Pub Date : 2026-06-01Epub Date: 2026-01-16DOI: 10.1016/j.synbio.2026.01.001
Xinyu Liu , Qihang Chen , Wenbao Zhao , Qi Li , Liushuting Xiao , Qian He , Weizhu Zeng , Jingwen Zhou
Ergosterol is the key precursor of steroid drug synthesis. In this experiment, we systematically modified the synthesis of ergosterol. Firstly, we identified key rate-limiting enzymes through systematic screening of the post-squalene pathway. Combinatorial overexpression of IDI1, tHMG1, ERG4, ERG5, ERG27, ERG1 and ERG11 achieved an ergosterol titer of 94.2 mg/L. Molecular dynamics guided mutagenesis of key substrate channel residues, particularly S372V Erg11, enhanced local flexibility and significantly increased ergosterol production. Introduction of the proton-donating mutations S372V-T305H-ERG11 established an artificial proton-dependent pathway, which, together with channel engineering, further increased the titer to 124 mg/L. Lipid droplet engineering and cellular compartmentalization strategies increased the titer to 148.3 mg/L. Ultimately, multi-copy integration of all ergosterol synthesis pathway genes increased the titer to 433.1 mg/L, and fed-batch fermentation in a 5-L bioreactor resulted in a final titer of 4.58 g/L. This study demonstrates a comprehensive hierarchical strategy for high-level sterol production.
{"title":"Engineering Yarrowia lipolytica for high-level ergosterol production","authors":"Xinyu Liu , Qihang Chen , Wenbao Zhao , Qi Li , Liushuting Xiao , Qian He , Weizhu Zeng , Jingwen Zhou","doi":"10.1016/j.synbio.2026.01.001","DOIUrl":"10.1016/j.synbio.2026.01.001","url":null,"abstract":"<div><div>Ergosterol is the key precursor of steroid drug synthesis. In this experiment, we systematically modified the synthesis of ergosterol. Firstly, we identified key rate-limiting enzymes through systematic screening of the post-squalene pathway. Combinatorial overexpression of <em>IDI1</em>, <em>tHMG1</em>, <em>ERG4</em>, <em>ERG5</em>, <em>ERG27</em>, <em>ERG1</em> and <em>ERG11</em> achieved an ergosterol titer of 94.2 mg/L. Molecular dynamics guided mutagenesis of key substrate channel residues, particularly S372V Erg11, enhanced local flexibility and significantly increased ergosterol production. Introduction of the proton-donating mutations S372V-T305H-<em>ERG11</em> established an artificial proton-dependent pathway, which, together with channel engineering, further increased the titer to 124 mg/L. Lipid droplet engineering and cellular compartmentalization strategies increased the titer to 148.3 mg/L. Ultimately, multi-copy integration of all ergosterol synthesis pathway genes increased the titer to 433.1 mg/L, and fed-batch fermentation in a 5-L bioreactor resulted in a final titer of 4.58 g/L. This study demonstrates a comprehensive hierarchical strategy for high-level sterol production.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 393-400"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145975972","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 : 2026-06-01Epub Date: 2025-12-31DOI: 10.1016/j.synbio.2025.12.013
Huixue Chen , Yan Gao , Shixue Jin , Qian Yun , Xinchen Ruan , Xudong Qu , Chun Lei
The azinothricin family of hybrid hexadepsipeptide-polyketide natural products exhibit remarkable bioactivities, including potent antibacterial, antitumor, antimalarial and anti-inflammatory activities. However, only a few azinothricin-type natural products are currently known, and the biosynthetic potential of microbes remains underexplored. In this work, 137 candidate biosynthetic gene clusters (BGCs) were identified using a genome-mining strategy based on cblaster homology screening. Furthermore, Streptomyces durmitorensis DSM 41863 was prioritized for in-depth experiment due to its unique PKS module expansion, leading to the discovery of kettapeptin, the azinothricin-type metabolite isolated from this species for the first time. Additionally, a putative biosynthetic pathway for kettapeptin was proposed. This work expands the azinothricin-type BGC landscape and establishes S. durmitorensis DSM 41863 as a genetically tractable platform for bioengineering novel derivatives.
{"title":"Molecular landscape analysis of azinothricin-type natural products enables the identification of kettapeptin from Streptomyces durmitorensis","authors":"Huixue Chen , Yan Gao , Shixue Jin , Qian Yun , Xinchen Ruan , Xudong Qu , Chun Lei","doi":"10.1016/j.synbio.2025.12.013","DOIUrl":"10.1016/j.synbio.2025.12.013","url":null,"abstract":"<div><div>The azinothricin family of hybrid hexadepsipeptide-polyketide natural products exhibit remarkable bioactivities, including potent antibacterial, antitumor, antimalarial and anti-inflammatory activities. However, only a few azinothricin-type natural products are currently known, and the biosynthetic potential of microbes remains underexplored. In this work, 137 candidate biosynthetic gene clusters (BGCs) were identified using a genome-mining strategy based on cblaster homology screening. Furthermore, <em>Streptomyces durmitorensis</em> DSM 41863 was prioritized for in-depth experiment due to its unique PKS module expansion, leading to the discovery of kettapeptin, the azinothricin-type metabolite isolated from this species for the first time. Additionally, a putative biosynthetic pathway for kettapeptin was proposed. This work expands the azinothricin-type BGC landscape and establishes <em>S</em>. <em>durmitorensis</em> DSM 41863 as a genetically tractable platform for bioengineering novel derivatives.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 265-273"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883631","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 : 2026-06-01Epub Date: 2025-12-04DOI: 10.1016/j.synbio.2025.11.005
Yu Qin , Jiaxiang Hu , Kezhen Su
Synthetic biology, as an emerging field that integrates life sciences and engineering technology, is driving profound transformations in global science, ethics, and legal systems. In international legal framework, the Biological Weapons Convention (BWC) and the Convention on Biological Diversity (CBD) have established initial hard law governance systems. However, these frameworks still face structural limitations in terms of technical adaptability, the scope of provisions, and institutional coordination. Soft law, with its flexibility, non-binding nature, and ability to build consensus, is increasingly becoming an essential supplement to the international response to the ethical risks of synthetic biology. International organizations, industry alliances, and non-governmental actors are constructing a multi-layered soft law governance network through ethical guidelines, policy recommendations, and codes of conduct, providing institutional support for risk identification, technology classification, and behavioral guidance. Soft law is well-suited to perform the roles of guiding and providing feedback in governance, while hard law should focus on the construction of systems of rights and responsibilities and the establishment of obligations. There is a collaborative governance model that integrates both soft and hard law. This model, characterized by “soft law guidance, hard law consolidation, and soft law feedback,” aims to create a flexible and enforceable governance framework. This approach ensures that soft law provides a timely and adaptive starting point, hard law offers a uniform and accountable foundation, and a feedback loop allows for continuous adjustment based on practical experience.
{"title":"Functions and optimization of soft law in the international governance of synthetic biology: The predicament of hard law vs. the rise of soft law","authors":"Yu Qin , Jiaxiang Hu , Kezhen Su","doi":"10.1016/j.synbio.2025.11.005","DOIUrl":"10.1016/j.synbio.2025.11.005","url":null,"abstract":"<div><div>Synthetic biology, as an emerging field that integrates life sciences and engineering technology, is driving profound transformations in global science, ethics, and legal systems. In international legal framework, the Biological Weapons Convention (BWC) and the Convention on Biological Diversity (CBD) have established initial hard law governance systems. However, these frameworks still face structural limitations in terms of technical adaptability, the scope of provisions, and institutional coordination. Soft law, with its flexibility, non-binding nature, and ability to build consensus, is increasingly becoming an essential supplement to the international response to the ethical risks of synthetic biology. International organizations, industry alliances, and non-governmental actors are constructing a multi-layered soft law governance network through ethical guidelines, policy recommendations, and codes of conduct, providing institutional support for risk identification, technology classification, and behavioral guidance. Soft law is well-suited to perform the roles of guiding and providing feedback in governance, while hard law should focus on the construction of systems of rights and responsibilities and the establishment of obligations. There is a collaborative governance model that integrates both soft and hard law. This model, characterized by “soft law guidance, hard law consolidation, and soft law feedback,” aims to create a flexible and enforceable governance framework. This approach ensures that soft law provides a timely and adaptive starting point, hard law offers a uniform and accountable foundation, and a feedback loop allows for continuous adjustment based on practical experience.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 134-151"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145690538","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 : 2026-06-01Epub Date: 2026-01-10DOI: 10.1016/j.synbio.2025.12.015
Zong-jie Wang , Haibo Zhou , Youming Zhang , Fu Yan , Liujie Huo , Xiaotong Wang
Marine microorganisms possess vast biosynthetic potential, yet most of their biosynthetic gene clusters (BGCs) remain transcriptionally silent under laboratory conditions. Genetic intractability has been a major barrier to activating these cryptic pathways. Here, we present RECC, an integrated Red/ET–CRISPR/Cas9 system that enables seamless, marker-free genome editing in marine bacteria. RECC couples Red/ET recombineering with CRISPR/Cas9-mediated cleavage, allowing the incorporation of homology arms and protospacers into a single construct through one-step Gibson assembly, thereby substantially simplifying the engineering process. Using Pseudoalteromonas flavipulchra DSM 14401 as a model, we employed RECC to replace the native promoter of a silent nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) hybrid gene cluster with a strong constitutive promoter. This targeted activation led to the production of a series of previously unknown cyclolipopeptides, designated flavipulchrins. Structural characterization and bioinformatic analysis revealed a plausible biosynthetic pathway for these metabolites. Collectively, RECC provides a robust and generalizable genome-editing platform that facilitates the systematic exploration of biosynthetic potential in genetically recalcitrant marine microorganisms.
{"title":"RECC: A Red/ET–CRISPR/Cas9-based system enabling genome mining of marine Pseudoalteromonas for novel natural products","authors":"Zong-jie Wang , Haibo Zhou , Youming Zhang , Fu Yan , Liujie Huo , Xiaotong Wang","doi":"10.1016/j.synbio.2025.12.015","DOIUrl":"10.1016/j.synbio.2025.12.015","url":null,"abstract":"<div><div>Marine microorganisms possess vast biosynthetic potential, yet most of their biosynthetic gene clusters (BGCs) remain transcriptionally silent under laboratory conditions. Genetic intractability has been a major barrier to activating these cryptic pathways. Here, we present RECC, an integrated <u>R</u>ed/<u>E</u>T–<u>C</u>RISPR/<u>C</u>as9 system that enables seamless, marker-free genome editing in marine bacteria. RECC couples Red/ET recombineering with CRISPR/Cas9-mediated cleavage, allowing the incorporation of homology arms and protospacers into a single construct through one-step Gibson assembly, thereby substantially simplifying the engineering process. Using <em>Pseudoalteromonas flavipulchra</em> DSM 14401 as a model, we employed RECC to replace the native promoter of a silent nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) hybrid gene cluster with a strong constitutive promoter. This targeted activation led to the production of a series of previously unknown cyclolipopeptides, designated flavipulchrins. Structural characterization and bioinformatic analysis revealed a plausible biosynthetic pathway for these metabolites. Collectively, RECC provides a robust and generalizable genome-editing platform that facilitates the systematic exploration of biosynthetic potential in genetically recalcitrant marine microorganisms.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 342-351"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145938534","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 : 2026-06-01Epub Date: 2026-01-15DOI: 10.1016/j.synbio.2026.01.004
Zhenqiang Zhao , Yizheng Liu , Rongshuai Zhu , Fengyu Yang , Zhifei Liu , Jiajia You , Xuewei Pan , Jianming Yang , Zhiming Rao
L-2-Aminobutyric acid (L-2-ABA) is a non-proteinogenic amino acid and an important chiral intermediate widely used in pharmaceuticals and fine chemicals. However, its fermentative production is limited by intermediate toxicity and imbalanced metabolic flux. In this study, Escherichia coli was systematically engineered for efficient de novo synthesis of L-2-ABA using a multi-layer metabolic engineering strategy. A quorum-sensing–based dynamic control circuit was introduced to decouple cell growth from 2-oxobutyric acid formation, thereby alleviating precursor toxicity and improving flux coordination. Combined with optimization of the L-2-ABA conversion pathway, model-guided carbon flux redistribution, cofactor regeneration, and tuning of global transcriptional regulation, a high-performance production strain was obtained without the need for antibiotics or inducers. The final engineered strain ABA40 achieved 45.3 g/L L-2-ABA with a yield of 0.31 g/g glucose in a 72 h fed-batch fermentation. This study demonstrates the effectiveness of dynamic and integrated metabolic engineering strategies for the biosynthesis of non-natural amino acids.
l -2-氨基丁酸（L-2-ABA）是一种非蛋白质原性氨基酸，是一种重要的手性中间体，广泛应用于医药和精细化工领域。然而，其发酵生产受到中间毒性和代谢通量不平衡的限制。在这项研究中，利用多层代谢工程策略，系统地改造大肠杆菌，使其高效地从头合成L-2-ABA。引入基于群体感应的动态控制电路，将细胞生长与2-氧丁酸形成解耦，从而减轻前体毒性并改善通量协调。通过对L-2-ABA转化途径的优化、模型引导的碳通量再分配、辅因子再生和全局转录调控的调整，获得了无需抗生素和诱导剂的高效生产菌株。最终工程菌株ABA40在补料批发酵72 h后，L-2- aba产量为45.3 g/L，葡萄糖产量为0.31 g/g。该研究证明了动态和综合代谢工程策略在非天然氨基酸生物合成中的有效性。
{"title":"De novo synthesis of L-2-aminobutyric acid in Escherichia coli based on multi-layered metabolic engineering strategies","authors":"Zhenqiang Zhao , Yizheng Liu , Rongshuai Zhu , Fengyu Yang , Zhifei Liu , Jiajia You , Xuewei Pan , Jianming Yang , Zhiming Rao","doi":"10.1016/j.synbio.2026.01.004","DOIUrl":"10.1016/j.synbio.2026.01.004","url":null,"abstract":"<div><div>L-2-Aminobutyric acid (L-2-ABA) is a non-proteinogenic amino acid and an important chiral intermediate widely used in pharmaceuticals and fine chemicals. However, its fermentative production is limited by intermediate toxicity and imbalanced metabolic flux. In this study, <em>Escherichia coli</em> was systematically engineered for efficient <em>de novo</em> synthesis of L-2-ABA using a multi-layer metabolic engineering strategy. A quorum-sensing–based dynamic control circuit was introduced to decouple cell growth from 2-oxobutyric acid formation, thereby alleviating precursor toxicity and improving flux coordination. Combined with optimization of the L-2-ABA conversion pathway, model-guided carbon flux redistribution, cofactor regeneration, and tuning of global transcriptional regulation, a high-performance production strain was obtained without the need for antibiotics or inducers. The final engineered strain ABA40 achieved 45.3 g/L L-2-ABA with a yield of 0.31 g/g glucose in a 72 h fed-batch fermentation. This study demonstrates the effectiveness of dynamic and integrated metabolic engineering strategies for the biosynthesis of non-natural amino acids.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 374-382"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145975970","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 : 2026-06-01Epub Date: 2026-01-17DOI: 10.1016/j.synbio.2025.12.014
Weiwei Bao , Qiqun Peng , Hongxiang Yu , Hongwei Yang , Shihui Yang
Acetoin, a valuable platform chemical, faces sustainability challenges in its traditional energy-intensive synthesis. Microbial fermentation using microorganisms such as Zymomonas mobilis offers a promising alternative. To overcome metabolic limitations and process inefficiencies of economic acetoin production, we integrated strategies of metabolic engineering, transcriptomic-guided analysis, flocculation-based cell recycling, and non-food feedstock utilization. A dominant metabolism compromised intermediate (DMCI) chassis of Z. mobilis was constructed by deleting ethanol production and acetoin degradation pathways. Transcriptomics was then employed to identify and knockout latent competing pathway genes of ZMO0318 and ZMO1576 to enhance acetoin production. The engineered strain also tolerated to inhibitors in lignocellulosic hydrolysates, and fed-batch fermentation achieved an acetoin titer of 73 g/L. Furthermore, self-flocculating phenotype was engineered via ZMO1082 modification to enable efficient cell recycling over multiple batches for production cost reduction. This study thus establishes a synergistic strategy to enhance acetoin production, highlighting the role of combining metabolic engineering, omics analyses, and processing engineering for economic biochemical production.
{"title":"A synergistic strategy of metabolic engineering and flocculation recycling for enhanced acetoin production in Zymomonas mobilis","authors":"Weiwei Bao , Qiqun Peng , Hongxiang Yu , Hongwei Yang , Shihui Yang","doi":"10.1016/j.synbio.2025.12.014","DOIUrl":"10.1016/j.synbio.2025.12.014","url":null,"abstract":"<div><div>Acetoin, a valuable platform chemical, faces sustainability challenges in its traditional energy-intensive synthesis. Microbial fermentation using microorganisms such as <em>Zymomonas mobilis</em> offers a promising alternative. To overcome metabolic limitations and process inefficiencies of economic acetoin production, we integrated strategies of metabolic engineering, transcriptomic-guided analysis, flocculation-based cell recycling, and non-food feedstock utilization. A dominant metabolism compromised intermediate (DMCI) chassis of <em>Z. mobilis</em> was constructed by deleting ethanol production and acetoin degradation pathways. Transcriptomics was then employed to identify and knockout latent competing pathway genes of <em>ZMO0318</em> and <em>ZMO1576</em> to enhance acetoin production. The engineered strain also tolerated to inhibitors in lignocellulosic hydrolysates, and fed-batch fermentation achieved an acetoin titer of 73 g/L. Furthermore, self-flocculating phenotype was engineered via <em>ZMO1082</em> modification to enable efficient cell recycling over multiple batches for production cost reduction. This study thus establishes a synergistic strategy to enhance acetoin production, highlighting the role of combining metabolic engineering, omics analyses, and processing engineering for economic biochemical production.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 401-410"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145975888","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 : 2026-06-01Epub Date: 2026-01-02DOI: 10.1016/j.synbio.2025.12.004
Yucheng Hu , Jinde Chen , Shaofang Tian , Yang Zhang , Zhiqian Zhang , Ao Jiang , Yi-Rui Wu , Baoshun Zhang
Poor aqueous solubility of steroid precursors, such as pregnenolone and progesterone, limits microbial biotransformation and high-throughput strain screening, representing a bottleneck for strain improvement and potential industrial applications.
To address this, we developed a growth-coupled progesterone-responsive biosensor in Saccharomyces cerevisiae, integrated with a hydroxypropyl-β-cyclodextrin (HP-β-CD) system to enhance intracellular steroid availability. The biosensor links progesterone formation to cell growth and fluorescence, with selection stringency finely tuned via an IPTG-inducible lac operator and 3-aminotriazole (3-AT) to suppress low-producing cells. Coupled with atmospheric and room temperature plasma (ARTP) mutagenesis, the growth-coupled biosensor–FADS platform identified five yeast variants capable of improved conversion of pregnenolone to progesterone while expressing 3β-hydroxysteroid dehydrogenase (3β-HSD) without altering the enzyme itself. The progesterone production of these selected variants was subsequently validated using 1 mM pregnenolone as the substrate, showing 2.0–3.37-fold higher titers than the wild-type strain, demonstrating proof-of-concept. Microfluidic droplet encapsulation allowed clear separation of high-producers, highlighting the platform's selectivity, robustness, and scalability. This synthetic biology–driven system integration platform provides a practical, modular, and high-throughput strategy for screening poorly water-soluble steroid-producing yeast. It is adaptable to other bioactive molecules, can support future enzyme evolution, and demonstrates potential for broader biotechnological applications.
{"title":"A growth-coupled progesterone-responsive biosensor for high-throughput microfluidic screening in Saccharomyces cerevisiae","authors":"Yucheng Hu , Jinde Chen , Shaofang Tian , Yang Zhang , Zhiqian Zhang , Ao Jiang , Yi-Rui Wu , Baoshun Zhang","doi":"10.1016/j.synbio.2025.12.004","DOIUrl":"10.1016/j.synbio.2025.12.004","url":null,"abstract":"<div><div>Poor aqueous solubility of steroid precursors, such as pregnenolone and progesterone, limits microbial biotransformation and high-throughput strain screening, representing a bottleneck for strain improvement and potential industrial applications.</div><div>To address this, we developed a growth-coupled progesterone-responsive biosensor in <em>Saccharomyces cerevisiae</em>, integrated with a hydroxypropyl-β-cyclodextrin (HP-β-CD) system to enhance intracellular steroid availability. The biosensor links progesterone formation to cell growth and fluorescence, with selection stringency finely tuned via an IPTG-inducible lac operator and 3-aminotriazole (3-AT) to suppress low-producing cells. Coupled with atmospheric and room temperature plasma (ARTP) mutagenesis, the growth-coupled biosensor–FADS platform identified five yeast variants capable of improved conversion of pregnenolone to progesterone while expressing 3β-hydroxysteroid dehydrogenase (3β-HSD) without altering the enzyme itself. The progesterone production of these selected variants was subsequently validated using 1 mM pregnenolone as the substrate, showing 2.0–3.37-fold higher titers than the wild-type strain, demonstrating proof-of-concept. Microfluidic droplet encapsulation allowed clear separation of high-producers, highlighting the platform's selectivity, robustness, and scalability. This synthetic biology–driven system integration platform provides a practical, modular, and high-throughput strategy for screening poorly water-soluble steroid-producing yeast. It is adaptable to other bioactive molecules, can support future enzyme evolution, and demonstrates potential for broader biotechnological applications.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 301-311"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883696","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 : 2026-06-01Epub Date: 2025-11-18DOI: 10.1016/j.synbio.2025.11.004
Ya Wu , Chonghao Guo , Lizhen Deng , Derui Zhang , Yutong Bie , Yuxin He , Gen Lu , Shewei Hu , Ruiqi Zeng , Zeyang Li , Xudong Xu , Longjiang Yu
Serinol (2-amino-1,3-propanediol) is an important pharmaceutical intermediate, but conventional chemical or microbial routes are hampered by high energy demand, product toxicity, or complex regulation. Here, we report a modular cell-free enzyme cascade, termed the methanol-to-serinol pathway (MSP), that efficiently converts methanol—a low-cost C1 feedstock—into serinol with high carbon yield. The cascade comprises two modules: Module 1 employs an alcohol oxidase and an engineered formolase to generate dihydroxyacetone (DHA), while Module 2 uses a tailored ω-transaminase for direct one-step amination. To overcome the rate-limiting DHA amination, we applied an “ALF” scanning strategy and identified a triple-mutant Cv-ωTA (Y153F/Y168F/C418F) with 6.3-fold higher specific activity than the wild type. Fitness landscape analysis revealed strong non-additive interactions, highlighting the synergistic effect of these three mutations. Molecular dynamics simulations revealed structural changes underlying the activity boost. By incorporating a pyruvate-removal system to drive the equilibrium toward product formation, the integrated cascade achieved 43.86 mM (4 g/L) serinol from 150 mM methanol in 7 h, corresponding to 87.7 % carbon yield and a productivity of 0.57 g/L/h. This work establishes a carbon-efficient route for serinol biosynthesis and provides a generalizable strategy for sustainable C1 biomanufacturing.
丝氨酸醇（2-氨基-1,3-丙二醇）是一种重要的医药中间体，但传统的化学或微生物途径受到高能量需求、产品毒性或复杂调控的阻碍。在这里，我们报道了一个模块化的无细胞酶级联，称为甲醇-丝氨酸醇途径（MSP），它有效地将甲醇（低成本的C1原料）转化为高碳产量的丝氨酸醇。该级联包括两个模块：模块1使用酒精氧化酶和工程甲酰基酶生成二羟基丙酮（DHA），而模块2使用定制的ω-转氨酶进行直接一步胺化。为了克服DHA胺化的限速，我们采用了“ALF”扫描策略，鉴定出了一个比野生型高6.3倍的三突变Cv-ωTA （Y153F/Y168F/C418F）。适应度景观分析显示，这3个突变具有较强的非加性相互作用，突出了协同效应。分子动力学模拟揭示了活动增强背后的结构变化。通过加入丙酮酸脱除系统来驱动平衡生成产物，集成级联在7小时内从150 mM甲醇中获得43.86 mM （4 g/L）丝氨酸醇，对应的碳收率为87.7%，生产率为0.57 g/L/h。这项工作建立了丝氨酸醇生物合成的碳高效途径，并为可持续的C1生物制造提供了一种可推广的策略。
{"title":"Engineering ω-transaminase for efficient dihydroxyacetone transamination in serinol biosynthesis starting from methanol","authors":"Ya Wu , Chonghao Guo , Lizhen Deng , Derui Zhang , Yutong Bie , Yuxin He , Gen Lu , Shewei Hu , Ruiqi Zeng , Zeyang Li , Xudong Xu , Longjiang Yu","doi":"10.1016/j.synbio.2025.11.004","DOIUrl":"10.1016/j.synbio.2025.11.004","url":null,"abstract":"<div><div>Serinol (2-amino-1,3-propanediol) is an important pharmaceutical intermediate, but conventional chemical or microbial routes are hampered by high energy demand, product toxicity, or complex regulation. Here, we report a modular cell-free enzyme cascade, termed the methanol-to-serinol pathway (MSP), that efficiently converts methanol—a low-cost C1 feedstock—into serinol with high carbon yield. The cascade comprises two modules: Module 1 employs an alcohol oxidase and an engineered formolase to generate dihydroxyacetone (DHA), while Module 2 uses a tailored ω-transaminase for direct one-step amination. To overcome the rate-limiting DHA amination, we applied an “ALF” scanning strategy and identified a triple-mutant Cv-ωTA (Y153F/Y168F/C418F) with 6.3-fold higher specific activity than the wild type. Fitness landscape analysis revealed strong non-additive interactions, highlighting the synergistic effect of these three mutations. Molecular dynamics simulations revealed structural changes underlying the activity boost. By incorporating a pyruvate-removal system to drive the equilibrium toward product formation, the integrated cascade achieved 43.86 mM (4 g/L) serinol from 150 mM methanol in 7 h, corresponding to 87.7 % carbon yield and a productivity of 0.57 g/L/h. This work establishes a carbon-efficient route for serinol biosynthesis and provides a generalizable strategy for sustainable C1 biomanufacturing.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 71-81"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145577445","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 : 2026-06-01Epub Date: 2025-11-11DOI: 10.1016/j.synbio.2025.10.016
Hongyang Chen , Liqiu Su , Zhen Yao , Kaizhi Jia , Zongjie Dai , Qinhong Wang
β-farnesene, a natural sesquiterpene compound, has gained significant attention due to its versatile applications in agriculture, industry, biofuels, and related fields. Microbial biosynthesis offers an environmentally sustainable approach for its commercial-scale production. In order to enhance its production efficiency, further exploration of key rate-limiting steps is required. Here, through directed evolution of the essential β-farnesene synthase, we obtained an optimal variant (AaFST196A/M356T/E380G), demonstrating 2.29-fold enhancement in β-farnesene titer relative to wild-type. Structural elucidation revealed that the distal mutations mediate allosteric modulation of the catalytic core significantly improving the conversion efficiency of farnesyl diphosphate (FPP) to β-farnesene. Then comprehensive pathway engineering, including the mevalonate pathway amplification, acetyl-CoA precursor enhancement, competitive pathway elimination, and auxotrophic restoration, were carried out in Yarrowia lipolytica, resulting in a high-performance strain FS18 capable of producing 3.41 g/L β-farnesene in shake-flask cultures. Notably, scale up fermentation in 5 L bioreactors yielded a titer of 45.69 g/L, the highest concentration reported in Y. lipolytica to date. This study provided mechanistic insights into terpene synthase engineering and a practical framework for high-level terpenoid biosynthesis in Y. lipolytica.
{"title":"Combinatorial engineering of enzyme and pathway for efficient β-farnesene bioproduction in Yarrowia lipolytica","authors":"Hongyang Chen , Liqiu Su , Zhen Yao , Kaizhi Jia , Zongjie Dai , Qinhong Wang","doi":"10.1016/j.synbio.2025.10.016","DOIUrl":"10.1016/j.synbio.2025.10.016","url":null,"abstract":"<div><div>β-farnesene, a natural sesquiterpene compound, has gained significant attention due to its versatile applications in agriculture, industry, biofuels, and related fields. Microbial biosynthesis offers an environmentally sustainable approach for its commercial-scale production. In order to enhance its production efficiency, further exploration of key rate-limiting steps is required. Here, through directed evolution of the essential β-farnesene synthase, we obtained an optimal variant (AaFS<sup>T196A/M356T/E380G</sup>), demonstrating 2.29-fold enhancement in β-farnesene titer relative to wild-type. Structural elucidation revealed that the distal mutations mediate allosteric modulation of the catalytic core significantly improving the conversion efficiency of farnesyl diphosphate (FPP) to β-farnesene. Then comprehensive pathway engineering, including the mevalonate pathway amplification, acetyl-CoA precursor enhancement, competitive pathway elimination, and auxotrophic restoration, were carried out in <em>Yarrowia lipolytica</em>, resulting in a high-performance strain FS18 capable of producing 3.41 g/L β-farnesene in shake-flask cultures. Notably, scale up fermentation in 5 L bioreactors yielded a titer of 45.69 g/L, the highest concentration reported in <em>Y</em>. <em>lipolytica</em> to date. This study provided mechanistic insights into terpene synthase engineering and a practical framework for high-level terpenoid biosynthesis in <em>Y. lipolytica</em>.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 32-41"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145527392","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 : 2026-06-01Epub Date: 2025-11-13DOI: 10.1016/j.synbio.2025.11.003
Wen Tian , Songcheng Yu , Kaiyang Zhang , Tao Liu , Lihua Ding , Peng Zhang
The CRISPR/Cas12a system holds significant promise for biomedical applications. Nevertheless, the commonly used reporter, fluorophore–quencher-labeled substrates, is hindered by labor-intensive synthesis procedures and high costs, while also relying on a single-photon method and being vulnerable to environmental interference. Herein, a label-free dual-fluorescent functional nucleic acid (DFFNA) was engineered, comprising an aptamer domain for auramine O (AO) recognition and a dSpacer-integrated DNA duplex region for 5,6,7-trimethyl-1,8-naphthyridin-2-amine (ATMND) binding. The fluorescence of AO and ATMND can be enhanced and quenched, respectively, when bound to DFFNAs. The fluorescence intensity ratio between ATMND and AO increased significantly following the cleavage of DFFNAs by activated Cas12a, thus offering a universal, label-free, ratiometric fluorescent reporter for the CRISPR/Cas12a system. To explore the application of the DFFNA-based CRISPR/Cas12a system, a novel biosensor was developed to detect site-specific DNA methylation. It employs a methylation-sensitive restriction enzyme to recognize methylation sites, Cas12a for site-specific DNA identification and signal amplification, and DFFNAs to produce ratiometric fluorescence. The assay demonstrated remarkable specificity and sensitivity, with a limit of detection of 152 pM, due to the high resolution and trans-cleavage activity of Cas12a. The rationally designed and label-free DFFNAs enhance stability, increase flexibility, and reduce cost. The observable color change and smartphone imaging capability facilitate portable, point-of-care testing. Specifically, the biosensor demonstrated excellent specificity by differentiating colorectal cancer patients from healthy individuals. Consequently, this work presents a superior label-free and ratiometric fluorescent reporter for the CRISPR/Cas12a system, which offers a promising strategy for DNA methylation detection in clinical settings.
CRISPR/Cas12a系统在生物医学应用方面具有重大前景。然而，常用的报告材料，荧光团猝灭剂标记的衬底，受到劳动密集型合成程序和高成本的阻碍，同时也依赖于单光子方法，容易受到环境干扰。本文构建了一种无标记的双荧光功能核酸（DFFNA），包括一个用于auramine O （AO）识别的适配体结构域和一个用于5,6,7-三甲基-1,8-萘啶-2-胺（ATMND）结合的dspacer -整合DNA双链区域。AO和ATMND与DFFNAs结合后，其荧光分别增强和减弱。活化的Cas12a切割dffna后，ATMND和AO之间的荧光强度比显著增加，从而为CRISPR/Cas12a系统提供了一种通用的、无标记的比例荧光报告基因。为了探索基于dffna的CRISPR/Cas12a系统的应用，开发了一种新型生物传感器来检测位点特异性DNA甲基化。它使用甲基化敏感的限制性内切酶来识别甲基化位点，使用Cas12a进行位点特异性DNA鉴定和信号扩增，使用dffna产生比例荧光。由于Cas12a的高分辨率和反式裂解活性，该方法具有显著的特异性和敏感性，检测限为152 pM。合理设计和无标签的dffna提高了稳定性，增加了灵活性，降低了成本。可观察的颜色变化和智能手机成像功能便于便携式，即时检测。具体来说，该生物传感器在区分结直肠癌患者和健康个体方面表现出了出色的特异性。因此，这项工作为CRISPR/Cas12a系统提供了一种优越的无标记和比例荧光报告，为临床环境中的DNA甲基化检测提供了一种有前途的策略。
{"title":"Engineered dual-fluorescence functional nucleic acid-based CRISPR/Cas12a biosensor for label-free ratiometric detection of site-specific DNA methylation","authors":"Wen Tian , Songcheng Yu , Kaiyang Zhang , Tao Liu , Lihua Ding , Peng Zhang","doi":"10.1016/j.synbio.2025.11.003","DOIUrl":"10.1016/j.synbio.2025.11.003","url":null,"abstract":"<div><div>The CRISPR/Cas12a system holds significant promise for biomedical applications. Nevertheless, the commonly used reporter, fluorophore–quencher-labeled substrates, is hindered by labor-intensive synthesis procedures and high costs, while also relying on a single-photon method and being vulnerable to environmental interference. Herein, a label-free dual-fluorescent functional nucleic acid (DFFNA) was engineered, comprising an aptamer domain for auramine O (AO) recognition and a dSpacer-integrated DNA duplex region for 5,6,7-trimethyl-1,8-naphthyridin-2-amine (ATMND) binding. The fluorescence of AO and ATMND can be enhanced and quenched, respectively, when bound to DFFNAs. The fluorescence intensity ratio between ATMND and AO increased significantly following the cleavage of DFFNAs by activated Cas12a, thus offering a universal, label-free, ratiometric fluorescent reporter for the CRISPR/Cas12a system. To explore the application of the DFFNA-based CRISPR/Cas12a system, a novel biosensor was developed to detect site-specific DNA methylation. It employs a methylation-sensitive restriction enzyme to recognize methylation sites, Cas12a for site-specific DNA identification and signal amplification, and DFFNAs to produce ratiometric fluorescence. The assay demonstrated remarkable specificity and sensitivity, with a limit of detection of 152 pM, due to the high resolution and <em>trans</em>-cleavage activity of Cas12a. The rationally designed and label-free DFFNAs enhance stability, increase flexibility, and reduce cost. The observable color change and smartphone imaging capability facilitate portable, point-of-care testing. Specifically, the biosensor demonstrated excellent specificity by differentiating colorectal cancer patients from healthy individuals. Consequently, this work presents a superior label-free and ratiometric fluorescent reporter for the CRISPR/Cas12a system, which offers a promising strategy for DNA methylation detection in clinical settings.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 52-58"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145527393","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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