em> O157:H7) was evaluated based on bactericidal efficacy, cell membrane damage, DNA and protein damage, motility changes, biofilm formation, and extracellular polysaccharide production. In addition, gene expression analysis by RT-qPCR showed its significant inhibition on the two-component system (TCS) and quorum sensing system (QS). The results indicated that FIT-β-CD exhibited efficient, sensitive photodynamic, and antibacterial properties against <em>E.coli</em> O157:H7, making it a novel photosensitizer. These findings imply potential new applications of fisetin in the field of food antimicrobial agents.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 145-152"},"PeriodicalIF":3.9,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145708122","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sodium alginate-immobilized epoxide hydrolase: A multifaceted strategy for enhanced stability, reusability, and catalytic performance","authors":"Zongzhong Yu ,&nbsp;Meinan Zhu ,&nbsp;Xiao Gu ,&nbsp;Zhongkun Wu ,&nbsp;Peiqin Chen ,&nbsp;Chunying Jin ,&nbsp;Junning Zhao ,&nbsp;Guangya Zhang ,&nbsp;Wei Jiang","doi":"10.1016/j.jbiotec.2025.11.017","DOIUrl":"10.1016/j.jbiotec.2025.11.017","url":null,"abstract":"<div><div>Immobilized macromolecular enzymes effectively address critical challenges including environmental sensitivity, thermal instability, and non-reusability, thereby establishing the fundamental and practical significance of enzyme immobilization research. This study employed sodium alginate (SA), ZIF-8, and Bi-EA materials to immobilize <em>Aspergillus carlsbadensis</em>-derived epoxide hydrolase (AcEH). Comparative analysis revealed SA's superior immobilization efficiency. Material characterization via SEM, FTIR, and XRD was performed, followed by optimization of SA immobilization parameters. The immobilized enzyme maintained activity over 6 reuse cycles. Incorporating 0.5 wt% carboxymethyl cellulose (CMC) extended AcEH@SA-CMC reusability to 8 cycles. Magnetic modification through Fe₃O₄ powder incorporation yielded AcEH@SA-CMC- Fe₃O₄ hydrogel with enhanced recoverability. Results demonstrate SA's exceptional biocompatibility underpins its immobilization efficacy, while CMC co-entanglement creates a porous network facilitating mass transfer and mechanical reinforcement. Fe₃O₄ powder plays a promoting role in improving enzyme activity. This methodology not only evaluates immobilization matrices but establishes an optimized protocol for macromolecular AcEH immobilization.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 84-95"},"PeriodicalIF":3.9,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145622129","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optical, structural, and antifungal properties of nanosilver borate bioactive glass synthesized using gamma rays on the survival of Candida albicans and Candida tropicalis","authors":"E.M. Abou Hussein ,&nbsp;Rasha Mohammad Fathy","doi":"10.1016/j.jbiotec.2025.11.013","DOIUrl":"10.1016/j.jbiotec.2025.11.013","url":null,"abstract":"<div><div>A New glass system with a composition of; 50.3 B₂O₃+ 21 CaO + 8 MgO + 8 K₂O + 8 Na₂O + 4 P₂O₅ + 0.7 Nano Ag₂O, was prepared by the traditional melting technique. Some of its structural, optical, and physical characteristics were investigated regarding the effect of gamma radiation. The XRD pattern revealed the amorphous nature of the fabricated glass sample. UV–visible absorption spectra revealed an obvious peak before 400 nm due to the absorption of Ag^2 + ions, and a photo-reduction reaction to Ag⁰ was expected due to the impact of gamma irradiation. FTIR absorption spectra displayed structural bands in the region 800–1600 cm⁻¹ recognized to the specific vibrations of BO₃, BO₄ and PO₂. Results displayed optical and structural stability against gamma irradiation doses of 25 and 50 kGy, and revealed a positive effect of irradiation to enhance the antifungal efficiency of the glass or in sterilization processes before usage. The antifungal activity of borate bioglass doped with silver nanoparticles (BG/Ag) against <em>Candida albicans</em> and <em>Candida tropicalis</em> was assessed. BG/Ag was mostly effective against <em>C. albicans</em>. Furthermore, at a dosage of 40.0 mg/mL, BG/Ag considerably reduced biofilm formation of <em>C. albicans</em> and <em>C. tropicalis</em> (90.79 and 75.07 %). At 40 mg/mL, BG/Ag significantly reduced <em>Candida</em> viability, colony counts, and phospholipase activity. BG/Ag can be successfully applied as an attractive biomaterial for the medication of serious fungal infections. The novelty in the study is the synthesis of borate bioactive glass containing nanosilver- and the use of gamma irradiation to improve its biological properties.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 33-44"},"PeriodicalIF":3.9,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145587571","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High specificity of MsmCAR toward 4-hydroxyvaleric acid enables efficient 1,4-pentanediol production from biomass-derived levulinic acid","authors":"Suhye Choi ,&nbsp;Seung Hun Lee ,&nbsp;Yunyeong Kim ,&nbsp;Woo Young Jeon ,&nbsp;Jung oh Ahn ,&nbsp;Shashi Kant Bhatia ,&nbsp;Kwon-Young Choi ,&nbsp;Jeong Chan Joo ,&nbsp;Yung-Hun Yang","doi":"10.1016/j.jbiotec.2025.11.020","DOIUrl":"10.1016/j.jbiotec.2025.11.020","url":null,"abstract":"<div><div>Alkanediols are industrial compounds with diverse applications. However, the biological production of C5 diols is limited by the low substrate specificity and efficiency of carboxylic acid reductases (CARs) toward short to medium-chain substrates. We developed a whole-cell biotransformation system for efficient 1,4-pentanediol (1,4-PDO) production, aimed at developing a platform pathway for diol biosynthesis. By screening 10 CARs, we identified <em>Msm</em>CAR from <em>Mycolicibacterium smegmatis</em> MC2 155 as the most effective for C5 diol biosynthesis, with high specificity toward 4-hydroxyvaleric acid (4-HV). We constructed a whole-cell catalyst expressing <em>Msm</em>CAR and enhanced its performance by introducing <em>ppk2b</em> from <em>Corynebacterium glutamicum</em> ATCC 13032 and <em>chnD</em> from <em>Acinetobacter</em> sp., which encode proteins involved in cofactor regeneration. Systematic optimization of the reaction conditions including buffers, cofactors, metal ions, and cultivation parameters led to a maximum titer of 78.10 mM 1,4-PDO, with a productivity of 1.86 mM/h, when using 4-HV as a substrate. To expand the substrate scope to levulinic acid, 3-hydroxybutyrate dehydrogenase and formate dehydrogenase biosynthetic genes were introduced. This system yielded 22.10 mM 1,4-PDO within 10 h. This work highlights the potential of <em>Msm</em>CAR as a versatile biocatalyst and provides a sustainable strategy for producing short- to medium-chain diols from biomass-derived feedstocks.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 115-124"},"PeriodicalIF":3.9,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145668412","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human beta-defensin 3-functionalized Fe/GMP nanozyme for multifunctional antimicrobial and anticancer activity against Helicobacter pylori-associated gastrointestinal cancer","authors":"Yoganathan Kamaraj ,&nbsp;Veenayohini Kumaresan ,&nbsp;Jinhao Hu ,&nbsp;Daochen Zhu","doi":"10.1016/j.jbiotec.2025.11.018","DOIUrl":"10.1016/j.jbiotec.2025.11.018","url":null,"abstract":"<div><h3>Background</h3><div>The rising incidence of <em>Helicobacter pylori</em>-associated infections and colorectal cancer necessitates multifunctional therapeutics targeting both microbial and tumor pathologies. However, antimicrobial peptides (AMPs) like human β-defensin 3 (hBD-3) faces challenges such as proteolytic degradation and poor bioavailability. To address these limitations, we developed a novel hybrid nanozyme by immobilizing hBD-3 onto a guanosine monophosphate-coordinated iron framework (Fe/GMP), known for its biocompatibility and intrinsic peroxidase-like activity.</div></div><div><h3>Methods</h3><div>The hBD-3@Fe/GMP nanozyme was synthesized and characterized for its physicochemical and catalytic properties. Its antibacterial activity against <em>H. pylori</em> was assessed via agar diffusion, biofilm inhibition, and virulence gene expression. Anti-colon cancer potential was evaluated in HT-29 cells using assays for cytotoxicity, ROS generation, apoptosis, migration inhibition, and redox biomarkers. <em>in vivo</em> antitumor efficacy was studied using a colon carcinoma xenograft mice model.</div></div><div><h3>Results</h3><div>hBD-3@Fe/GMP showed enhanced peroxidase-like activity with elevated ROS production than Fe/GMP alone. It significantly inhibited <em>H. pylori</em> growth, biofilm formation, and virulence gene expression (<em>babA, alpA, alpB, flaA, flaB</em>). In HT-29 cells, it induced cytotoxicity, suppressed antioxidant enzymes, and triggered apoptosis via p53 and Bax upregulation and Bcl-2 downregulation. In tumor xenograft model, it reduced tumor volume and weight.</div></div><div><h3>Conclusion</h3><div>This study reports, for the first time, a hybrid nanozyme that synergistically integrates the antimicrobial potency of hBD-3 with the catalytic redox functionality of Fe/GMP, offering dual action against <em>H. pylori</em> infection and colorectal cancer. The multifunctional therapeutic potential and biocompatibility of hBD-3@Fe/GMP mark a novel paradigm in the development of next-generation nanozymes for combinatorial infectious and oncological interventions.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 67-83"},"PeriodicalIF":3.9,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145622128","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineered BDH–NOx co-expression in Escherichia coli enables highly efficient in vivo cascade catalysis for the transformation of racemic epoxides to α-hydroxyketones","authors":"Bochun Hu ,&nbsp;Xiaoqi Xi ,&nbsp;Qianjin Hou ,&nbsp;Jiaxin Ou ,&nbsp;Xiaoxuan Shen ,&nbsp;Zining Wang ,&nbsp;Jiahui Zhao ,&nbsp;Cunduo Tang ,&nbsp;Jihong Huang","doi":"10.1016/j.jbiotec.2025.12.012","DOIUrl":"10.1016/j.jbiotec.2025.12.012","url":null,"abstract":"<div><div>To eliminate the requirement for pyruvate supplementation and to avoid lactate accumulation, a multi-enzyme cascade was systematically restructured by replacing lactate dehydrogenase (LDH) with water-forming NADH oxidase (<em>Ef</em>NOx). In this revised system, <em>Rp</em>EH^L360V catalyzed the enantioconvergent hydrolysis of <em>rac</em>-4-chlorostyrene oxide (<em>rac</em>-<strong>1a</strong>) to (<em>R</em>)-1-(4-chlorophenyl)-1,2-ethanediol (<strong>1b</strong>), followed by stereoselective oxidation of (<em>R</em>)-<strong>1b</strong> to 4’-chloro-2-hydroxyacetophenone (<strong>1c</strong>) by <em>Bs</em>BDHA^{I49L/V266L/G292A}. Concurrently, <em>Ef</em>NOx reduced molecular oxygen to water, regenerating NAD⁺ and sustaining redox balance. To further enhance intracellular cofactor turnover, <em>Bs</em>BDHA^{I49L/V266L/G292A} and <em>Ef</em>NOx were co-expressed in <em>Escherichia coli</em>, generating the recombinant strain <em>E. coli</em>/C<em>bn</em>. Through single-factor and L9(3³) orthogonal array optimization, the one-pot <em>in vivo</em> cascade achieved maximal efficiency with lyophilized whole cells of <em>E. coli/rpeh</em>^L360V (5 mg DCW/mL) and <em>E. coli</em>/C<em>bn</em> (35 mg DCW/mL) in sodium phosphate buffer (10 mM, pH 7.0) containing 5 mM NAD⁺ and 5 % (v/v) Tween-20 at 25 °C for 8 h under controlled agitation and aeration. Under these conditions, 25 mM <em>rac</em>-<strong>1a</strong> was converted to <strong>1c</strong> with a yield of 92.1 % and a space-time yield (STY) of 2.9 mmol/L/h, representing a 3.7-fold increase relative to the original LDH-based system. The applicability of the cascade was further demonstrated using additional substrates (<em>rac</em>-<strong>2a</strong> to <em>rac</em>-<strong>7a</strong>), yielding the corresponding α-hydroxyketones at 39.1–94.5 % with STYs of 1.2–3.0 mmol/L/h. These findings establish BDH–NOx co-expression as a robust strategy for cofactor self-sufficiency and provide a scalable framework for the efficient <em>in vivo</em> synthesis of structurally diverse α-hydroxyketones.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 184-193"},"PeriodicalIF":3.9,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145781375","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Increasing the chiral purity of cellulosic L-lactic acid by D-Lactate oxidase-catalyzed oxidation in engineered lactic acid bacterium Pediococcus acidilactici","authors":"Jiao Liu,&nbsp;Chaolong Qu,&nbsp;Bin Zhang,&nbsp;Jie Bao","doi":"10.1016/j.jbiotec.2025.12.002","DOIUrl":"10.1016/j.jbiotec.2025.12.002","url":null,"abstract":"<div><div>Trace amounts of <span>D</span>-lactic acid derived from lignocellulose feedstock and nitrogen additives frequently reduce the chiral purity of <span>L</span>-lactic acid below the polymerization standard during biorefinery fermentations. The spontaneous <span>D</span>-lactic acid degradation catalyzed by the highly substrate specific <span>D</span>-lactate oxidase in lactic acid bacterium provides an ideal solution. This study used a <span>D</span>-lactate oxidase GOX2071 with relatively high activity for construction of <span>D</span>-lactic acid oxidation pathway in the <span>L</span>-lactic acid producing strain <em>Pediococcus acidilactici</em> ZY271. The engineered <em>P</em>. <em>acidilactici</em> LJ2071 strain demonstrated an active <span>D</span>-lactic acid oxidation performance while the high <span>L</span>-lactic acid production property was well maintained. A <span>L</span>-lactic acid chiral purity of 99.63 % was obtained using the <span>D</span>-lactic acid containing wheat straw as feedstock and 40 % of corn steep liquor (CSL) as nitrogen additive to alternate expensive yeast extract. This study provided a practical method for upgrading the chirality of cellulosic <span>L</span>-lactic acid using lignocellulose feedstock and cheap nitrogen additives.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 137-144"},"PeriodicalIF":3.9,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145701077","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}