Pub Date : 2025-12-04DOI: 10.1016/j.synbio.2025.11.012
Chunhe Yang , YuLing Zhao , Ruoyu Wang , Haoran Li , Xiaoping Liao , Hongwu Ma
Accurately predicting recombinant protein expression in Escherichia coli remains a long-standing challenge due to the multifactorial nature of gene regulation and translation. Existing computational approaches typically emphasize either codon usage or protein sequence features, limiting predictive accuracy and generalizability. Here we present TLCP-EPE, a transfer learning framework that, for the first time, fuses codon- and protein-level pre-trained language models to jointly capture determinants of expression. By fine-tuning CaLM and ProtT5 with low-rank adaptation (LoRA) and integrating their embeddings through a BiGRU-MLP predictor, TLCP-EPE learns expression-aware representations that outperform state-of-the-art methods. Across two independent test datasets, TLCP-EPE achieved robust performance (AUC 0.835 on codon data; AUC 0.713 on protein data), consistently surpassing conventional codon-based metrics and deep learning baselines. Our results demonstrate that dual-modal modeling of codon and protein sequences enables more accurate and generalizable prediction of expression levels, providing a powerful foundation for rational protein design and biomanufacturing applications.
{"title":"Transfer learning with pre-trained language models for protein expression level prediction in Escherichia coli","authors":"Chunhe Yang , YuLing Zhao , Ruoyu Wang , Haoran Li , Xiaoping Liao , Hongwu Ma","doi":"10.1016/j.synbio.2025.11.012","DOIUrl":"10.1016/j.synbio.2025.11.012","url":null,"abstract":"<div><div>Accurately predicting recombinant protein expression in <em>Escherichia coli</em> remains a long-standing challenge due to the multifactorial nature of gene regulation and translation. Existing computational approaches typically emphasize either codon usage or protein sequence features, limiting predictive accuracy and generalizability. Here we present TLCP-EPE, a transfer learning framework that, for the first time, fuses codon- and protein-level pre-trained language models to jointly capture determinants of expression. By fine-tuning CaLM and ProtT5 with low-rank adaptation (LoRA) and integrating their embeddings through a BiGRU-MLP predictor, TLCP-EPE learns expression-aware representations that outperform state-of-the-art methods. Across two independent test datasets, TLCP-EPE achieved robust performance (AUC 0.835 on codon data; AUC 0.713 on protein data), consistently surpassing conventional codon-based metrics and deep learning baselines. Our results demonstrate that dual-modal modeling of codon and protein sequences enables more accurate and generalizable prediction of expression levels, providing a powerful foundation for rational protein design and biomanufacturing applications.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 115-123"},"PeriodicalIF":4.4,"publicationDate":"2025-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145690540","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-02DOI: 10.1016/j.synbio.2025.11.008
Lifang Yu , Mario Andrea Marchisio
The emergence of base and prime editors—genome editing tools that avoid double-strand breaks (DSBs)—has enabled precise point mutations, insertions, inversions, deletions, and substitutions, which accelerates the development of single-intervention therapies and advances individualized genomic medicine. However, their limited efficiency in inserting large DNA fragments has restricted applications for correcting diverse pathogenic mutations within a single gene. In this review, we explore three recently developed strategies for efficient large DNA cargo insertion (>1 kb): CRISPR-associated Tn7-like transposases (CASTs), PE-integrase systems, and R2 retrotransposon fusions (nCas9-R2). We examine the applications of these systems in both bacterial and mammalian contexts and discuss their respective advantages and current limitations. Finally, we address persistent challenges and propose potential directions to guide future research.
{"title":"Programmable large-cargo integration: Overcoming size constraints for next-generation gene therapy","authors":"Lifang Yu , Mario Andrea Marchisio","doi":"10.1016/j.synbio.2025.11.008","DOIUrl":"10.1016/j.synbio.2025.11.008","url":null,"abstract":"<div><div>The emergence of base and prime editors—genome editing tools that avoid double-strand breaks (DSBs)—has enabled precise point mutations, insertions, inversions, deletions, and substitutions, which accelerates the development of single-intervention therapies and advances individualized genomic medicine. However, their limited efficiency in inserting large DNA fragments has restricted applications for correcting diverse pathogenic mutations within a single gene. In this review, we explore three recently developed strategies for efficient large DNA cargo insertion (>1 kb): CRISPR-associated Tn7-like transposases (CASTs), PE-integrase systems, and R2 retrotransposon fusions (nCas9-R2). We examine the applications of these systems in both bacterial and mammalian contexts and discuss their respective advantages and current limitations. Finally, we address persistent challenges and propose potential directions to guide future research.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 101-114"},"PeriodicalIF":4.4,"publicationDate":"2025-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145690541","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-27DOI: 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":"2025-11-27","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 : 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":"2025-11-18","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 : 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":"2025-11-18","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 : 2025-11-17DOI: 10.1016/j.synbio.2025.11.002
Hailin Zhang , Yueyue Song , Wenyue Liu , Xiaoqing Zheng , Xiaodong An , Chao Li , Weihua Chen , Hailong Wang , Yuran Zhang
Engineered bacteriophages (phages) have been developed to overcome the limitations of natural phage therapies and serve as precision-targeted agents against drug-resistant bacterial infections. However, their application has been constrained by the low efficiency of existing genome-editing tools, largely because of the absence of effective selection markers. This study proposed a novel strategy, termed defect-complementation homologous recombination (DCHR), for precise phage genome editing. In this approach, CRISPR-Cas9 cleaves a donor plasmid in host cells to release a linear donor template carrying homology arms, an essential phage gene used as a selection marker, and two lox sites. The donor template undergoes homologous recombination with the genome of essential gene-deficient phage, thereby enabling targeted genome modifications. Using DCHR, we successfully generated large genomic deletions (1.48-kb gp0.4–0.7 and 1.02-kb gp4.3–4.7), achieved gene insertion (3.08-kb lacZ), and introduced a single-base substitution (TGA to TAA) in the stop codon of gp9 within the same T7 phage genome, all with 100 % accuracy. The significant advantages of DCHR are as follows: (i) High-efficiency screening: Only progeny phages derived from successful homologous recombination retain viability and replicative capacity, thereby greatly simplifying recombinant isolation. (ii) Editing flexibility: Unlike CRISPR-Cas systems, DCHR cannot be constrained by protospacer adjacent motif dependence and allows modifications across diverse genomic loci. (iii) High recombination efficiency: DCHR can achieve a recombinant phage titer of 3.1 × 105 PFU mL−1 (plaque-forming units per mL) without relying on exogenous homologous recombination systems. In summary, DCHR demonstrates potential as a precise and efficient general genome-editing tool that facilitates design of engineered phages and advances functional genomic studies.
{"title":"Defect-complementation homologous recombination: A novel strategy for precise genome engineering of virulent phages","authors":"Hailin Zhang , Yueyue Song , Wenyue Liu , Xiaoqing Zheng , Xiaodong An , Chao Li , Weihua Chen , Hailong Wang , Yuran Zhang","doi":"10.1016/j.synbio.2025.11.002","DOIUrl":"10.1016/j.synbio.2025.11.002","url":null,"abstract":"<div><div>Engineered bacteriophages (phages) have been developed to overcome the limitations of natural phage therapies and serve as precision-targeted agents against drug-resistant bacterial infections. However, their application has been constrained by the low efficiency of existing genome-editing tools, largely because of the absence of effective selection markers. This study proposed a novel strategy, termed <u>d</u>efect-<u>c</u>omplementation <u>h</u>omologous <u>r</u>ecombination (DCHR), for precise phage genome editing. In this approach, CRISPR-Cas9 cleaves a donor plasmid in host cells to release a linear donor template carrying homology arms, an essential phage gene used as a selection marker, and two <em>lox</em> sites. The donor template undergoes homologous recombination with the genome of essential gene-deficient phage, thereby enabling targeted genome modifications. Using DCHR, we successfully generated large genomic deletions (1.48-kb <em>gp0.4–0.7</em> and 1.02-kb <em>gp4.3–4.7</em>), achieved gene insertion (3.08-kb <em>lacZ</em>), and introduced a single-base substitution (T<u>G</u>A to T<u>A</u>A) in the stop codon of <em>gp9</em> within the same T7 phage genome, all with 100 % accuracy. The significant advantages of DCHR are as follows: (i) High-efficiency screening: Only progeny phages derived from successful homologous recombination retain viability and replicative capacity, thereby greatly simplifying recombinant isolation. (ii) Editing flexibility: Unlike CRISPR-Cas systems, DCHR cannot be constrained by protospacer adjacent motif dependence and allows modifications across diverse genomic loci. (iii) High recombination efficiency: DCHR can achieve a recombinant phage titer of 3.1 × 10<sup>5</sup> PFU mL<sup>−1</sup> (plaque-forming units per mL) without relying on exogenous homologous recombination systems. In summary, DCHR demonstrates potential as a precise and efficient general genome-editing tool that facilitates design of engineered phages and advances functional genomic studies.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 59-70"},"PeriodicalIF":4.4,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145577484","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-13DOI: 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":"2025-11-13","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}
Pub 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":"2025-11-12","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 : 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":"2025-11-11","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 : 2025-11-11DOI: 10.1016/j.synbio.2025.10.015
Zhihao Liu , Linghong Zheng , Xiaofei Yu , Yingping Zhuang , Guan Wang
Ethanol, a high-demand clean energy source, is primarily produced via fed-batch fermentation in industrial settings. Although our previous study identified an optimal glucose concentration of 30 g/L for maximal ethanol yield, the mechanisms underlying glucose-dependent cellular adaptation remain unclear. Here, we performed an integrated multi-omics analysis, including transcriptomics, proteomics, metabolomics, and fluxomics, to compare yeast cells under glucose-controlled and uncontrolled conditions. Our results indicate that high glucose stress triggers the regulation of transporters with different affinities and the upregulation of heat shock proteins (HSPs), trehalose, and amino acids. In contrast, protein turnover was reduced under glucose-controlled conditions, suggesting more efficient resource allocation. This metabolic reallocation enhances carbon flux through glycolysis, potentially providing additional energy and NADH to support biomass growth and ethanol production. These findings advance our understanding of yeast regulatory mechanisms under glucose stress and provide insights for metabolic engineering and process optimization.
{"title":"Multi-omics elucidation of resource allocation for enhanced ethanol production via precise glucose control in anaerobic Saccharomyces cerevisiae fermentation","authors":"Zhihao Liu , Linghong Zheng , Xiaofei Yu , Yingping Zhuang , Guan Wang","doi":"10.1016/j.synbio.2025.10.015","DOIUrl":"10.1016/j.synbio.2025.10.015","url":null,"abstract":"<div><div>Ethanol, a high-demand clean energy source, is primarily produced via fed-batch fermentation in industrial settings. Although our previous study identified an optimal glucose concentration of 30 g/L for maximal ethanol yield, the mechanisms underlying glucose-dependent cellular adaptation remain unclear. Here, we performed an integrated multi-omics analysis, including transcriptomics, proteomics, metabolomics, and fluxomics, to compare yeast cells under glucose-controlled and uncontrolled conditions. Our results indicate that high glucose stress triggers the regulation of transporters with different affinities and the upregulation of heat shock proteins (HSPs), trehalose, and amino acids. In contrast, protein turnover was reduced under glucose-controlled conditions, suggesting more efficient resource allocation. This metabolic reallocation enhances carbon flux through glycolysis, potentially providing additional energy and NADH to support biomass growth and ethanol production. These findings advance our understanding of yeast regulatory mechanisms under glucose stress and provide insights for metabolic engineering and process optimization.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 20-31"},"PeriodicalIF":4.4,"publicationDate":"2025-11-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145527395","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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