Pub Date : 2025-11-19DOI: 10.1016/j.biotechadv.2025.108763
Yuki Uno , Yusuke Hayashi , Hirokazu Sugiyama , Jun Okuda , Tetsuji Nakamura , Masahiro Kino-oka
The projected expansion of the global market for cell manufacturing, which contributes to regenerative medicine and cell therapies, warrants the designing and development of scalable cryopreservation processes for cell-based products (CBPs) for use in both standard and personalized therapies. However, the change in scale causes variations in process parameters, which affects the stability of the CBP quality. Therefore, the cryopreservation process for CBPs needs to be designed based on the concept of cell manufacturability and consideration of both engineering and biological aspects. In this review, we discussed strategies to enhance the quality stability of CBPs during cryopreservation, focusing primarily on four key processes: dispensing, freezing, storage, and thawing. Additionally, we discussed the application of simulation technologies because they aid in constructing digital twins for the designing and development of the cryopreservation process and facilitate efficiency with limited time and resources.
{"title":"Strategies to Enhance Stability of Cryopreservation Processes for Cell-Based Products","authors":"Yuki Uno , Yusuke Hayashi , Hirokazu Sugiyama , Jun Okuda , Tetsuji Nakamura , Masahiro Kino-oka","doi":"10.1016/j.biotechadv.2025.108763","DOIUrl":"10.1016/j.biotechadv.2025.108763","url":null,"abstract":"<div><div>The projected expansion of the global market for cell manufacturing, which contributes to regenerative medicine and cell therapies, warrants the designing and development of scalable cryopreservation processes for cell-based products (CBPs) for use in both standard and personalized therapies. However, the change in scale causes variations in process parameters, which affects the stability of the CBP quality. Therefore, the cryopreservation process for CBPs needs to be designed based on the concept of cell manufacturability and consideration of both engineering and biological aspects. In this review, we discussed strategies to enhance the quality stability of CBPs during cryopreservation, focusing primarily on four key processes: dispensing, freezing, storage, and thawing. Additionally, we discussed the application of simulation technologies because they aid in constructing digital twins for the designing and development of the cryopreservation process and facilitate efficiency with limited time and resources.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"87 ","pages":"Article 108763"},"PeriodicalIF":12.5,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145553879","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-17DOI: 10.1016/j.biotechadv.2025.108760
Anna Pijnacker , Christine W. Bruggeman , Hendrik C. Korswagen , Geert Smant , José L. Lozano-Torres
Obligate parasites pose a significant threat to animal, human, and plant health by affecting host gene expression through mechanisms that are poorly understood. Spatial transcriptomic technologies are revolutionizing our understanding of animal-parasite interactions, revealing tissue reorganization, cellular responses, and infection dynamics at a microscopic scale. These technologies also accelerate the identification of potential targets for treating animal parasite infections. Despite their potential, the application of spatial transcriptomic technologies to plant-parasite interactions is limited. This review highlights key challenges in applying spatial transcriptomics to plants. By drawing parallels with advances in animal systems, we discuss how spatial transcriptomics could contribute to localize and identify effectors, uncover the molecular mechanisms of plant-parasite infections, and find novel disease control targets. This cross-disciplinary perspective provides a roadmap for future research in plant and animal parasitology.
{"title":"Harnessing spatial transcriptomics to understand host-parasite interactions in plants and animals","authors":"Anna Pijnacker , Christine W. Bruggeman , Hendrik C. Korswagen , Geert Smant , José L. Lozano-Torres","doi":"10.1016/j.biotechadv.2025.108760","DOIUrl":"10.1016/j.biotechadv.2025.108760","url":null,"abstract":"<div><div>Obligate parasites pose a significant threat to animal, human, and plant health by affecting host gene expression through mechanisms that are poorly understood. Spatial transcriptomic technologies are revolutionizing our understanding of animal-parasite interactions, revealing tissue reorganization, cellular responses, and infection dynamics at a microscopic scale. These technologies also accelerate the identification of potential targets for treating animal parasite infections. Despite their potential, the application of spatial transcriptomic technologies to plant-parasite interactions is limited. This review highlights key challenges in applying spatial transcriptomics to plants. By drawing parallels with advances in animal systems, we discuss how spatial transcriptomics could contribute to localize and identify effectors, uncover the molecular mechanisms of plant-parasite infections, and find novel disease control targets. This cross-disciplinary perspective provides a roadmap for future research in plant and animal parasitology.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"87 ","pages":"Article 108760"},"PeriodicalIF":12.5,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145554002","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-17DOI: 10.1016/j.biotechadv.2025.108749
Johnny Peng, Thanh Tung Khuat, Katarzyna Musial, Bogdan Gabrys
Data are crucial for machine learning (ML) applications, yet acquiring large datasets can be costly and timeconsuming, especially in complex, resource-intensive fields like biopharmaceuticals. A key process in this industry is upstream bioprocessing, where living cells are cultivated and optimised to produce therapeutic proteins and biologics. The intricate nature of these processes, combined with high resource demands, often limits data collection, resulting in smaller datasets. This comprehensive review explores ML methods designed to address the challenges posed by small data and classifies them into a taxonomy to guide practical applications. Furthermore, each method in the taxonomy was thoroughly analysed, with a detailed discussion of its core concepts and an evaluation of its effectiveness in tackling small data challenges, as demonstrated by application results in the upstream bioprocessing and other related domains. By analyzing how these methods tackle small data challenges from different perspectives, this review provides actionable insights, identifies current research gaps, and offers guidance for leveraging ML in data-constrained environments.
{"title":"Machine learning methods for small data and upstream bioprocessing applications: A comprehensive review","authors":"Johnny Peng, Thanh Tung Khuat, Katarzyna Musial, Bogdan Gabrys","doi":"10.1016/j.biotechadv.2025.108749","DOIUrl":"10.1016/j.biotechadv.2025.108749","url":null,"abstract":"<div><div>Data are crucial for machine learning (ML) applications, yet acquiring large datasets can be costly and timeconsuming, especially in complex, resource-intensive fields like biopharmaceuticals. A key process in this industry is upstream bioprocessing, where living cells are cultivated and optimised to produce therapeutic proteins and biologics. The intricate nature of these processes, combined with high resource demands, often limits data collection, resulting in smaller datasets. This comprehensive review explores ML methods designed to address the challenges posed by small data and classifies them into a taxonomy to guide practical applications. Furthermore, each method in the taxonomy was thoroughly analysed, with a detailed discussion of its core concepts and an evaluation of its effectiveness in tackling small data challenges, as demonstrated by application results in the upstream bioprocessing and other related domains. By analyzing how these methods tackle small data challenges from different perspectives, this review provides actionable insights, identifies current research gaps, and offers guidance for leveraging ML in data-constrained environments.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"87 ","pages":"Article 108749"},"PeriodicalIF":12.5,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145553993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-17DOI: 10.1016/j.biotechadv.2025.108761
Si Bin Chew, Emily Harjabrata, Cameron Jing Han Goh, Qunxiang Ong
Intracellular optogenetics represents a rapidly advancing biotechnology that enables precise, reversible control of protein activity, signaling dynamics, and cellular behaviours using genetically encoded, light-responsive systems. Originally pioneered in neuroscience through channelrhodopsins to manipulate neuronal excitability, the field has since expanded into diverse intracellular applications with broad implications for medicine, agriculture, and biomanufacturing. Key to these advances are photoreceptors such as cryptochrome 2 (CRY2), light–oxygen–voltage (LOV) domains, and phytochromes, which undergo conformational changes upon illumination to trigger conditional protein–protein interactions, localization shifts, or phase transitions. Recent engineering breakthroughs—including the creation of red-light responsive systems such as MagRed that exploit endogenous biliverdin—have enhanced tissue penetration, minimized phototoxicity, and expanded applicability to complex biological systems.
This review provides an overarching synthesis of the molecular principles underlying intracellular optogenetic actuators, including the photophysical basis of light-induced conformational changes, oligomerization, and signaling control. We highlight strategies that employ domain fusions, rational mutagenesis, and synthetic circuits to extend their utility across biological and industrial contexts. We also critically assess current limitations, such as chromophore dependence, light delivery challenges, and safety considerations, so as to frame realistic paths towards translation. Looking ahead, future opportunities include multi-colour and multiplexed systems, integration with high-throughput omics and artificial intelligence, and development of non-invasive modalities suited for in vivo and industrial applications.
Intracellular optogenetics is thus emerging as a versatile platform technology, with the potential to reshape how we interrogate biology and engineer cells for therapeutic, agricultural, and environmental solutions.
{"title":"Capitalizing on mechanistic insights to power design of future-ready intracellular optogenetics tools","authors":"Si Bin Chew, Emily Harjabrata, Cameron Jing Han Goh, Qunxiang Ong","doi":"10.1016/j.biotechadv.2025.108761","DOIUrl":"10.1016/j.biotechadv.2025.108761","url":null,"abstract":"<div><div>Intracellular optogenetics represents a rapidly advancing biotechnology that enables precise, reversible control of protein activity, signaling dynamics, and cellular behaviours using genetically encoded, light-responsive systems. Originally pioneered in neuroscience through channelrhodopsins to manipulate neuronal excitability, the field has since expanded into diverse intracellular applications with broad implications for medicine, agriculture, and biomanufacturing. Key to these advances are photoreceptors such as cryptochrome 2 (CRY2), light–oxygen–voltage (LOV) domains, and phytochromes, which undergo conformational changes upon illumination to trigger conditional protein–protein interactions, localization shifts, or phase transitions. Recent engineering breakthroughs—including the creation of red-light responsive systems such as MagRed that exploit endogenous biliverdin—have enhanced tissue penetration, minimized phototoxicity, and expanded applicability to complex biological systems.</div><div>This review provides an overarching synthesis of the molecular principles underlying intracellular optogenetic actuators, including the photophysical basis of light-induced conformational changes, oligomerization, and signaling control. We highlight strategies that employ domain fusions, rational mutagenesis, and synthetic circuits to extend their utility across biological and industrial contexts. We also critically assess current limitations, such as chromophore dependence, light delivery challenges, and safety considerations, so as to frame realistic paths towards translation. Looking ahead, future opportunities include multi-colour and multiplexed systems, integration with high-throughput omics and artificial intelligence, and development of non-invasive modalities suited for <em>in vivo</em> and industrial applications.</div><div>Intracellular optogenetics is thus emerging as a versatile platform technology, with the potential to reshape how we interrogate biology and engineer cells for therapeutic, agricultural, and environmental solutions.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"87 ","pages":"Article 108761"},"PeriodicalIF":12.5,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145554410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-14DOI: 10.1016/j.biotechadv.2025.108752
Fan Liu , Chunxiang Feng , Zirui Yin , Jingwen Zhou , Jianghua Li , Jian Chen , Guocheng Du , Xinrui Zhao
Hemoglobin is a functional protein with heme as a cofactor, playing a crucial role in transporting oxygen and maintaining nitric oxide metabolic balance. Besides its physiological functions, hemoglobin has broad potential applications in medicine and biotechnology. However, the widespread use of hemoglobin is constrained by limited natural sources, challenges in heterologous synthesis, and functional restrictions that hinder efficient application. In this review, we discuss the key challenges and solutions associated with microbial synthesis of hemoglobin. We systematically elucidate the engineering strategies to improve the stability, autoxidation rate, heme-binding capacity, oxygen transport efficiency, and nitric oxide scavenging rate of hemoglobin, with particular emphasis on the use of artificial intelligence algorithms to customize the function modification of hemoglobin. Also, we provide a comprehensive overview of the various applications of hemoglobin, including artificial oxygen carriers, medical treatments requiring enhanced oxygen supply, synthesis of high-value products, biocatalysis, artificial foods, agriculture, functional substance testing, and bioactive peptide production, with a special focus on the potential of hemoglobin mutants and derivatives in expanding its use across various fields. Finally, we explore the prospects for accelerating the resolution of hemoglobin synthesis and overcoming the application challenges by integrating Pareto-optimal and iterative bioengineering frameworks, deep learning, synthetic biology, and other advanced technologies.
{"title":"Engineering strategies for microbial synthesis, customized modification, and application of hemoglobin","authors":"Fan Liu , Chunxiang Feng , Zirui Yin , Jingwen Zhou , Jianghua Li , Jian Chen , Guocheng Du , Xinrui Zhao","doi":"10.1016/j.biotechadv.2025.108752","DOIUrl":"10.1016/j.biotechadv.2025.108752","url":null,"abstract":"<div><div>Hemoglobin is a functional protein with heme as a cofactor, playing a crucial role in transporting oxygen and maintaining nitric oxide metabolic balance. Besides its physiological functions, hemoglobin has broad potential applications in medicine and biotechnology. However, the widespread use of hemoglobin is constrained by limited natural sources, challenges in heterologous synthesis, and functional restrictions that hinder efficient application. In this review, we discuss the key challenges and solutions associated with microbial synthesis of hemoglobin. We systematically elucidate the engineering strategies to improve the stability, autoxidation rate, heme-binding capacity, oxygen transport efficiency, and nitric oxide scavenging rate of hemoglobin, with particular emphasis on the use of artificial intelligence algorithms to customize the function modification of hemoglobin. Also, we provide a comprehensive overview of the various applications of hemoglobin, including artificial oxygen carriers, medical treatments requiring enhanced oxygen supply, synthesis of high-value products, biocatalysis, artificial foods, agriculture, functional substance testing, and bioactive peptide production, with a special focus on the potential of hemoglobin mutants and derivatives in expanding its use across various fields. Finally, we explore the prospects for accelerating the resolution of hemoglobin synthesis and overcoming the application challenges by integrating Pareto-optimal and iterative bioengineering frameworks, deep learning, synthetic biology, and other advanced technologies.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"87 ","pages":"Article 108752"},"PeriodicalIF":12.5,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145531338","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-10DOI: 10.1016/j.biotechadv.2025.108751
Tosin Victor Adegoke , Sifan Lu , Ogedegbe Gloria Adegoke , Yufei Wang , Yan Wang
Mycotoxins, among the most extensively studied biological toxins, pose significant health risks to humans and animals, causing substantial economic losses in the agricultural sector. Numerous conventional enzymes isolated from microorganisms have been reported to detoxify mycotoxin, but their stability is questionable for detoxifying mycotoxin and the direct industrial production of enzymes. Currently, few commercial enzymes are available for the detoxification of mycotoxins. Enhancing enzyme stability is essential to ensure effective detoxification under feed-appropriate temperature and pH conditions. To overcome this challenge, the amalgamation of numerous fields, such as bioinformatics and protein engineering, is crucial for improving the enzyme for industrial production. Computational tools are crucial for determining the nucleotides of the sequence for modification using site-directed mutagenesis (SDM) for the existing conventional enzyme. The SDM technique offers a promising approach for modifying conventional enzymes for commercial purposes. Therefore, focusing on identifying, modifying, and producing enzymes that effectively detoxify mycotoxins is crucial for mitigating their effects on animals and preventing economic losses. Also, a fusion of modified enzymes involved in the cascade detoxification of mycotoxin and its derivatives should be focused on. This review provides an overview of the computational tools and protein engineering approaches, focusing on SDM and cascade catalysis for enhanced mycotoxin detoxification. We also discuss the future directions for incorporating these engineered enzyme systems on a commercial scale.
{"title":"Optimizing single and cascade microbial enzyme systems through site-directed mutagenesis for enhancing mycotoxin detoxification","authors":"Tosin Victor Adegoke , Sifan Lu , Ogedegbe Gloria Adegoke , Yufei Wang , Yan Wang","doi":"10.1016/j.biotechadv.2025.108751","DOIUrl":"10.1016/j.biotechadv.2025.108751","url":null,"abstract":"<div><div>Mycotoxins, among the most extensively studied biological toxins, pose significant health risks to humans and animals, causing substantial economic losses in the agricultural sector. Numerous conventional enzymes isolated from microorganisms have been reported to detoxify mycotoxin, but their stability is questionable for detoxifying mycotoxin and the direct industrial production of enzymes. Currently, few commercial enzymes are available for the detoxification of mycotoxins. Enhancing enzyme stability is essential to ensure effective detoxification under feed-appropriate temperature and pH conditions. To overcome this challenge, the amalgamation of numerous fields, such as bioinformatics and protein engineering, is crucial for improving the enzyme for industrial production. Computational tools are crucial for determining the nucleotides of the sequence for modification using site-directed mutagenesis (SDM) for the existing conventional enzyme. The SDM technique offers a promising approach for modifying conventional enzymes for commercial purposes. Therefore, focusing on identifying, modifying, and producing enzymes that effectively detoxify mycotoxins is crucial for mitigating their effects on animals and preventing economic losses. Also, a fusion of modified enzymes involved in the cascade detoxification of mycotoxin and its derivatives should be focused on. This review provides an overview of the computational tools and protein engineering approaches, focusing on SDM and cascade catalysis for enhanced mycotoxin detoxification. We also discuss the future directions for incorporating these engineered enzyme systems on a commercial scale.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"87 ","pages":"Article 108751"},"PeriodicalIF":12.5,"publicationDate":"2025-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145485498","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-08DOI: 10.1016/j.biotechadv.2025.108750
Ying-ying Xu , Sheng-mei Zhou , Lu-yan Wang , Rong Zhang , Kai Li , Zhi-yuan Qian , Li Xiao
The CRISPR/Cas9 system has emerged as a revolutionary tool for gene editing, widely used in the biomedical field due to its simplicity, efficiency, and cost-effectiveness. However, evidence suggests that CRISPR/Cas9 can induce off-target effects, leading to unintended mutations that may compromise the precision of gene modifications. Consequently, predicting，detecting and evaluating these off-target effects is crucial for optimizing the accuracy and reliability of CRISPR/Cas9 system. This paper provides an overview of the various methodologies and strategies, used or to be used for identifying off-target effects in CRISPR/Cas9-based genome editing, offering insights to improve the precision and safety of CRISPR applications in research and therapeutics.
{"title":"Methods for detecting off-target effects of CRISPR/Cas9","authors":"Ying-ying Xu , Sheng-mei Zhou , Lu-yan Wang , Rong Zhang , Kai Li , Zhi-yuan Qian , Li Xiao","doi":"10.1016/j.biotechadv.2025.108750","DOIUrl":"10.1016/j.biotechadv.2025.108750","url":null,"abstract":"<div><div>The CRISPR/Cas9 system has emerged as a revolutionary tool for gene editing, widely used in the biomedical field due to its simplicity, efficiency, and cost-effectiveness. However, evidence suggests that CRISPR/Cas9 can induce off-target effects, leading to unintended mutations that may compromise the precision of gene modifications. Consequently, predicting，detecting and evaluating these off-target effects is crucial for optimizing the accuracy and reliability of CRISPR/Cas9 system. This paper provides an overview of the various methodologies and strategies, used or to be used for identifying off-target effects in CRISPR/Cas9-based genome editing, offering insights to improve the precision and safety of CRISPR applications in research and therapeutics.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"86 ","pages":"Article 108750"},"PeriodicalIF":12.5,"publicationDate":"2025-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145473181","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-02DOI: 10.1016/j.biotechadv.2025.108748
Duodong Wang , Na Wang , Houhui Song , Chenggang Xu
Bacteria exhibit remarkable precision in controlling the stoichiometry of protein subunits within metabolic pathways and macromolecular complexes—a requirement for optimal function and fitness. This review explores the RNA-level mechanisms that enable bacteria to maintain precise subunit ratios, moving beyond canonical transcriptional regulation to highlight the role of post-transcriptional fine-tuning. We discuss how internal transcriptional terminators serve as tunable attenuators, creating expression gradients within polycistronic operons, and how selective RNA processing and stabilization (SRPS) systems generate differential mRNA stability to shape proteomic stoichiometry. Furthermore, we outline how these native strategies have inspired the design of synthetic genetic circuits—including promoter libraries, engineered terminators, and RNase-based processing modules—that allow programmable control of gene expression levels. By leveraging modular and layered regulatory elements, synthetic biologists can now construct robust systems with user-defined stoichiometric outputs, facilitating the engineering of complex metabolic pathways and protein assemblies for biotechnological and biomedical applications.
{"title":"Precise control of transcriptional stoichiometry in bacteria: From mechanisms to synthetic biology applications","authors":"Duodong Wang , Na Wang , Houhui Song , Chenggang Xu","doi":"10.1016/j.biotechadv.2025.108748","DOIUrl":"10.1016/j.biotechadv.2025.108748","url":null,"abstract":"<div><div>Bacteria exhibit remarkable precision in controlling the stoichiometry of protein subunits within metabolic pathways and macromolecular complexes—a requirement for optimal function and fitness. This review explores the RNA-level mechanisms that enable bacteria to maintain precise subunit ratios, moving beyond canonical transcriptional regulation to highlight the role of post-transcriptional fine-tuning. We discuss how internal transcriptional terminators serve as tunable attenuators, creating expression gradients within polycistronic operons, and how selective RNA processing and stabilization (SRPS) systems generate differential mRNA stability to shape proteomic stoichiometry. Furthermore, we outline how these native strategies have inspired the design of synthetic genetic circuits—including promoter libraries, engineered terminators, and RNase-based processing modules—that allow programmable control of gene expression levels. By leveraging modular and layered regulatory elements, synthetic biologists can now construct robust systems with user-defined stoichiometric outputs, facilitating the engineering of complex metabolic pathways and protein assemblies for biotechnological and biomedical applications.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"86 ","pages":"Article 108748"},"PeriodicalIF":12.5,"publicationDate":"2025-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145434684","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-01DOI: 10.1016/j.biotechadv.2025.108747
Cuifang Ye , Xiaoqian Li , Tao Liu , Shiyu Li , Mengyu Zhang , Yao Zhao , Jintao Cheng , Guiling Yang , Peiwu Li
Peroxisome engineering in yeast has emerged as a promising strategy for biomanufacturing, as it enables the compartmentalization of biosynthetic pathways and thus alleviates key bottlenecks in natural product biosynthesis. By sequestering specific metabolic pathways within peroxisomes, this strategy effectively reduces product cytotoxicity, enhances intracellular product storage, and allows precise redirection of metabolic fluxes. Nevertheless, its broader application remains limited by several unresolved challenges, including the insufficient understanding of peroxisomal membrane permeability, inadequate cofactor supply, and glucose-mediated repression of peroxisomal capacity. To overcome these obstacles, a range of conventional and emerging approaches—such as engineering peroxisomal targeting signal type 1 (PTS1), regulation of peroxisome proliferation, development of orthogonal artificial peroxisomal protein transport systems, and applying machine learning to predict gene overexpression for optimizing peroxisomal functional capacity—have expanded the toolkit for peroxisome engineering in yeast. This review summarizes recent advances in peroxisomal surface display engineering, peroxisomal matrix engineering, and multi-organelle spatial combination coordination, highlighting the importance of peroxisome engineering in optimizing yeast-based cell factories for natural product biosynthesis. Moreover, it critically evaluates current limitations, along with a comprehensive discussion of both conventional and emerging approaches aimed at further optimizing peroxisome engineering. In the future, integrating peroxisome engineering with advanced machine learning will be crucial for addressing remaining challenges and fully realizing the potential of sustainable and scalable yeast-based biomanufacturing.
{"title":"Peroxisome engineering in yeast: Advances, challenges, and prospects","authors":"Cuifang Ye , Xiaoqian Li , Tao Liu , Shiyu Li , Mengyu Zhang , Yao Zhao , Jintao Cheng , Guiling Yang , Peiwu Li","doi":"10.1016/j.biotechadv.2025.108747","DOIUrl":"10.1016/j.biotechadv.2025.108747","url":null,"abstract":"<div><div>Peroxisome engineering in yeast has emerged as a promising strategy for biomanufacturing, as it enables the compartmentalization of biosynthetic pathways and thus alleviates key bottlenecks in natural product biosynthesis. By sequestering specific metabolic pathways within peroxisomes, this strategy effectively reduces product cytotoxicity, enhances intracellular product storage, and allows precise redirection of metabolic fluxes. Nevertheless, its broader application remains limited by several unresolved challenges, including the insufficient understanding of peroxisomal membrane permeability, inadequate cofactor supply, and glucose-mediated repression of peroxisomal capacity. To overcome these obstacles, a range of conventional and emerging approaches—such as engineering peroxisomal targeting signal type 1 (PTS1), regulation of peroxisome proliferation, development of orthogonal artificial peroxisomal protein transport systems, and applying machine learning to predict gene overexpression for optimizing peroxisomal functional capacity—have expanded the toolkit for peroxisome engineering in yeast. This review summarizes recent advances in peroxisomal surface display engineering, peroxisomal matrix engineering, and multi-organelle spatial combination coordination, highlighting the importance of peroxisome engineering in optimizing yeast-based cell factories for natural product biosynthesis. Moreover, it critically evaluates current limitations, along with a comprehensive discussion of both conventional and emerging approaches aimed at further optimizing peroxisome engineering. In the future, integrating peroxisome engineering with advanced machine learning will be crucial for addressing remaining challenges and fully realizing the potential of sustainable and scalable yeast-based biomanufacturing.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"86 ","pages":"Article 108747"},"PeriodicalIF":12.5,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145423995","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-01Epub Date: 2025-08-06DOI: 10.1016/j.biotechadv.2025.108684
Anastasia E C Rumpl, Joshua R Goodhew, Paul F Kelly, Mika Hirano, Michael E Pyne
The Ehrlich pathway is a catabolic process that imparts Saccharomyces cerevisiae and other yeasts with the ability to utilize branched-chain and aromatic amino acids as a source of nitrogen. Using this route, amino acids are transaminated to α-keto acids and the liberated ammonia is utilized for assimilatory reactions. This process leaves behind an array of aliphatic and aromatic carbon skeletons (fusel metabolites) that have found a multitude of uses in the production of flavors, chemicals, and pharmaceuticals. This review provides an update on the genetics and biochemistry of the Ehrlich pathway with an emphasis on the biotechnological valorization of fusel metabolites. We outline the impact of fusel metabolism on the organoleptic properties of fermented beverages and recap ongoing efforts to repurpose the Ehrlich pathway for production of advanced biofuels. We also highlight recent activity directed at producing opioids and other plant benzylisoquinolines, as well as engineering new-to-nature alkaloids by rewiring the yeast Ehrlich pathway. Collectively, these efforts have stimulated a deeper understanding of yeast fusel metabolism and opened new opportunities for biomanufacturing using conventional and non-conventional yeasts.
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