{"title":"Enzyme and cofactor engineering to increase d-xylonate dehydratase activity for improved d-1,2,4-butanetriol production from d-xylose","authors":"Jingwen Chen, Zhangyu Liu, Dandan Mai, Sheng Xu, Xin Wang, Kequan Chen","doi":"10.1016/j.synbio.2025.07.003","DOIUrl":"10.1016/j.synbio.2025.07.003","url":null,"abstract":"<div><div>d-1,2,4-Butanetriol (BTO), a C4 platform compound, is widely used in fields such as military and pharmaceuticals. Biosynthesis of d-1,2,4-BTO from lignocellulose-derived <span>d</span>-xylose presents a promising production route. However, the low catalytic activity of <span>d</span>-xylonate dehydratase leading to the accumulation of <span>d</span>-xylonic acid remains a key bottleneck for the efficient production of d-1,2,4-BTO. In this study, we aimed to enhance the catalytic activity of <span>d</span>-xylonate dehydratase through an integrated enzyme and cofactor engineering approach. Firstly, we evolved the <span>d</span>-xylonate dehydratase YjhG by using both random mutagenesis and site-directed saturation mutagenesis. Among the generated variants, YjhG<sup>T325F</sup> showed an 1.82-fold increase in <span>d</span>-xylonic acid consumption compared to the wild-type enzyme. When introduced into the producing strain, this variant increased d-1,2,4-BTO production by 1.34-fold compared to the original strain. Further enhancement was achieved by modifying the iron-sulfur [Fe–S] cluster synthesis system, which was critical for <span>d</span>-xylonate dehydratase activity. We systematically evaluated three [Fe–S] assembly systems, including SUF (encoded by <em>sufABCDSE</em>), ISC (encoded by <em>iscSUA-hscBA-fdx</em>), and CSD (encoded by <em>csdAE</em>). Comparative analysis revealed that the overexpression of SUF system conferred the highest catalytic efficiency of YjhG. The recombinant strain of BT-YjhG<sup>T325F</sup>-SUF produced 10.36 g/L of d-1,2,4-BTO from <span>d</span>-xylose, achieving a molar yield of 73.6 %, which was 1.88-fold that of the original strain. This study provided a robust foundation for high-efficiency d-1,2,4-BTO production through enzyme and cofactor engineering.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 4","pages":"Pages 1234-1241"},"PeriodicalIF":4.4,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144694925","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}
Po Li , Xueying Lei , Xiaoying Niu , Wen Tian , Zhehuang Li , Songcheng Yu , Peng Zhang
{"title":"A multifunctional switch for label-free CRISPR/Cas12a sensor with self-driven amplification","authors":"Po Li , Xueying Lei , Xiaoying Niu , Wen Tian , Zhehuang Li , Songcheng Yu , Peng Zhang","doi":"10.1016/j.synbio.2025.07.002","DOIUrl":"10.1016/j.synbio.2025.07.002","url":null,"abstract":"<div><div>MicroRNA (miRNA) is promising candidate for non-invasive diagnostic biomarker. Conventional CRISPR/Cas12a-based miRNA detection systems are constrained by reliance on reverse transcription, nucleic acid pre-amplification and costly fluorescently labeled reporters which introduce chemical modification complexity and background noise. To address these limitations, we herein developed a multifunctional switch that integrated target recognition, CRISPR/Cas12a system activation, intrinsic fluorescence signaling, and autonomous signal amplification within a single molecular architecture. As a proof of concept, this switch enabled a label-free CRISPR/Cas12a biosensing for miR-21 detection with a detection limit of 4.8 nM and robust performance in accuracy, precision, and selectivity. This proposed label-free CRISPR/Cas12a platform could be applied for real sample and is a promising candidate for point-of-care miRNA detection.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 4","pages":"Pages 1208-1214"},"PeriodicalIF":4.4,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144680405","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}
Pingping Yuan , Shijie Zhou , Qianqian Li , Lin Li , Shaoqi Qu
{"title":"Extracellular vesicles enhance the efficacy of ceftiofur against intracellular bacterial infections","authors":"Pingping Yuan , Shijie Zhou , Qianqian Li , Lin Li , Shaoqi Qu","doi":"10.1016/j.synbio.2025.07.001","DOIUrl":"10.1016/j.synbio.2025.07.001","url":null,"abstract":"<div><div>Bacterial infections pose a major threat to human health and economic stability. In particular, intracellular bacterial infections present significant clinical challenges due to antibiotic resistance and poor drug penetration. Therefore, there is an urgent need to develop novel therapeutic strategies to address the problem of intracellular bacterial infections. Extracellular bacterial vesicles become ideal delivery systems due to their natural targeting properties. Here, we developed a bacteria-derived extracellular vesicles (EVs)-based drug delivery platform to enhance the therapeutic efficacy of antibiotics against intracellular infections. EVs were successfully isolated and increased production from <em>S. aureus</em> by ultracentrifugation, then loaded with ceftiofur (CEF) via co-incubation. <em>In vitro</em> tests demonstrated the potent antibacterial activity of CEF, achieving complete growth inhibition within 24 h and a 4-log viability reduction in 4 h. Furthermore, confocal microscopy revealed efficient CEV internalization in IEC-6 cells, with 12-fold greater intracellular bacterial clearance than free CEF. <em>In vivo</em>, CEV-incorporated hydrogel (CEVH) significantly reduced both intra- and extracellular bacterial loads and accelerated wound healing. These findings demonstrate that bacterial EVs serve as a universal delivery platform to significantly enhance the efficacy of existing antibiotics against intracellular infections.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 4","pages":"Pages 1200-1207"},"PeriodicalIF":4.4,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144663487","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}
Jie Gao, Hengyi Wang, Jingtao Sun, Hongjie Tang, Yuhan Yang, Qi Li
{"title":"Highly efficient genome editing in Bacillus subtilis via miniature DNA nucleases IscB","authors":"Jie Gao, Hengyi Wang, Jingtao Sun, Hongjie Tang, Yuhan Yang, Qi Li","doi":"10.1016/j.synbio.2025.06.012","DOIUrl":"10.1016/j.synbio.2025.06.012","url":null,"abstract":"<div><div>Existing CRISPR-based genome editing techniques for <em>Bacillus subtilis</em> (<em>B. subtilis</em>) are limited due to the large size of the <em>cas</em> gene. IscB, a recently reported DNA nuclease, is one-third the size of Cas9, making it a potential tool for genome editing; however, its application in <em>B. subtilis</em> remains unexplored. In this study, two IscB and enhanced IscB (enIscB)-based genome editing systems, named pBsuIscB and pBsuenIscB were established in <em>B. subtilis</em> SCK6, and their deletion efficiencies ranging from 13.3 % to 100 %. Compared to the pBsuIscB system, the pBsuenIscB system showed higher deletion efficiency, inducing the deletion of a large genomic fragment with a single ωRNA. Additionally, the pBsuenIscB system could integrate both single-copy and multi-copy <em>mCherry</em> genes in the <em>B. subtilis</em> SCK6 genome. Lastly, the pBsuenIscB system could efficiently conduct a second round of genome editing in <em>B. subtilis</em> SCK6. This study indicates that IscB can be used for genome editing in <em>B. subtilis</em>, enabling the efficient construction of engineered <em>B. subtilis</em> strains for large-scale biomolecule production.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 4","pages":"Pages 1215-1223"},"PeriodicalIF":4.4,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144680403","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}
{"title":"Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium stationis and its application in hypoxanthine biosynthesis","authors":"Zhilin Ouyang , Xinyu Zhang , Xinyi Hou , Jiabei Huang , Ying Lin , Suiping Zheng","doi":"10.1016/j.synbio.2025.06.010","DOIUrl":"10.1016/j.synbio.2025.06.010","url":null,"abstract":"<div><div><em>Corynebacterium stationis</em>, a high-GC Gram-positive bacterium with significant industrial potential, has faced limitations due to the lack of efficient genetic tools. In this study, we developed a CRISPR/Cas9-based genome editing platform specifically tailored for <em>C. stationis</em>. First, electroporation efficiency was optimized to 1.81 ± 0.16 × 10<sup>5</sup> CFU (colony forming units)/μg plasmid DNA through medium selection, pulse parameter adjustments (2.5 kV, 2 pulses), and concentration optimization of cell wall-weakening agents (3.0 % glycine, 0.25 % isoniazid). Three functional shuttle vectors (p99E-pCG1, p19-Kan, p19-Spe) were constructed, enabling stable heterologous gene expression. By engineering a tightly regulated Cas9 expression system (P<em>lac</em> promoter with dual LacO∗ operators), we achieved high-efficiency genome editing, with deletion efficiencies of 81.2–98.6 % for 1.7–50 kb fragments and insertion efficiencies of 27.5–65.2 % for 1–5 kb fragments. CRISPR/Cas9-assisted ssDNA recombineering facilitated single/triple nucleotide changes with >90 % efficiency. Applying this toolbox, we engineered <em>C. stationis</em> for hypoxanthine biosynthesis by combining <em>purA</em> deletion with integration of heterologous feedback-resistant <em>prs</em><sup>D128A</sup> and endogenous <em>purF</em> deregulation (<em>purF</em><sup>K334Q</sup>), achieving a titer of 0.047 g/L. This study establishes a robust genetic platform for <em>C. stationis</em>, accelerating its industrial application in the production of biochemicals and biofuels.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 4","pages":"Pages 1190-1199"},"PeriodicalIF":4.4,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144535761","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}
Mingyu Dai , Qingyi Cao , Shuanglong Jia, Jiang Chen, Hui-Ying Xu, Bang-Ce Ye, Wei-Bing Liu, Ying Zhou
{"title":"Engineering Yarrowia lipolytica for high-level β-elemene production via combinatorial metabolic strategies","authors":"Mingyu Dai , Qingyi Cao , Shuanglong Jia, Jiang Chen, Hui-Ying Xu, Bang-Ce Ye, Wei-Bing Liu, Ying Zhou","doi":"10.1016/j.synbio.2025.06.007","DOIUrl":"10.1016/j.synbio.2025.06.007","url":null,"abstract":"<div><div>β-Elemene, a pharmacologically active sesquiterpene derived from the traditional Chinese medicinal herb <em>Curcuma wenyujin</em>, exhibits broad-spectrum anti-tumor and anti-inflammatory activities. Currently, its commercial production relies heavily on plant extraction, a process associated with high costs and environmental burdens, limiting industrial scalability. To address these challenges, we developed a metabolically engineered <em>Yarrowia lipolytica</em> strain as an efficient microbial cell factory for de novo Germacrene A biosynthesis, the direct precursor of β-elemene. Through combinatorial optimization strategies—including (1) mevalonate (MVA) pathway enhancement, (2) copy number amplification of germacrene A synthase (GAS), (3) β-oxidation pathway reinforcement, and (4) introduction of the isopentenyl utilization pathway (IUP)—we significantly improved β-elemene production. The engineered strain achieved a titer of 3.08 ± 0.05 g/L in shake-flask cultures, with a yield of 51.27 ± 0.75 mg/g glucose, representing a 3.5-fold increase over the parental strain. Our work highlights the potential of <em>Y. lipolytica</em> as a sustainable and scalable platform for high-value sesquiterpene production, offering a viable alternative to plant-derived extraction.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 4","pages":"Pages 1172-1179"},"PeriodicalIF":4.4,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144522717","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}
Xiaoyi Zou , Jiaqi Miao , Hongbiao Li , Yanshan Ke , Yan Chen , Weizhu Zeng , Jingwen Zhou
{"title":"Development of precise genome editing and multi-copy integration tools in Hansenula polymorpha DL-1","authors":"Xiaoyi Zou , Jiaqi Miao , Hongbiao Li , Yanshan Ke , Yan Chen , Weizhu Zeng , Jingwen Zhou","doi":"10.1016/j.synbio.2025.06.009","DOIUrl":"10.1016/j.synbio.2025.06.009","url":null,"abstract":"<div><div><em>Hansenula polymorpha</em> DL-1 is a thermotolerant yeast capable of utilizing multiple renewable carbon sources, making it a promising microbial cell factory for sustainable manufacturing. However, advanced metabolic engineering efforts have been constrained by its strong non-homologous end joining (NHEJ) mechanism and limited choice of suitable genetic tools. This study presents an optimized synthetic biology toolkit to address these limitations. A high-efficiency CRISPR-Cas9-based genome editing system was established, achieving an editing efficiency of 97.2 %. To further enhance homologous recombination (HR), the NHEJ pathway was partially suppressed by knocking out <em>KU80</em> and overexpressing HR-related genes from <em>Saccharomyces cerevisiae</em>. This increased HR rates to 88.9 %. In addition, 36 neutral sites were identified for stable single-copy gene integration without disrupting native gene expression cassettes. Finally, multi-copy integration tools were developed by targeting rDNA and Ty elements, leading to a ∼60-fold increase in β-carotene production compared with single-copy integrants. Furthermore, squalene titers were increased from 0.1 mg/L in the wild-type strain to 187.2 mg/L through iterative multi-copy integration. These advances significantly expand the genetic tractability of <em>H. polymorpha</em> DL-1, underscoring its potential as a versatile platform for efficient and sustainable production of value-added compounds.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 4","pages":"Pages 1224-1233"},"PeriodicalIF":4.4,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144687427","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}
{"title":"From consultors to collaborators – An SOP for advancing ethics engagement in science","authors":"Varsha Aravind Paleri, Kristien Hens","doi":"10.1016/j.synbio.2025.06.006","DOIUrl":"10.1016/j.synbio.2025.06.006","url":null,"abstract":"<div><div>Synthetic Biology (SynBio) is an interdisciplinary field that tackles global challenges in healthcare, sustainability, food production, among others. However, it also presents critical ethical concerns, including biosecurity, justice, and the unintended consequences of research biases. Addressing these concerns requires timely and systematic ethical evaluation. Conventional approaches often relegate ethics to an external, procedural role rather than embedding it within the core of scientific inquiry, leading to delayed engagement and associated challenges. This article introduces a structured, stage-wise Standard Operating Procedure (SOP) to support the systematic integration of ethics into scientific research. Particularly beneficial for early-career researchers and ethicists, this framework offers a practical method for incorporating ethical reflection throughout the research process.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 4","pages":"Pages 1180-1189"},"PeriodicalIF":4.4,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144535762","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}
Ying Huang , Kang Ma , Yan Li , Qingyan Li , Fuping Lu , Xueli Zhang , Zhe Sun
{"title":"Engineering T7 RNA polymerase-cascaded systems controlled by nisin and theophylline for protein overexpression and targeted gene mutagenesis in Lactococcus lactis","authors":"Ying Huang , Kang Ma , Yan Li , Qingyan Li , Fuping Lu , Xueli Zhang , Zhe Sun","doi":"10.1016/j.synbio.2025.06.008","DOIUrl":"10.1016/j.synbio.2025.06.008","url":null,"abstract":"<div><div><em>Lactococcus lactis</em> serves as an important platform for heterologous protein production, with the nisin-controlled gene expression (NICE) system being widely employed for regulated protein overexpression. However, the NICE system relies on the native RNA polymerase, which limits transcriptional efficiency, and there remains a lack of tools enabling continuous target gene mutagenesis in <em>L. lactis</em>. In this study, we enhanced the NICE system by integrating the highly processive T7 RNA polymerase (T7RNAP) to boost protein expression. A theophylline-dependent riboswitch, RbxE, was incorporated into the nisin-induced promoter to mitigate the toxicity caused by basal T7RNAP expression in <em>Escherichia coli</em>. Directed mutagenesis of the riboswitch region between the stem-loop and the ribosome binding site optimized T7RNAP expression, leading to a 2.4-fold increase upon nisin and theophylline induction in <em>L. lactis.</em> The resulting NICE-T7 system achieved a 2.8-fold increase in GFP compared to the original NICE system. Furthermore, adenosine deaminase TadA8e was fused to T7RNAP to generate the MutaT7LL system, facilitating targeted A-to-G mutagenesis and successfully reactivated an erythromycin resistance gene with a mutation efficiency of 1.33 × 10<sup>−6</sup>. Overall, this study presents an upgraded NICE system that enhances protein production and enables continuous in vivo mutagenesis of target genes in <em>L. lactis</em>.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 4","pages":"Pages 1150-1159"},"PeriodicalIF":4.4,"publicationDate":"2025-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144501319","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}
Xingcun Fan , Guangming Xiang , Wenbin Liao , Luchi Xiao , Siwei He , Na Luo , Hongzhong Lu , Xuefeng Yan
{"title":"Decoding yeast transcriptional regulation via a data-and mechanism-driven distributed large-scale network model","authors":"Xingcun Fan , Guangming Xiang , Wenbin Liao , Luchi Xiao , Siwei He , Na Luo , Hongzhong Lu , Xuefeng Yan","doi":"10.1016/j.synbio.2025.06.005","DOIUrl":"10.1016/j.synbio.2025.06.005","url":null,"abstract":"<div><div>The complex transcriptional regulatory relationships among genes influence gene expression levels and play a crucial role in determining cellular phenotypes. In this study, we propose a novel, distributed, large-scale transcriptional regulatory neural network model (DLTRNM), which integrates prior knowledge into the reconstruction of pre-trained machine learning models, followed by fine-tuning. Using <em>Saccharomyces cerevisiae</em> as a case study, the curated transcriptional regulatory relationships are used to define the interactions between transcription factors (TFs) and their target genes (TGs). Subsequently, DLTRNM is pre-trained on pan-transcriptomic data and fine-tuned with time-series data, enabling it to accurately predict regulatory correlations. Additionally, DLTRNM can help identify potential key TFs, thereby simplifying the complex and interrelated transcriptional regulatory networks (TRNs). It can also complement previously reported transcriptional regulatory subnetworks. DLTRNM provides a powerful tool for studying transcriptional regulation with reduced computational demands and enhanced interpretability. Thus, this study marks a significant advancement in systems biology for understanding the complex transcriptional regulation within cells.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 4","pages":"Pages 1140-1149"},"PeriodicalIF":4.4,"publicationDate":"2025-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144492172","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}