Pub Date : 2026-01-06DOI: 10.1016/j.jbiotec.2026.01.002
Keegan Orzechowski , Johanna Vappiani-Korben , Daniel C. Sevin , Juan Aon
Metabolomics analyses of cell culture processes can provide valuable insight into cellular physiology that can be leveraged to develop more productive processes. In this work, we applied metabolomics to interrogate CHO cell behavior in two industrial chemically-defined media in cultures co-fed with glucose and lactic acid. We previously reported that secreted acylcarnitines are indicative of altered mitochondrial metabolism when cultures are fed lactic acid and serve to maintain homeostasis between free CoA, acetyl-CoA, free carnitines, and acylcarnitines (Vappiani et al., 2021). One of the two media (“Medium B”) increased significantly viable-cell count and antibody titer than Medium A. Here, we report that CHO’s mitochondrial dysfunctionality based on the secretion of acylcarnitines in lactic acid-fed cultures depends on the overall medium composition. We hypothesize that in order to achieve better growth and titer, Medium B exhibited an increased oxidative phosphorylation based on the lower secretion of acylcarnitines and a differential utilization of riboflavin and thiamine, precursors of coenzymes required to enhance mitochondrial pyruvate incorporation and TCA cycle function. Therefore, our data provides further evidence that non-obvious changes to medium composition can have substantial effects on CHO-based production processes by altering the activity of oxidative phosphorylation required for the proper functioning of mitochondria but also for better antibody production.
{"title":"Two industrial media reveal a mitochondrial disfunction in CHO cell cultures co-fed with glucose and lactic acid","authors":"Keegan Orzechowski , Johanna Vappiani-Korben , Daniel C. Sevin , Juan Aon","doi":"10.1016/j.jbiotec.2026.01.002","DOIUrl":"10.1016/j.jbiotec.2026.01.002","url":null,"abstract":"<div><div>Metabolomics analyses of cell culture processes can provide valuable insight into cellular physiology that can be leveraged to develop more productive processes. In this work, we applied metabolomics to interrogate CHO cell behavior in two industrial chemically-defined media in cultures co-fed with glucose and lactic acid. We previously reported that secreted acylcarnitines are indicative of altered mitochondrial metabolism when cultures are fed lactic acid and serve to maintain homeostasis between free CoA, acetyl-CoA, free carnitines, and acylcarnitines (Vappiani <em>et al</em>., 2021). One of the two media (“Medium B”) increased significantly viable-cell count and antibody titer than Medium A. Here, we report that CHO’s mitochondrial dysfunctionality based on the secretion of acylcarnitines in lactic acid-fed cultures depends on the overall medium composition. We hypothesize that in order to achieve better growth and titer, Medium B exhibited an increased oxidative phosphorylation based on the lower secretion of acylcarnitines and a differential utilization of riboflavin and thiamine, precursors of coenzymes required to enhance mitochondrial pyruvate incorporation and TCA cycle function. Therefore, our data provides further evidence that non-obvious changes to medium composition can have substantial effects on CHO-based production processes by altering the activity of oxidative phosphorylation required for the proper functioning of mitochondria but also for better antibody production.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"411 ","pages":"Pages 1-11"},"PeriodicalIF":3.9,"publicationDate":"2026-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145933438","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}
<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
{"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":"2026-01-06","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}
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 Geobacillus zalihae and H1-Pol from Aeribacillus pallidus, 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.
{"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 , Eisuke Inoue , Yuto Murakami , Yuri Ishii , Yukiko Nakura , Itaru Yanagihara , Kiyoshi Yasukawa , 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":"2026-01-02","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}
Pub Date : 2026-01-02DOI: 10.1016/j.jbiotec.2025.12.019
Mingyang Hu , Chongpeng Bi , Yuwen Li, Yutong Xue, Sina Cha, Lu Zhao, Chenyu Xue, Na Dong
The rising prevalence of antibiotic resistance underscores the urgent need for alternative strategies to manage pathogenic bacteria. Engineered probiotics offer a promising platform for delivering antimicrobial peptides (AMPs); however, their practical application remains constrained by challenges related to maintaining viability and in vivo functionality. This study focused on two main aspects: (1) optimizing a freeze-drying strategy for Lactococcus lactis/pNZC-Usp45-H-6 ×His (L. L/HI), which expresses the AMP HI targeting Escherichia coli, and (2) evaluating its protective efficacy against enterotoxigenic Escherichia coli (ETEC) infection in a murine model. Sorbitol at a concentration of 6 % (w/v) was identified as the most effective cryoprotectant for preserving bacterial viability after freeze-drying. In the ETEC infection model, oral administration of L. L/HI significantly alleviated intestinal injury by reducing bacterial colonization and lipopolysaccharide levels, alleviating inflammation, and restoring the expression of tight junction genes. Moreover, L. L/HI downregulated the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and upregulated the anti-inflammatory cytokine IL-10 in ileal tissues. These findings demonstrate that oral administration of L. L/HI reduced the bacterial burden in the ileum of ETEC–infected mice and indirectly alleviated inflammation and intestinal barrier damage caused by ETEC infection. This study provides a novel approach for the translational application of engineered probiotics.
抗生素耐药性的日益流行突出表明迫切需要采取其他战略来管理致病菌。工程益生菌为提供抗菌肽（amp）提供了一个有前途的平台；然而，它们的实际应用仍然受到与维持活力和体内功能相关的挑战的限制。本研究主要集中在两个方面：(1)优化以大肠杆菌为靶点表达AMP HI的乳酸乳球菌/pNZC-Usp45-H-6×His （L. L/HI）的冷冻干燥策略；(2)在小鼠模型上评价其对产肠毒素大肠杆菌（ETEC）感染的保护作用。山梨醇在6% （w/v）的浓度下被确定为冷冻干燥后保持细菌活力最有效的冷冻保护剂。在ETEC感染模型中，口服L. L. /HI通过减少细菌定植和脂多糖水平、减轻炎症、恢复紧密连接基因的表达，显著减轻肠道损伤。L. L/HI下调回肠组织中促炎因子（TNF-α、IL-1β、IL-6）的表达，上调抗炎因子IL-10的表达。上述结果表明，口服L. L/HI可减轻ETEC感染小鼠回肠细菌负荷，间接减轻ETEC感染引起的炎症和肠屏障损伤。本研究为工程益生菌的转化应用提供了新的途径。
{"title":"Engineered Lactococcus lactis expressing antimicrobial peptide HI: Enhanced survival and protection against ETEC in mice","authors":"Mingyang Hu , Chongpeng Bi , Yuwen Li, Yutong Xue, Sina Cha, Lu Zhao, Chenyu Xue, Na Dong","doi":"10.1016/j.jbiotec.2025.12.019","DOIUrl":"10.1016/j.jbiotec.2025.12.019","url":null,"abstract":"<div><div>The rising prevalence of antibiotic resistance underscores the urgent need for alternative strategies to manage pathogenic bacteria. Engineered probiotics offer a promising platform for delivering antimicrobial peptides (AMPs); however, their practical application remains constrained by challenges related to maintaining viability and <em>in vivo</em> functionality. This study focused on two main aspects: (1) optimizing a freeze-drying strategy for <em>Lactococcus lactis/</em>pNZC-Usp45-H-6 ×His (<em>L. L</em>/HI), which expresses the AMP HI targeting <em>Escherichia coli</em>, and (2) evaluating its protective efficacy against enterotoxigenic <em>Escherichia coli</em> (ETEC) infection in a murine model. Sorbitol at a concentration of 6 % (w/v) was identified as the most effective cryoprotectant for preserving bacterial viability after freeze-drying. In the ETEC infection model, oral administration of <em>L. L</em>/HI significantly alleviated intestinal injury by reducing bacterial colonization and lipopolysaccharide levels, alleviating inflammation, and restoring the expression of tight junction genes. Moreover, <em>L. L</em>/HI downregulated the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and upregulated the anti-inflammatory cytokine IL-10 in ileal tissues. These findings demonstrate that oral administration of <em>L. L</em>/HI reduced the bacterial burden in the ileum of ETEC–infected mice and indirectly alleviated inflammation and intestinal barrier damage caused by ETEC infection. This study provides a novel approach for the translational application of engineered probiotics.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 331-340"},"PeriodicalIF":3.9,"publicationDate":"2026-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145900309","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}
Pub Date : 2025-12-29DOI: 10.1016/j.jbiotec.2025.12.018
Xiaoyu Zhang , Jiayue Guan , Yanqi Yang , Shangci Zuo , Chang Liu , Pengchao Wang
Ergothioneine (EGT) is a rare sulfur-containing derivative of methionine with potent antioxidant, anti-inflammatory, and neuroprotective properties. Its unique bioactivities make it a promising ingredient for applications in functional foods, nutraceuticals, and cosmetics. Microbial fermentation offers a sustainable alternative to extraction from natural sources, yet challenges such as precursor limitations, cofactor imbalances, and pathway complexity continue to restrict industrial-scale production. In this study, we engineered Escherichia coli (E. coli) as a microbial chassis for efficient de novo synthesis of EGT. By co-expressing key enzymes derived from bacteria and fungi, enhancing cysteine biosynthesis, and improving methionine utilization, we addressed key bottlenecks in precursor supply. Furthermore, the introduction of a methylation cycle significantly improved the regeneration of S-adenosylmethionine (SAM), relieving cofactor limitations. These combined metabolic engineering strategies led to a substantial increase in EGT production. The final engineered strain achieved a titer of 141.3 mg/L in shake flasks, representing a sixfold improvement over the base strain. In a 5-liter fed-batch fermentation, the titer reached 1.95 g/L without precursor supplementation and further increased to 2.52 g/L upon low-dose amino acid feeding. This work establishes a cost-effective and scalable biosynthetic platform for EGT production in E. coli, offering a viable route for its application in food and health-related industries.
{"title":"Construction of an E. coli cell factory for ergothioneine through SAM-cycle enhancement and pathway reconstruction","authors":"Xiaoyu Zhang , Jiayue Guan , Yanqi Yang , Shangci Zuo , Chang Liu , Pengchao Wang","doi":"10.1016/j.jbiotec.2025.12.018","DOIUrl":"10.1016/j.jbiotec.2025.12.018","url":null,"abstract":"<div><div>Ergothioneine (EGT) is a rare sulfur-containing derivative of methionine with potent antioxidant, anti-inflammatory, and neuroprotective properties. Its unique bioactivities make it a promising ingredient for applications in functional foods, nutraceuticals, and cosmetics. Microbial fermentation offers a sustainable alternative to extraction from natural sources, yet challenges such as precursor limitations, cofactor imbalances, and pathway complexity continue to restrict industrial-scale production. In this study, we engineered <em>Escherichia coli</em> (<em>E. coli</em>) as a microbial chassis for efficient de novo synthesis of EGT. By co-expressing key enzymes derived from bacteria and fungi, enhancing cysteine biosynthesis, and improving methionine utilization, we addressed key bottlenecks in precursor supply. Furthermore, the introduction of a methylation cycle significantly improved the regeneration of S-adenosylmethionine (SAM), relieving cofactor limitations. These combined metabolic engineering strategies led to a substantial increase in EGT production. The final engineered strain achieved a titer of 141.3 mg/L in shake flasks, representing a sixfold improvement over the base strain. In a 5-liter fed-batch fermentation, the titer reached 1.95 g/L without precursor supplementation and further increased to 2.52 g/L upon low-dose amino acid feeding. This work establishes a cost-effective and scalable biosynthetic platform for EGT production in <em>E. coli</em>, offering a viable route for its application in food and health-related industries.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 298-308"},"PeriodicalIF":3.9,"publicationDate":"2025-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145878348","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}
Pub Date : 2025-12-24DOI: 10.1016/j.jbiotec.2025.12.017
Fei-yan Cheng , Long-wei Lou , Zong-lin Li , Zhi-min Li
Cyanophycin, also known as cyanophycin granule polypeptide (CGP), was a natural polyamide synthesized via a nonribosomal pathway from L-aspartic acid and L-arginine. Its derived dipeptide, β-Asp-Arg, holds potential applications in various fields requiring arginine supplementation in feed or food industries. However, conventional CGP production methods were constrained by strict substrate specificity and high costs. To address these challenges, two-step enzymatic cascade system was constructed integrating CGP biosynthesis with dipeptide production. Central to this system is a novel cyanophycin synthetase (CphA1) from Acidobacteria bacterium, which exhibits remarkable substrate promiscuity. This enzyme enables both primer-independent polymerization of poly-L-Arg/L-Lys-poly(L-Asp) backbones and primer-dependent incorporation of non-canonical substrates (L-ornithine and L-citrulline), thereby expanding the repertoire of cyanophycin derivatives. Furthermore, polyphosphate kinase synergizes with CphA1 to regenerate ATP and promote CGP synthesis, reducing ATP consumption by 90 %. Subsequently, the polymer was efficiently hydrolyzed by CphB from Geminocystis herdmanii to yield the target dipeptides. This method achieved complete conversion of CGP into classical dipeptides β-Asp-Arg and β-Asp-Lys, with the highest titer reaching 80 mM, while also generating non-classical products β-Asp-Orn and β-Asp-Cit. These results highlight the robustness and versatility of this strategy, offering a promising route for the scalable synthesis of β-Asp-basic amino acid dipeptides.
藻青素又称藻青素颗粒多肽（CGP），是由l -天冬氨酸和l -精氨酸经非核糖体途径合成的天然聚酰胺。其衍生的二肽β-Asp-Arg在饲料或食品工业中需要精氨酸补充的各种领域具有潜在的应用前景。然而，传统的CGP生产方法受到严格的底物特异性和高成本的限制。为了解决这些问题，我们构建了将CGP生物合成与二肽生产相结合的两步酶级联系统。该系统的核心是一种来自酸杆菌的新型蓝藻素合成酶（CphA1），它表现出显著的底物混杂性。该酶既可以独立于引物的聚l -精氨酸/ l -赖氨酸聚（L-Asp）骨架聚合，也可以依赖于引物的非规范底物（l -鸟氨酸和l -瓜氨酸）的结合，从而扩大了蓝藻素衍生物的范围。此外，多磷酸激酶与CphA1协同再生ATP，促进CGP合成，减少90%的ATP消耗。随后，该聚合物被来自herdmanii的CphB高效水解，以产生目标二肽。该方法实现了CGP完全转化为经典二肽β-Asp-Arg和β-Asp-Lys，最高滴度达到80mM，同时还生成了非经典产物β-Asp-Orn和β-Asp-Cit。这些结果突出了该策略的稳健性和多功能性，为大规模合成β- asp -碱性氨基酸二肽提供了一条有前途的途径。
{"title":"In vitro synthesis of β-aspartyl-basic amino acid dipeptides via a multi-enzyme cascade system with ATP regeneration","authors":"Fei-yan Cheng , Long-wei Lou , Zong-lin Li , Zhi-min Li","doi":"10.1016/j.jbiotec.2025.12.017","DOIUrl":"10.1016/j.jbiotec.2025.12.017","url":null,"abstract":"<div><div>Cyanophycin, also known as cyanophycin granule polypeptide (CGP), was a natural polyamide synthesized via a nonribosomal pathway from <span>L</span>-aspartic acid and <span>L</span>-arginine. Its derived dipeptide, β-Asp-Arg, holds potential applications in various fields requiring arginine supplementation in feed or food industries. However, conventional CGP production methods were constrained by strict substrate specificity and high costs. To address these challenges, two-step enzymatic cascade system was constructed integrating CGP biosynthesis with dipeptide production. Central to this system is a novel cyanophycin synthetase (CphA1) from <em>Acidobacteria bacterium</em>, which exhibits remarkable substrate promiscuity. This enzyme enables both primer-independent polymerization of poly-<span>L</span>-Arg/<span>L</span>-Lys-poly(<span>L</span>-Asp) backbones and primer-dependent incorporation of non-canonical substrates (<span>L</span>-ornithine and <span>L</span>-citrulline), thereby expanding the repertoire of cyanophycin derivatives. Furthermore, polyphosphate kinase synergizes with CphA1 to regenerate ATP and promote CGP synthesis, reducing ATP consumption by 90 %. Subsequently, the polymer was efficiently hydrolyzed by CphB from <em>Geminocystis herdmanii</em> to yield the target dipeptides. This method achieved complete conversion of CGP into classical dipeptides β-Asp-Arg and β-Asp-Lys, with the highest titer reaching 80 mM, while also generating non-classical products β-Asp-Orn and β-Asp-Cit. These results highlight the robustness and versatility of this strategy, offering a promising route for the scalable synthesis of β-Asp-basic amino acid dipeptides.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 276-284"},"PeriodicalIF":3.9,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145843647","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}
Pub Date : 2025-12-23DOI: 10.1016/j.jbiotec.2025.12.016
Maryam Ehsasatvatan , Bahram Baghban Kohnehrouz
The growing world population and the increasing demand for proteins present significant challenges to food security. Advances in synthetic biology have enabled the development of transplastomic plants engineered to produce milk proteins, offering a promising solution for the large-scale, economical production of alternative protein sources. This study demonstrated the extended stability of human α-lactalbumin (hLA) in freeze-dried transplastomic tobacco chloroplasts. hLA was expressed at high levels, constituting 39.1 % of the total soluble protein in chloroplasts (66.5 mg/g isolated chloroplasts). Western blot analysis of lyophilized tobacco chloroplasts stored at room temperature for up to six months indicated that the hLA protein remained stable, soluble, and properly folded over extended periods. The freeze-drying process of isolated chloroplasts resulted in a 34-fold increase in hLA protein concentrations. Importantly, the biological functionality of purified hLA from lyophilized chloroplasts was confirmed through an in vitro lactose synthesis assay, where recombinant hLA successfully activated galactosyltransferase, leading to efficient lactose production at level comparable to commercial standard. Lyophilized chloroplasts, free from nicotine and bacterial contamination, are suitable candidates for the oral administration of the dietary protein alpha-lactalbumin. These findings suggest that recombinant protein production in transplastomic plants could serve as a viable food source and alternative to conventional sources. Lyophilization further reduces expenses and streamlines downstream processing, purification, and storage. These methods facilitate relevant formulation practices for these compounds to meet the demand-oriented requirements. Future research should prioritize translation of this technology to edible plant hosts to enable direct human consumption and undertake comprehensive in vivo and clinical studies to validate safety, bioavailability, and efficacy.
{"title":"Lyophilized chloroplasts as molecular capsules: A scalable platform for stable and functional human α-lactalbumin production","authors":"Maryam Ehsasatvatan , Bahram Baghban Kohnehrouz","doi":"10.1016/j.jbiotec.2025.12.016","DOIUrl":"10.1016/j.jbiotec.2025.12.016","url":null,"abstract":"<div><div>The growing world population and the increasing demand for proteins present significant challenges to food security. Advances in synthetic biology have enabled the development of transplastomic plants engineered to produce milk proteins, offering a promising solution for the large-scale, economical production of alternative protein sources. This study demonstrated the extended stability of human α-lactalbumin (hLA) in freeze-dried transplastomic tobacco chloroplasts. hLA was expressed at high levels, constituting 39.1 % of the total soluble protein in chloroplasts (66.5 mg/g isolated chloroplasts). Western blot analysis of lyophilized tobacco chloroplasts stored at room temperature for up to six months indicated that the hLA protein remained stable, soluble, and properly folded over extended periods. The freeze-drying process of isolated chloroplasts resulted in a 34-fold increase in hLA protein concentrations. Importantly, the biological functionality of purified hLA from lyophilized chloroplasts was confirmed through an <em>in vitro</em> lactose synthesis assay, where recombinant hLA successfully activated galactosyltransferase, leading to efficient lactose production at level comparable to commercial standard. Lyophilized chloroplasts, free from nicotine and bacterial contamination, are suitable candidates for the oral administration of the dietary protein alpha-lactalbumin. These findings suggest that recombinant protein production in transplastomic plants could serve as a viable food source and alternative to conventional sources. Lyophilization further reduces expenses and streamlines downstream processing, purification, and storage. These methods facilitate relevant formulation practices for these compounds to meet the demand-oriented requirements. Future research should prioritize translation of this technology to edible plant hosts to enable direct human consumption and undertake comprehensive <em>in vivo</em> and clinical studies to validate safety, bioavailability, and efficacy.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 266-275"},"PeriodicalIF":3.9,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145834083","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}
Pub Date : 2025-12-19DOI: 10.1016/j.jbiotec.2025.12.007
Nazgul Wagner, Jonas Austerjost, Julia Niemann
Adeno-associated virus (AAV) vectors are recognized as highly efficient carriers for genetic material. Among the various manufacturing platforms, transient production is the most established one for AAV production. Despite this, the transient production is influenced by numerous variables that can significantly impact the outcome. In this study, we employed 15 ml multi-parallel scale-down bioreactor system to evaluate critical process parameters like cell densities and plasmid DNA concentration. Additionally, a 250 ml bioreactor system was used to explore factors, such as bioprocess mode, comparing batch versus perfusion processes under optimized conditions. Results showed that in low cell density AAV production, where cell specific productivity is highest, the perfusion process yielded over three-fold lower functional titers compared to the batch process at 37°C. Furthermore, the impact of temperature-shifts, a yield improvement strategy that is rarely explored in the context of AAV production, was examined, resulting in increased functional AAV yields - almost two-fold in batch process and over three-fold increase in perfusion process. The temperature-shift not only increased total number of functional AAV particles but also improved the ratio of functional to capsid titers, suggesting enhanced encapsidation or reduced genomic loss from filled particles. After identifying the optimal setup, the process was successfully scaled up to a benchtop bioreactor, demonstrating the scalability and reproducibility of the optimized process. Production results were rapidly assessed using high-throughput analytical techniques, evaluating capsid titers via the biolayer interferometry (BLI)-based Octet® platform and functional titers through an in vitro assay using the high-throughput imaging-based Incucyte® system.
{"title":"Multi-parameter process optimization for high yield AAV2 vector production using scale-down multi-parallel bioreactor systems and high-throughput analytical tools","authors":"Nazgul Wagner, Jonas Austerjost, Julia Niemann","doi":"10.1016/j.jbiotec.2025.12.007","DOIUrl":"10.1016/j.jbiotec.2025.12.007","url":null,"abstract":"<div><div>Adeno-associated virus (AAV) vectors are recognized as highly efficient carriers for genetic material. Among the various manufacturing platforms, transient production is the most established one for AAV production. Despite this, the transient production is influenced by numerous variables that can significantly impact the outcome. In this study, we employed 15 ml multi-parallel scale-down bioreactor system to evaluate critical process parameters like cell densities and plasmid DNA concentration. Additionally, a 250 ml bioreactor system was used to explore factors, such as bioprocess mode, comparing batch versus perfusion processes under optimized conditions. Results showed that in low cell density AAV production, where cell specific productivity is highest, the perfusion process yielded over three-fold lower functional titers compared to the batch process at 37°C. Furthermore, the impact of temperature-shifts, a yield improvement strategy that is rarely explored in the context of AAV production, was examined, resulting in increased functional AAV yields - almost two-fold in batch process and over three-fold increase in perfusion process. The temperature-shift not only increased total number of functional AAV particles but also improved the ratio of functional to capsid titers, suggesting enhanced encapsidation or reduced genomic loss from filled particles. After identifying the optimal setup, the process was successfully scaled up to a benchtop bioreactor, demonstrating the scalability and reproducibility of the optimized process. Production results were rapidly assessed using high-throughput analytical techniques, evaluating capsid titers via the biolayer interferometry (BLI)-based Octet® platform and functional titers through an <em>in vitro</em> assay using the high-throughput imaging-based Incucyte® system.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 228-235"},"PeriodicalIF":3.9,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145804653","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}
Pub Date : 2025-12-19DOI: 10.1016/j.jbiotec.2025.12.015
Xiaopeng Guo , Xiuyue Xiao , Xuexia Dong , Runsheng Yin , Shengli Zhang , Miaomiao Zhang , Guanghong Luo , Yonggang Wang , Dong Lu
Efficient microbial mutagenesis using heavy-ion beam (HIB) radiation is crucial for breeding. Here, Saccharomyces cerevisiae was irradiated with HIB across medium and high doses. Based on 65 randomly selected isolates, we systematically characterized the mutagenic features and preliminarily explored the influence of gene transcriptional activity on mutation susceptibility, while also modulating the intracellular state to optimize strain breeding. High-dose irradiation (120–210 Gy) resulted in a mutation frequency more than double that of the medium-dose (90 Gy), with minimal overlapping mutations between doses. Although mutation site numbers correlated with chromosome length broadly, they were not uniformly distributed at a finer scale. The overall expression of genes associated with mutation sites moderately exceeded the genome-wide background level (p < 0.05). By coupling radiation with osmotic stress, osmoregulatory-related genes were induced to express highly during irradiation. The proportion of osmotolerant mutants obtained from each coupled treatment group (averaging 27.62 %) was higher than that from the radiation-only group (11.43 %). Inference and validation indicated that early selection pressure alone could not fully account for this improvement, highlighting the importance of the intracellular state. Compared to radiation alone, coupled radiation-osmotic stress increased the distribution of mutations in osmotically inducible osmoregulatory-related genes. We propose that the enhanced transcriptional activity may alter local chromatin conformation, together with pre-activation of shared osmotic-radiation response genes, reshape the damage-repair-mutagenesis balance. The coupled treatment produced genetically stable, highly osmotolerant mutants with mutations synergistically regulating carbon metabolism, ion homeostasis, cell adhesion, and DNA replication. This work supports developing high-efficiency microbial breeding strategies.
{"title":"Heavy ion beam irradiation-induced mutational profiles in Saccharomyces cerevisiae and their dependencies on dose and intracellular state inform an enhanced microbial breeding strategy","authors":"Xiaopeng Guo , Xiuyue Xiao , Xuexia Dong , Runsheng Yin , Shengli Zhang , Miaomiao Zhang , Guanghong Luo , Yonggang Wang , Dong Lu","doi":"10.1016/j.jbiotec.2025.12.015","DOIUrl":"10.1016/j.jbiotec.2025.12.015","url":null,"abstract":"<div><div>Efficient microbial mutagenesis using heavy-ion beam (HIB) radiation is crucial for breeding. Here, <em>Saccharomyces cerevisiae</em> was irradiated with HIB across medium and high doses. Based on 65 randomly selected isolates, we systematically characterized the mutagenic features and preliminarily explored the influence of gene transcriptional activity on mutation susceptibility, while also modulating the intracellular state to optimize strain breeding. High-dose irradiation (120–210 Gy) resulted in a mutation frequency more than double that of the medium-dose (90 Gy), with minimal overlapping mutations between doses. Although mutation site numbers correlated with chromosome length broadly, they were not uniformly distributed at a finer scale. The overall expression of genes associated with mutation sites moderately exceeded the genome-wide background level (<em>p</em> < 0.05). By coupling radiation with osmotic stress, osmoregulatory-related genes were induced to express highly during irradiation. The proportion of osmotolerant mutants obtained from each coupled treatment group (averaging 27.62 %) was higher than that from the radiation-only group (11.43 %). Inference and validation indicated that early selection pressure alone could not fully account for this improvement, highlighting the importance of the intracellular state. Compared to radiation alone, coupled radiation-osmotic stress increased the distribution of mutations in osmotically inducible osmoregulatory-related genes. We propose that the enhanced transcriptional activity may alter local chromatin conformation, together with pre-activation of shared osmotic-radiation response genes, reshape the damage-repair-mutagenesis balance. The coupled treatment produced genetically stable, highly osmotolerant mutants with mutations synergistically regulating carbon metabolism, ion homeostasis, cell adhesion, and DNA replication. This work supports developing high-efficiency microbial breeding strategies.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 236-257"},"PeriodicalIF":3.9,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145804626","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}
Glycosylation of natural products significantly enhances their physicochemical properties, with glycosyltransferases (GTs) serving as the catalytic core of this biotransformation. Rational design of tailored GTs aligned with green chemistry principles is pivotal for the targeted synthesis of high-value glycosides, yet precise functional engineering remains challenging. This study employed a multi-scale computational strategy (molecular docking, multiple sequence alignment, molecular dynamics simulations) to systematically re-engineer the substrate recognition and stability modules of the bacterial GT BsGT-1. By reverse-engineering the active pocket of a plant-derived hyperpromiscuous GT (FiGT-2), we orthogonally mapped and identified six functional hotspots in BsGT-1. Site-directed mutagenesis and screening yielded the double variant S128T/T229S, achieving dual optimization: improved substrate scope (121–140 % increase in conversion with UDP-Gal/UDP-Rha compared to wild-type) and enhanced thermostability (>70 % residual activity after 4 h at 50℃). Structural dynamics analyses revealed that mutation-induced global conformational rigidity and localized hydrogen-bond network optimization primarily drove thermostability improvement and substrate affinity enhancement, respectively. This work establishes a closed-loop engineering paradigm of "computational prediction → rational mutagenesis → mechanistic decoding", providing a scalable framework for precision engineering of GTs and glycoside biomanufacturing.
{"title":"Rational design of a bifunctional glycosyltransferase for enhanced substrate promiscuity and thermostability","authors":"Dekai Liu , Ruifang Zhang , Peiqin Chen , Chunying Jin , Lingzhi Zhang , Xiao Gu , Li Wen , Lijuan Zhang , Guangya Zhang , Zhongkun Wu , Wei Jiang","doi":"10.1016/j.jbiotec.2025.12.009","DOIUrl":"10.1016/j.jbiotec.2025.12.009","url":null,"abstract":"<div><div>Glycosylation of natural products significantly enhances their physicochemical properties, with glycosyltransferases (GTs) serving as the catalytic core of this biotransformation. Rational design of tailored GTs aligned with green chemistry principles is pivotal for the targeted synthesis of high-value glycosides, yet precise functional engineering remains challenging. This study employed a multi-scale computational strategy (molecular docking, multiple sequence alignment, molecular dynamics simulations) to systematically re-engineer the substrate recognition and stability modules of the bacterial GT BsGT-1. By reverse-engineering the active pocket of a plant-derived hyperpromiscuous GT (FiGT-2), we orthogonally mapped and identified six functional hotspots in BsGT-1. Site-directed mutagenesis and screening yielded the double variant S128T/T229S, achieving dual optimization: improved substrate scope (121–140 % increase in conversion with UDP-Gal/UDP-Rha compared to wild-type) and enhanced thermostability (>70 % residual activity after 4 h at 50℃). Structural dynamics analyses revealed that mutation-induced global conformational rigidity and localized hydrogen-bond network optimization primarily drove thermostability improvement and substrate affinity enhancement, respectively. This work establishes a closed-loop engineering paradigm of \"computational prediction → rational mutagenesis → mechanistic decoding\", providing a scalable framework for precision engineering of GTs and glycoside biomanufacturing.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 285-297"},"PeriodicalIF":3.9,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145804717","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}
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