Pub Date : 2026-06-01Epub Date: 2025-12-31DOI: 10.1016/j.synbio.2025.12.006
Yunyue Chen , Siyifei Wang , Leiying Xie , Luhao Zhang , Min Zhu , Yingke Xu
Precise regulation of protein abundance is essential for understanding dynamic cellular processes and for advancing therapeutic development. However, existing approaches lack the spatiotemporal resolution required to these cellular processes. Recent advances in optogenetics have enabled the design of optogenetic targeted protein degradation systems (Opto-TPD) allowing reversible and non-invasive control of protein stability with high spatiotemporal precision. In this review, we systematically summarize the design principles of Opto-TPD tools, including those based on light-oxygen-voltage (LOV)-domain conformational systems, light-inducible dimerization systems, and light-controlled degradation tool expression systems. We further highlight their applications in probing protein function, modulating signaling pathways, and therapeutic translations. By comparing the mechanistic features, performance, and limitations of each platform, we aim to provide a comprehensive resource for guiding future tool optimization. Altogether, these Opto-TPD tools represent a powerful and versatile complement to existing protein manipulation technologies, expanding the toolbox for precise control of protein homeostasis in living systems.
{"title":"Design principles for optogenetic-based targeted protein degradation","authors":"Yunyue Chen , Siyifei Wang , Leiying Xie , Luhao Zhang , Min Zhu , Yingke Xu","doi":"10.1016/j.synbio.2025.12.006","DOIUrl":"10.1016/j.synbio.2025.12.006","url":null,"abstract":"<div><div>Precise regulation of protein abundance is essential for understanding dynamic cellular processes and for advancing therapeutic development. However, existing approaches lack the spatiotemporal resolution required to these cellular processes. Recent advances in optogenetics have enabled the design of optogenetic targeted protein degradation systems (Opto-TPD) allowing reversible and non-invasive control of protein stability with high spatiotemporal precision. In this review, we systematically summarize the design principles of Opto-TPD tools, including those based on light-oxygen-voltage (LOV)-domain conformational systems, light-inducible dimerization systems, and light-controlled degradation tool expression systems. We further highlight their applications in probing protein function, modulating signaling pathways, and therapeutic translations. By comparing the mechanistic features, performance, and limitations of each platform, we aim to provide a comprehensive resource for guiding future tool optimization. Altogether, these Opto-TPD tools represent a powerful and versatile complement to existing protein manipulation technologies, expanding the toolbox for precise control of protein homeostasis in living systems.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 255-264"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883629","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.2025.12.008
Rui Liu , Lu-Wei Wang , Zi-Han Gao , Xiao-Tong Sun , Shu-Ran Lv , Huan Liu , Sa-ouk Kang , Bo Sun
Escherichia coli (E. coli) has long served as a versatile workhorse for recombinant protein production. As synthetic biology expands the demand for coordinated expression of multiple genes, co-expression systems in E. coli have evolved from basic dual-gene constructs to programmable, polygenic expression platforms. This review critically examines the major strategies enabling multigene co-expression in E. coli, including internal ribosome entry sites (IRES), 2A self-cleaving peptides, dual-promoter cassettes, multicistronic operons, and multi-plasmid configurations. We highlight the mechanistic principles, design trade-offs, and regulatory bottlenecks associated with each approach, such as translational imbalance, inclusion body formation, and plasmid compatibility. Real-world applications in metabolic engineering, complex protein assembly, and biomanufacturing are analyzed to demonstrate the functional advantages of these systems. Finally, we explore emerging programmable toolkits that integrate modular architecture, expression modeling, and AI-assisted design, paving the way for next-generation synthetic expression control in microbial chassis. This review offers a comprehensive and strategic roadmap for researchers engineering multi-gene systems in E. coli and beyond.
{"title":"Multi-gene Co-expression systems in E. coli: From single-vector designs to programmable expression platforms","authors":"Rui Liu , Lu-Wei Wang , Zi-Han Gao , Xiao-Tong Sun , Shu-Ran Lv , Huan Liu , Sa-ouk Kang , Bo Sun","doi":"10.1016/j.synbio.2025.12.008","DOIUrl":"10.1016/j.synbio.2025.12.008","url":null,"abstract":"<div><div><em>Escherichia coli</em> (<em>E. coli</em>) has long served as a versatile workhorse for recombinant protein production. As synthetic biology expands the demand for coordinated expression of multiple genes, co-expression systems in <em>E. coli</em> have evolved from basic dual-gene constructs to programmable, polygenic expression platforms. This review critically examines the major strategies enabling multigene co-expression in <em>E. coli,</em> including internal ribosome entry sites (IRES), 2A self-cleaving peptides, dual-promoter cassettes, multicistronic operons, and multi-plasmid configurations. We highlight the mechanistic principles, design trade-offs, and regulatory bottlenecks associated with each approach, such as translational imbalance, inclusion body formation, and plasmid compatibility. Real-world applications in metabolic engineering, complex protein assembly, and biomanufacturing are analyzed to demonstrate the functional advantages of these systems. Finally, we explore emerging programmable toolkits that integrate modular architecture, expression modeling, and AI-assisted design, paving the way for next-generation synthetic expression control in microbial chassis. This review offers a comprehensive and strategic roadmap for researchers engineering multi-gene systems in <em>E. coli</em> and beyond.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 330-341"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145938533","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: 2025-10-24DOI: 10.1016/j.synbio.2025.10.009
Heng Zhang , Fuqiang Song , Ke Wang , Faqing Wu , Lihao Deng , Kun Qiu , Jingwen Zhou
l-Isoleucine (L-Ile), a critical branched-chain amino acid with diverse applications in food, pharmaceutical, and cosmetic industries, is difficult to produce efficiently at scale in microbial systems due to metabolic bottlenecks and cofactor limitations. This study metabolically engineered Escherichia coli BL21(DE3) to develop a whole-cell biocatalyst for efficient L-Ile biosynthesis. Key strategies included screening acetohydroxy acid synthase (AHAS) isoenzymes, identifying ilvGM-encoded AHAS II as the optimal enzyme, relieving feedback inhibition of ilvA (encoding l-threonine dehydratase) through mutant screening, and optimizing genetic circuits (promoter tuning, plasmid copy number). Dual-precursor supplementation revealed l-threonine as a critical factor for suppressing l-valine byproduct. Fed-batch fermentation in a 5 L bioreactor achieved a peak molar conversion rate of 98.4 %, yielding 40.1 g/L L-Ile within 36 h. The mass conversion rate (L-Ile/glucose) achieved 0.36 g/g and the production efficiency achieved 1.11 g/L/h, demonstrating the feasibility of whole-cell catalysis. This work provides a robust framework for industrial L-Ile production and transferable strategies for branched-chain amino acid pathway optimization.
l-异亮氨酸（L-Ile）是一种重要的支链氨基酸，在食品、制药和化妆品行业有着广泛的应用，但由于代谢瓶颈和辅助因素的限制，在微生物系统中难以大规模高效地生产。本研究对大肠杆菌BL21（DE3）进行代谢工程，以开发一种高效的L-Ile生物合成的全细胞生物催化剂。关键策略包括筛选乙酰羟基酸合成酶（AHAS）同工酶，确定ilvgm编码的AHAS II为最佳酶，通过突变体筛选缓解ilvA（编码l-苏氨酸脱水酶）的反馈抑制，以及优化遗传回路（启动子调谐，质粒拷贝数）。双前体补充表明l-苏氨酸是抑制l-缬氨酸副产物的关键因素。在5 L的生物反应器中分批补料发酵，峰值摩尔转化率达到98.4%，36 h内L- ile转化率达到40.1 g/L，质量转化率（L- ile /glucose）达到0.36 g/g，生产效率达到1.11 g/L/h，证明了全细胞催化的可行性。这项工作为工业L-Ile生产和支链氨基酸途径优化的可转移策略提供了一个强大的框架。
{"title":"Precise l-threonine-to-l-isoleucine pathway regulation for engineering high-efficiency whole-cell biocatalysts","authors":"Heng Zhang , Fuqiang Song , Ke Wang , Faqing Wu , Lihao Deng , Kun Qiu , Jingwen Zhou","doi":"10.1016/j.synbio.2025.10.009","DOIUrl":"10.1016/j.synbio.2025.10.009","url":null,"abstract":"<div><div><span>l</span>-Isoleucine (L-Ile), a critical branched-chain amino acid with diverse applications in food, pharmaceutical, and cosmetic industries, is difficult to produce efficiently at scale in microbial systems due to metabolic bottlenecks and cofactor limitations. This study metabolically engineered <em>Escherichia coli</em> BL21(DE3) to develop a whole-cell biocatalyst for efficient L-Ile biosynthesis. Key strategies included screening acetohydroxy acid synthase (AHAS) isoenzymes, identifying <em>ilvGM</em>-encoded AHAS II as the optimal enzyme, relieving feedback inhibition of <em>ilvA</em> (encoding <span>l</span>-threonine dehydratase) through mutant screening, and optimizing genetic circuits (promoter tuning, plasmid copy number). Dual-precursor supplementation revealed <span>l</span>-threonine as a critical factor for suppressing <span>l</span>-valine byproduct. Fed-batch fermentation in a 5 L bioreactor achieved a peak molar conversion rate of 98.4 %, yielding 40.1 g/L L-Ile within 36 h. The mass conversion rate (L-Ile/glucose) achieved 0.36 g/g and the production efficiency achieved 1.11 g/L/h, demonstrating the feasibility of whole-cell catalysis. This work provides a robust framework for industrial L-Ile production and transferable strategies for branched-chain amino acid pathway optimization.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 1-9"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145449296","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: 2025-11-27DOI: 10.1016/j.synbio.2025.11.007
Shaowei Li , Jinghui Wang , Yaoyao Zhang , Kaixin Du , Jiangnan Chen , Jianping Sun , Huan Wang , Pengfei Ouyang , Xuanming Xu , Fuqing Wu , Fang Yang , Guo-Qiang Chen
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a 0–30 mol% controllable range of 3HV ratios was produced by Halomonas bluephagenesis (H. bluephagenesis) and characterized. An endogenous plasmid containing scpA and scpB encoding methylmalonyl-CoA epimerase and methylmalonyl-CoA decarboxylase, respectively, redirects succinyl-CoA toward propionyl-CoA, enabling de novo PHBV synthesis with a 1.7 mol% 3HV. Deletion of sdhE and prpC encoding succinate dehydrogenase and 2-methylcitrate synthase, respectively, further enhanced the 3HV to 4 mol%. H. bluephagenesis GZ05 was engineered for late-phase-specific MreB (a cytoskeletal protein) degradation, enlarged intracellular PHBV granules for enhanced PHBV synthesis, and convenient downstream. A series of growth experiments was conducted in a 7 L bioreactor fed with valerate to produce PHBV with various 3HV molar ratios (2–27 mol%). A quantitative relationship between valerate concentration and the final 3HV molar ratio was established with an R2 = 0.9833, enabling precise control of the 3HV ratio in PHBV. H. bluephagenesis GZ05 was grown to 100 g L−1 cell dry weight (CDW) containing 73 wt% PHBV consisting of 1.6 mol% 3HV in a 5000 L bioreactor. Thermal analysis demonstrated enhanced flexibility with higher 3HV content in PHBV.
{"title":"Synthesis of tunable copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate by engineered Halomonas bluephagenesis and their characterizations","authors":"Shaowei Li , Jinghui Wang , Yaoyao Zhang , Kaixin Du , Jiangnan Chen , Jianping Sun , Huan Wang , Pengfei Ouyang , Xuanming Xu , Fuqing Wu , Fang Yang , Guo-Qiang Chen","doi":"10.1016/j.synbio.2025.11.007","DOIUrl":"10.1016/j.synbio.2025.11.007","url":null,"abstract":"<div><div>Poly(3-hydroxybutyrate-<em>co</em>-3-hydroxyvalerate) (PHBV) with a 0–30 mol% controllable range of 3HV ratios was produced by <em>Halomonas bluephagenesis</em> (<em>H. bluephagenesis</em>) and characterized. An endogenous plasmid containing <em>scpA</em> and <em>scpB</em> encoding methylmalonyl-CoA epimerase and methylmalonyl-CoA decarboxylase, respectively, redirects succinyl-CoA toward propionyl-CoA, enabling <em>de novo</em> PHBV synthesis with a 1.7 mol% 3HV. Deletion of <em>sdhE</em> and <em>prpC</em> encoding succinate dehydrogenase and 2-methylcitrate synthase, respectively, further enhanced the 3HV to 4 mol%. <em>H. bluephagenesis</em> GZ05 was engineered for late-phase-specific MreB (a cytoskeletal protein) degradation, enlarged intracellular PHBV granules for enhanced PHBV synthesis, and convenient downstream. A series of growth experiments was conducted in a 7 L bioreactor fed with valerate to produce PHBV with various 3HV molar ratios (2–27 mol%). A quantitative relationship between valerate concentration and the final 3HV molar ratio was established with an R<sup>2</sup> = 0.9833, enabling precise control of the 3HV ratio in PHBV. <em>H. bluephagenesis</em> GZ05 was grown to 100 g L<sup>−1</sup> cell dry weight (CDW) containing 73 wt% PHBV consisting of 1.6 mol% 3HV in a 5000 L bioreactor. Thermal analysis demonstrated enhanced flexibility with higher 3HV content in PHBV.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 91-100"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145621007","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.2025.10.014
Yachao Xin , Jingping Du , Weiqiang Zhang , Haoran Bi , Limin Ba , Kai Wang , Yanhui Liu
Longifolene is a sesquiterpene commonly found in the heavy turpentine oil of pine plants, with various applications ranging from pest control and fragrance production to synthetic biofuels. While S. cerevisiae cell factories can effectively accumulate longifolene, further optimization and refinement of the metabolic modifications are still needed to improve the yield and conversion efficiency of longifolene production. In this study, we first explored enzyme fusion technology to enhance the efficiency of longifolene synthase catalysis, and improved acetyl-CoA availability by adjusting the pyruvate bypass pathway and introducing the synthetic chimeric citrate lyase pathway. The introduction of the formate dehydrogenase module was also used to supplement reducing power. By combining these strategies, the yield of longifolene reached 78.637 mg/L in shake flasks and 2063.7 mg/L in a 5 L bioreactor through fed-batch cultivation. This is the highest reported yield of longifolene to date. This study has important fundamental significance for the construction of biosynthetic factories for longifolene and other terpenes.
{"title":"Combining multiple metabolic strategies for efficient production of longifolene in Saccharomyces cerevisiae","authors":"Yachao Xin , Jingping Du , Weiqiang Zhang , Haoran Bi , Limin Ba , Kai Wang , Yanhui Liu","doi":"10.1016/j.synbio.2025.10.014","DOIUrl":"10.1016/j.synbio.2025.10.014","url":null,"abstract":"<div><div>Longifolene is a sesquiterpene commonly found in the heavy turpentine oil of pine plants, with various applications ranging from pest control and fragrance production to synthetic biofuels. While <em>S. cerevisiae</em> cell factories can effectively accumulate longifolene, further optimization and refinement of the metabolic modifications are still needed to improve the yield and conversion efficiency of longifolene production. In this study, we first explored enzyme fusion technology to enhance the efficiency of longifolene synthase catalysis, and improved acetyl-CoA availability by adjusting the pyruvate bypass pathway and introducing the synthetic chimeric citrate lyase pathway. The introduction of the formate dehydrogenase module was also used to supplement reducing power. By combining these strategies, the yield of longifolene reached 78.637 mg/L in shake flasks and 2063.7 mg/L in a 5 L bioreactor through fed-batch cultivation. This is the highest reported yield of longifolene to date. This study has important fundamental significance for the construction of biosynthetic factories for longifolene and other terpenes.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 82-90"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145577446","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: 2025-11-08DOI: 10.1016/j.synbio.2025.10.013
Ling Qin , Shoujie He , Dan Yuan, Yuyang Pan, Zhibo Yan, Mingtao Huang
Acetyl-CoA is a central metabolic intermediate that serves as a key precursor for the biosynthesis of high-value compounds such as terpenoids. However, its compartmentalization within Saccharomyces cerevisiae limits its availability in the cytosol, constraining production of cytosol-derived metabolites. In this study, we aimed to redirect carbon flux toward cytosolic acetyl-CoA synthesis by reducing entry into the tricarboxylic acid cycle. To achieve this, we attenuated LPD1 expression by deleting the noncoding RNA SUT526, which is located within the LPD1 promoter region and overlaps an upstream regulatory element. This intervention impaired cell growth and hindered the utilization of non-fermentable carbon sources such as ethanol. To address this limitation, adaptive laboratory evolution was performed in ethanol-based medium, leading to rapid recovery of growth and extended cell viability. The evolved strains exhibited enhanced acetyl-CoA synthetase activity and elevated squalene production, suggesting an increased cytosolic acetyl-CoA supply. These improvements reflect enhanced flux through acetyl-CoA-dependent biosynthetic pathways. This work presents a targeted strategy for modulating central carbon metabolism to increase cytosolic acetyl-CoA supply, providing a framework for efficient production of acetyl-CoA derived compounds in yeast.
{"title":"Truncation of LPD1 promoter and adaptive evolution increase cytosolic acetyl-CoA supply in yeast","authors":"Ling Qin , Shoujie He , Dan Yuan, Yuyang Pan, Zhibo Yan, Mingtao Huang","doi":"10.1016/j.synbio.2025.10.013","DOIUrl":"10.1016/j.synbio.2025.10.013","url":null,"abstract":"<div><div>Acetyl-CoA is a central metabolic intermediate that serves as a key precursor for the biosynthesis of high-value compounds such as terpenoids. However, its compartmentalization within <em>Saccharomyces cerevisiae</em> limits its availability in the cytosol, constraining production of cytosol-derived metabolites. In this study, we aimed to redirect carbon flux toward cytosolic acetyl-CoA synthesis by reducing entry into the tricarboxylic acid cycle. To achieve this, we attenuated <em>LPD1</em> expression by deleting the noncoding RNA SUT526, which is located within the <em>LPD1</em> promoter region and overlaps an upstream regulatory element. This intervention impaired cell growth and hindered the utilization of non-fermentable carbon sources such as ethanol. To address this limitation, adaptive laboratory evolution was performed in ethanol-based medium, leading to rapid recovery of growth and extended cell viability. The evolved strains exhibited enhanced acetyl-CoA synthetase activity and elevated squalene production, suggesting an increased cytosolic acetyl-CoA supply. These improvements reflect enhanced flux through acetyl-CoA-dependent biosynthetic pathways. This work presents a targeted strategy for modulating central carbon metabolism to increase cytosolic acetyl-CoA supply, providing a framework for efficient production of acetyl-CoA derived compounds in yeast.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 10-19"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145475940","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: 2025-11-12DOI: 10.1016/j.synbio.2025.11.001
Zelin Lu , Zhongshi Huang , Zhengyin Wu , Zhengwen Zhu , Yibo Zhu , Xiaonuo Teng , Huyang Chen , Jingwen Zhou , Fuqiang Ma , Xinglong Wang
Menaquinone-7 (MK-7), a key form of vitamin K2 with wide-ranging nutritional and pharmaceutical applications, has attracted increasing interest for microbial production. Here, we developed an integrated modular metabolic engineering strategy in Escherichia coli to enhance MK-7 biosynthesis. Cellular membrane capacity and acetate metabolism were rewired to improve precursor supply for the mevalonate (MVA) pathway, while arabinose induction was applied to overexpress three critical enzymes, including BsHepPPS (Bacillus subtilis), EcMenA (E. coli), and BsUbiE (B. subtilis). Among them, EcMenA was identified as a major bottleneck. Rational protein engineering based on folding free energy analysis and consensus design yielded the EcMenA mutant G110W, which produced 102.55 mg/L MK-7 in shake-flask fermentation, a 57.2 % increase compared with the wild-type (WT) enzyme. Further active-site hotspot random mutagenesis generated a G110W-Q57T double mutant, raising MK-7 production to 176.38 mg/L, a 72 % increase compared to the single mutant. Optimization of EcMenA expression cassette by ribosome binding site redesign using a generative network further improved MK-7 titer to 227.53 mg/L in shake flasks. Finally, scale-up fermentation in a 50-L bioreactor, combined with optimized fermentation strategies, achieved a maximum MK-7 titer of 2.18 g/L. This study establishes a systematic framework integrating metabolic rewiring, enzyme engineering, and expression optimization, providing a robust platform for industrial-scale MK-7 production in microbial hosts.
{"title":"High-level production of vitamin K2 in Escherichia coli via modular molecular engineering","authors":"Zelin Lu , Zhongshi Huang , Zhengyin Wu , Zhengwen Zhu , Yibo Zhu , Xiaonuo Teng , Huyang Chen , Jingwen Zhou , Fuqiang Ma , Xinglong Wang","doi":"10.1016/j.synbio.2025.11.001","DOIUrl":"10.1016/j.synbio.2025.11.001","url":null,"abstract":"<div><div>Menaquinone-7 (MK-7), a key form of vitamin K2 with wide-ranging nutritional and pharmaceutical applications, has attracted increasing interest for microbial production. Here, we developed an integrated modular metabolic engineering strategy in <em>Escherichia coli</em> to enhance MK-7 biosynthesis. Cellular membrane capacity and acetate metabolism were rewired to improve precursor supply for the mevalonate (MVA) pathway, while arabinose induction was applied to overexpress three critical enzymes, including BsHepPPS (<em>Bacillus subtilis</em>), EcMenA (<em>E. coli</em>), and BsUbiE (<em>B. subtilis</em>). Among them, EcMenA was identified as a major bottleneck. Rational protein engineering based on folding free energy analysis and consensus design yielded the EcMenA mutant G110W, which produced 102.55 mg/L MK-7 in shake-flask fermentation, a 57.2 % increase compared with the wild-type (WT) enzyme. Further active-site hotspot random mutagenesis generated a G110W-Q57T double mutant, raising MK-7 production to 176.38 mg/L, a 72 % increase compared to the single mutant. Optimization of EcMenA expression cassette by ribosome binding site redesign using a generative network further improved MK-7 titer to 227.53 mg/L in shake flasks. Finally, scale-up fermentation in a 50-L bioreactor, combined with optimized fermentation strategies, achieved a maximum MK-7 titer of 2.18 g/L. This study establishes a systematic framework integrating metabolic rewiring, enzyme engineering, and expression optimization, providing a robust platform for industrial-scale MK-7 production in microbial hosts.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 42-51"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145527394","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: 2025-12-29DOI: 10.1016/j.synbio.2025.11.016
Yong Feng , Xihua Chen , Zeyang Li , Zhong Ni , Zhengfen Wu , Zhongjian Guo , Fubao Sun , Huiqing Chen , Huayou Chen
Glucose oxidase (GOD) is a widely used enzyme in biotechnology, yet its narrow substrate specificity limits its application in complex bioconversion processes such as agricultural waste valorization. In this study, we employed synthetic biology and protein engineering strategies to engineer a broad-spectrum glucose oxidase from Aureobasidium sp. (AreGOD). Initially, site-directed mutagenesis at N82, a key gatekeeper at the dimer interface, modulated substrate channel geometry, leading to increased catalytic activity towards various sugars, particularly stachyose and xylose. Furthermore, systematic linker engineering between the spore anchor protein CotG and AreGOD revealed that flexible linkers, particularly the (GGGGS)5 repeat (LK3), dramatically expanded the enzyme's substrate spectrum towards various mono-, di-, and oligosaccharides. The optimized spore-displayed AreGOD (CotG-LK3-AreGOD) exhibited strong synergistic effects with cellulase in wheat straw degradation, significantly enhancing the hydrolysis of cellulose, hemicellulose, and lignin. Our work demonstrates an effective and generalizable strategy for engineering substrate-promiscuous oxidases, highlighting the potential of integrative enzyme design for sustainable bioprocessing and agricultural biotechnology.
{"title":"Engineering a broad-spectrum glucose oxidase via substrate channel and linker design for enhanced lignocellulose bioconversion","authors":"Yong Feng , Xihua Chen , Zeyang Li , Zhong Ni , Zhengfen Wu , Zhongjian Guo , Fubao Sun , Huiqing Chen , Huayou Chen","doi":"10.1016/j.synbio.2025.11.016","DOIUrl":"10.1016/j.synbio.2025.11.016","url":null,"abstract":"<div><div>Glucose oxidase (GOD) is a widely used enzyme in biotechnology, yet its narrow substrate specificity limits its application in complex bioconversion processes such as agricultural waste valorization. In this study, we employed synthetic biology and protein engineering strategies to engineer a broad-spectrum glucose oxidase from <em>Aureobasidium</em> sp. (AreGOD). Initially, site-directed mutagenesis at N82, a key gatekeeper at the dimer interface, modulated substrate channel geometry, leading to increased catalytic activity towards various sugars, particularly stachyose and xylose. Furthermore, systematic linker engineering between the spore anchor protein CotG and AreGOD revealed that flexible linkers, particularly the (GGGGS)<sub>5</sub> repeat (LK3), dramatically expanded the enzyme's substrate spectrum towards various mono-, di-, and oligosaccharides. The optimized spore-displayed AreGOD (CotG-LK3-AreGOD) exhibited strong synergistic effects with cellulase in wheat straw degradation, significantly enhancing the hydrolysis of cellulose, hemicellulose, and lignin. Our work demonstrates an effective and generalizable strategy for engineering substrate-promiscuous oxidases, highlighting the potential of integrative enzyme design for sustainable bioprocessing and agricultural biotechnology.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 218-228"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883634","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.2025.12.018
He Ren , Jianqi Nie , Zichuan Song , Wanting Mo , Yankun Yang , Zhonghu Bai
Adeno-associated virus (AAV) vectors are widely used in gene therapy owing to their safety, stability, and broad tissue tropism. However, current plasmid-based AAV manufacturing platforms suffer from low yield and high manufacturing cost, limiting their scalability for clinical and commercial applications. Rational engineering of pHelper vector offers an effective strategy to enhance AAV production. In this study, we engineered a novel helper vector (UL12-ICP22-miniHelper) by integrating UL12 and ICP22 genes from herpes simplex virus type 1 (HSV-1) into a size-reduced pHelper backbone (mini-pHelper) with partial deletion of E2a and E4 regions. In a triple-plasmid transfection system, UL12-ICP22-miniHelper increased AAV5 vector yield from 1.35 × 1011 to 2.85 × 1011 vg/mL (2.11-fold) without altering the proportion of full capsids. Enhanced productivity was also observed across multiple serotypes, with increases of 2.24-fold for AAV1, 1.54-fold for AAV2, 1.88-fold for AAV6, and 2.03-fold for AAV9, while maintaining transduction efficiency. Mechanistic analysis indicated that the improved productivity was associated with elevated viral genome replication and increased Rep/Cap protein expression. Collectively, these results demonstrate that the novel UL12-ICP22-miniHelper provides a broadly applicable and cost-effective strategy for improving AAV vector manufacturing in both clinical and industrial applications.
{"title":"Enhanced AAV production via rational design of a novel pHelper vector integrated with HSV-1 helper genes","authors":"He Ren , Jianqi Nie , Zichuan Song , Wanting Mo , Yankun Yang , Zhonghu Bai","doi":"10.1016/j.synbio.2025.12.018","DOIUrl":"10.1016/j.synbio.2025.12.018","url":null,"abstract":"<div><div>Adeno-associated virus (AAV) vectors are widely used in gene therapy owing to their safety, stability, and broad tissue tropism. However, current plasmid-based AAV manufacturing platforms suffer from low yield and high manufacturing cost, limiting their scalability for clinical and commercial applications. Rational engineering of pHelper vector offers an effective strategy to enhance AAV production. In this study, we engineered a novel helper vector (UL12-ICP22-miniHelper) by integrating <em>UL12</em> and <em>ICP22</em> genes from herpes simplex virus type 1 (HSV-1) into a size-reduced pHelper backbone (mini-pHelper) with partial deletion of <em>E2a</em> and <em>E4</em> regions. In a triple-plasmid transfection system, UL12-ICP22-miniHelper increased AAV5 vector yield from 1.35 × 10<sup>11</sup> to 2.85 × 10<sup>11</sup> vg/mL (2.11-fold) without altering the proportion of full capsids. Enhanced productivity was also observed across multiple serotypes, with increases of 2.24-fold for AAV1, 1.54-fold for AAV2, 1.88-fold for AAV6, and 2.03-fold for AAV9, while maintaining transduction efficiency. Mechanistic analysis indicated that the improved productivity was associated with elevated viral genome replication and increased Rep/Cap protein expression. Collectively, these results demonstrate that the novel UL12-ICP22-miniHelper provides a broadly applicable and cost-effective strategy for improving AAV vector manufacturing in both clinical and industrial applications.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 352-363"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145976680","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.2025.12.009
Lu Liu , Xiangjun Zhang , Tengteng Zhu , Tong Ye , Zongqian Li , Wei Ding , Huiyan Liu , Haitian Fang
In Escherichia coli, cofactor imbalance serves as a crucial limiting factor in cytidine biosynthesis, with nicotinamide adenine dinucleotide phosphate (NADPH) insufficiency representing the principal metabolic barrier. To overcome this limitation, an integrated engineering strategy targeting the enhancement of NADPH metabolism was implemented. Via CRISPR-Cas9-mediated multiplex genomic editing and strong constitutive promoter replacement, three NADPH-regenerating modules were concurrently enhanced: the membrane-bound transhydrogenase (pntAB), the oxidative pentose phosphate pathway (zwf-encoded glucose-6-phosphate dehydrogenase), and the decarboxylation shunt (gnd-encoded 6-phosphogluconate dehydrogenase). After 54-hour fermentation in 500 mL shake flasks, the cytidine titer of the engineered strain NXBG-20 reached 7.83 g/L, representing a 9.10-fold increase compared to the start strain. Systematic multi-omics profiling revealed that the metabolic network had undergone substantial alterations. These alterations were characterized by the redirection of glycolytic flux towards nucleotide precursor substances and the enhancement of ribose-5-phosphate biosynthesis. This engineering approach not only establishes a novel microbial platform for cytidine bioproduction but also provides mechanistic insights into cofactor-driven metabolic flux control.
{"title":"Integrated amplification of NADPH-regenerating modules enhances cytidine biosynthesis in Escherichia coli","authors":"Lu Liu , Xiangjun Zhang , Tengteng Zhu , Tong Ye , Zongqian Li , Wei Ding , Huiyan Liu , Haitian Fang","doi":"10.1016/j.synbio.2025.12.009","DOIUrl":"10.1016/j.synbio.2025.12.009","url":null,"abstract":"<div><div>In <em>Escherichia coli</em>, cofactor imbalance serves as a crucial limiting factor in cytidine biosynthesis, with nicotinamide adenine dinucleotide phosphate (NADPH) insufficiency representing the principal metabolic barrier. To overcome this limitation, an integrated engineering strategy targeting the enhancement of NADPH metabolism was implemented. Via CRISPR-Cas9-mediated multiplex genomic editing and strong constitutive promoter replacement, three NADPH-regenerating modules were concurrently enhanced: the membrane-bound transhydrogenase (<em>pntAB</em>), the oxidative pentose phosphate pathway (<em>zwf</em>-encoded glucose-6-phosphate dehydrogenase), and the decarboxylation shunt (<em>gnd</em>-encoded 6-phosphogluconate dehydrogenase). After 54-hour fermentation in 500 mL shake flasks, the cytidine titer of the engineered strain NXBG-20 reached 7.83 g/L, representing a 9.10-fold increase compared to the start strain. Systematic multi-omics profiling revealed that the metabolic network had undergone substantial alterations. These alterations were characterized by the redirection of glycolytic flux towards nucleotide precursor substances and the enhancement of ribose-5-phosphate biosynthesis. This engineering approach not only establishes a novel microbial platform for cytidine bioproduction but also provides mechanistic insights into cofactor-driven metabolic flux control.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 320-329"},"PeriodicalIF":4.4,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145938532","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}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号