Synthetic and Systems Biotechnology最新文献

{"title":"Whole-cell biosensors with regulatory circuits based on a synthetic dual-input promoter enabling the highly sensitive detection of ultra-trace Cd2+","authors":"Yuke Qin ,&nbsp;Shuting Hu ,&nbsp;Yibin Yang ,&nbsp;Xiaoqiang Jia","doi":"10.1016/j.synbio.2025.09.008","DOIUrl":"10.1016/j.synbio.2025.09.008","url":null,"abstract":"<div><div>The research on whole-cell biosensors tailored for trace and ultra-trace detection remains limited and the biosensors based on natural bacterial heavy metal resistance mechanisms have common issues of low sensitivity. In this study, we designed a promoter P_{<em>T7-cadO</em>} using the Cadmium ions (Cd²⁺)-binding protein binding site <em>cadO</em> with the T7 promoter at first and constructed a single-input whole-cell biosensor, which was named CP100, whose detection limit for Cd²⁺ met the WHO requirement, yet its response and sensitivity were quite low. We further introduced the <em>lacI</em> and <em>lac operator</em> (<em>lacO</em>) as the signal amplifier to construct a dual-input promoter P_{<em>T7-cadO-lacO-cadO</em>} and developed a biosensor named LC100, whose response and sensitivity were significantly improved, but background leakage became a new problem. Then we redesigned the gene circuit based on the regulatory circuit LCPM-2, which has the structure of “CadR-P_{<em>J23100</em>}-P_{<em>T7-cadO-lacO-cadO</em>}-mRFP1-LacI”, with the LacI protein as the autoregulatory negative feedback model and finally obtained the biosensor named LC100-2, which achieved the detection of ultra-trace Cd²⁺ (0.00001–0.02 nM), with the sensitivity of 3748.22 times that of CP100. Moreover, LC100-2 demonstrated excellent specificity to Cd²⁺ among four other divalent metal ions and good anti-interference capability in the mixed divalent metal ions system. The results of the real water sample tests demonstrated that precise quantitative detection of Cd²⁺ with a final concentration of 0.001–0.02 nM could be achieved by adding only a small volume of the sample (1 μL). This finding showed promising application potential in the field of trace detection. Additionally, the regulatory circuit LCPM-2 based on the unique dual-input promoter enhanced responses and reduced background leakage simultaneously, thereby providing an innovative strategy for the design and simplification of biosensors’ circuits.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 181-192"},"PeriodicalIF":4.4,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145159022","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":"Engineering heterologous core promoters to optimize the SES expression system for Pichia pastoris","authors":"Bo Sun ,&nbsp;Yu Tan ,&nbsp;Nitesh Kumar Mund ,&nbsp;Ketao Wang ,&nbsp;Lirong Yang ,&nbsp;Jianping Wu ,&nbsp;Jixing Luo ,&nbsp;Yuanqiang Li ,&nbsp;Ling Jiang ,&nbsp;Hao Fang ,&nbsp;Haoran Yu","doi":"10.1016/j.synbio.2025.10.005","DOIUrl":"10.1016/j.synbio.2025.10.005","url":null,"abstract":"<div><div>Synthetic biology has revolutionized microbial production by broadening the range of substrates, products, and host organisms. <em>Pichia pastoris</em>, a pivotal system for industrial enzyme production and biopharmaceuticals, encounters limitations in production capacity and transcriptional regulation, which impede its wider application. To enhance the performance of <em>P. pastoris</em>, we optimized the synthetic expression system (SES) by engineering heterologous core promoters derived from <em>Trichoderma reesei</em>. Through RNA-seq analysis, we identified high-expression genes’ promoters from <em>T. reesei</em>, which were integrated into the SES system to improve its efficiency in <em>P. pastoris</em>. A total of 214 candidate promoters were synthesized and screened based on expression of mCherry, and 54 of them were found to function in <em>P. pastoris</em>. Detailed characterization based on fluorescence intensity identified 41 SES systems showing enhanced mCherry expression compared to the traditional SES-A by up to 5.4 times. The best SES-CP32 was successfully used to expresse Protease K (Pro K) in <em>P. pastoris</em>. Additionally, we employed a multi-copy strategy, utilizing CRISPR/Cas9 for rapid multi-targeted integration, to further enhance the expression efficiency of the SES system in producing Pro K, with the enzyme activity in the fermentation supernatant of 3 copy of Pro K being 4.6 times greater than 1 copy of Pro K. This study enhances the robustness and versatility of the SES system, establishing a foundation for expanding the utility of the SES system in <em>P. pastoris</em>.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 333-341"},"PeriodicalIF":4.4,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145416666","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":"Construction and analysis of a cell factory for terpenoid biosynthesis in Pichia pastoris via metabolic engineering and metabolomics","authors":"Chenfan Sun ,&nbsp;Cuifang Ye ,&nbsp;Xiaoqian Li ,&nbsp;Jiabin Xu ,&nbsp;Huiru Yu ,&nbsp;Jucan Gao ,&nbsp;Chengran Guan ,&nbsp;Jintao Cheng","doi":"10.1016/j.synbio.2025.08.015","DOIUrl":"10.1016/j.synbio.2025.08.015","url":null,"abstract":"<div><div>Terpenoids are widely distributed in nature and have various applications in health products, pharmaceuticals, and fragrances. Despite the tremendous potential of terpenoids, traditional production methods such as plant extraction and chemical synthesis face challenges in meeting current market demands. With the developments in synthetic biology and metabolic engineering, it has become feasible to construct efficient microbial cell factories for large-scale production for terpenoids. In this work, using the yeast <em>Pichia pastoris</em> as the host cell, a \"plug-and-play\" cell factory for universal terpenoid production was constructed by enhancing the expression of the MVA pathway for common precursor synthesis and reducing branch pathway diversion strategies. We have successfully and efficiently synthesized β-elemene, β-farnesene, (+)-valencene, (−)-α-bisabolol by this cell factory. Furthermore, by analyzing metabolites in different engineered strains in terms of system biology, it was discovered that an increase in key protein copy numbers enhanced the synthesis of arginine and other metabolic pathways. The robustness of the strains and the tightly regulated metabolic network constrain rational metabolic engineering transformations. These data provide important clues for the modification and optimization of production strains.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 68-75"},"PeriodicalIF":4.4,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145046582","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":"The coupling effect of Penicillium baileys W2 in the Aspergillus flavus inhibition and peanut growth promotion","authors":"Qianqian Wang ,&nbsp;Shihua Shan ,&nbsp;Quanxi Sun ,&nbsp;Xiaobo Zhao ,&nbsp;Cuiling Yuan ,&nbsp;Yifei Mou ,&nbsp;Juan Wang ,&nbsp;Caixia Yan ,&nbsp;Qi Wang ,&nbsp;Qingchen Rui ,&nbsp;Chunjuan Li","doi":"10.1016/j.synbio.2025.09.003","DOIUrl":"10.1016/j.synbio.2025.09.003","url":null,"abstract":"<div><div><em>Aspergillus flavus</em> is a significant plant pathogen, and peanut crops are particularly vulnerable to aflatoxin contamination. This vulnerability underscores the need for more effective control methods. In this study, the strain <em>Penicillium baileys</em> W2 was isolated from the rhizosphere soils of healthy peanut seedlings. The fermentation extract exhibited concentration-dependent inhibition of pathogenic <em>A. flavus</em> growth, with a minimum inhibitory concentration (MIC) of 55 % and a minimum fungicidal concentration (MFC) of 60 %. Physiological data and transcriptome analysis demonstrated that the W2 fermentation supernatant inhibited <em>A. flavus</em> growth by disrupting membrane permeability. Metabolomics analysis identified active compounds, including propylparaben, taxifolin, and phloretin, which exhibited significant antagonistic effects against <em>A. flavus</em>. Additionally, we evaluated the impact of the W2 fermentation broth on peanut growth promotion and on rhizosphere microbial community structure using metagenomic sequencing. The reduction of harmful soil microorganisms contributed to the maintenance of soil health, whereas the increased abundance of beneficial microorganisms enhanced peanut seedling growth by facilitating soil nutrient cycling. These findings indicate that the development and application of <em>P. baileys</em> strain W2 or its fermentation extract aligns with sustainable agricultural principles and offers a promising biological control approach.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 127-140"},"PeriodicalIF":4.4,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145096568","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":"Engineering genetic elements for microbial protein expression systems: Advances, challenges, applications, and prospects","authors":"Xiaoqian Li ,&nbsp;Cuifang Ye ,&nbsp;Tao Liu ,&nbsp;Shiyu Li ,&nbsp;Mengyu Zhang ,&nbsp;Yao Zhao ,&nbsp;Yuanxiang Jin ,&nbsp;Jintao Cheng ,&nbsp;Guiling Yang ,&nbsp;Peiwu Li","doi":"10.1016/j.synbio.2025.10.008","DOIUrl":"10.1016/j.synbio.2025.10.008","url":null,"abstract":"<div><div>The rising global demand for sustainable protein sources poses critical challenges across food, pharmaceutical, and industrial biotechnology sectors. Microbial expression systems provide scalable and versatile platforms for producing recombinant proteins, including enzymes, therapeutic molecules, and functional food ingredients. These platforms enable efficient biosynthesis of high-value proteins from renewable substrates often via precision fermentation, surpassing conventional methods in yield, cost-efficiency, and environmental sustainability. This review summarizes the genetic regulatory elements that govern gene expression in microbial hosts, with comparative coverage of prokaryotic and eukaryotic systems at transcriptional and translational levels. Key regulatory components, such as promoters, ribosome binding sites (RBS), untranslated regions (UTRs), signal peptides, and terminators, are discussed in the context of host-specific engineering strategies. We highlight advanced engineering approaches, including artificial intelligence (AI) assisted sequence design, CRISPR-Cas-based genome editing, and modular combinatorial optimization of genetic elements. Particular attention is given to the integration of high-throughput screening and predictive modeling tools that accelerate the rational design and optimization of microbial production systems. The review also discusses practical applications in food, pharmaceutical, and industrial enzyme production, emphasizing how genetic element engineering bridges fundamental research and biomanufacturing. Finally, key challenges and future prospects are analyzed to guide the development of next-generation microbial cell factories for sustainable protein production and industrial innovation.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 370-384"},"PeriodicalIF":4.4,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145465810","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":"Topoisomerase II-Like variants enhance acid tolerance and low-pH l-malic acid biosynthesis in metabolic engineered Aspergillus nidulans","authors":"Lingling Pei,&nbsp;Yan Dong,&nbsp;Ziqing Chen,&nbsp;Chi Zhang,&nbsp;Ling Lu","doi":"10.1016/j.synbio.2025.08.008","DOIUrl":"10.1016/j.synbio.2025.08.008","url":null,"abstract":"<div><div><span>l</span>-malic acid is a versatile compound having extensive applications across food, pharmaceutical, medical, and chemical industries while its production from microbial fermentation offers sustainable advantages including renewable feedstocks and environmental friendliness. However, a major challenge faced by microbial <span>l</span>-malic acid production is acid-induced pH drop during the fermentation process, requiring excessive neutralizers (e.g., CaCO₃) that increase costs and environmental burdens greatly. In this study, we firstly made an engineered <em>Aspergillus nidulans</em> strain as a production chassis through metabolic engineering by overexpression of fructose-6-phosphate kinase gene (<em>pfkA</em>) combined with deletion of pyruvate decarboxylase gene (<em>pdcA</em>) so that the titer of <span>l</span>-malic acid was increased by 20.3 %. Secondly, overexpression of glucose transporter gene (<em>mstE)</em> further boosted production to the highest reported shake-flask titer (44.3 g/L) for <em>A. nidulans</em>. Moreover, acid-tolerant isolates capable of growth at pH 2.8 has been successfully obtained by adaptive laboratory evolution in aforementioned engineered strains. They achieved 75 % neutralizer reduction (just adding 10 g/L CaCO₃) while maintaining original titers. Whole-genome sequencing comparison identified mutations in gene (<em>ANIA_04699</em>) encoding a protein with a topoisomerase II associated PAT I domain happened in acid-tolerance isolates. Functional validation via engineered point-mutants confirmed mutations of the gene (<em>ANIA_04699)</em> as a key acid-tolerance determinant, contributes a major role enabling CaCO3 reduction without titer loss. Meanwhile, phylogenetic studies showed there are about 71–75 % homology across selected <em>Aspergillus</em> spp. suggesting high conservation and broad applicability. Function prediction analysis based on previous verified PAT I domains imply that mutations of this topoisomerase II-Like protein might be tolerant to DNA damage induced by low pH environment. This work establishes <em>A. nidulans</em> as a platform for low-pH organic acid production and identifies topoisomerase II variants as new targeted candidates for acid tolerance engineering.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 19-27"},"PeriodicalIF":4.4,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144933822","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 novel transformation system using zhongshengmycin in Aspergillus oryzae and Aspergillus niger","authors":"Futi Bi ,&nbsp;Qichen Huang ,&nbsp;Baoxiang Pan ,&nbsp;Bin Wang ,&nbsp;Li Pan","doi":"10.1016/j.synbio.2025.08.014","DOIUrl":"10.1016/j.synbio.2025.08.014","url":null,"abstract":"<div><div>Filamentous fungi such as <em>Aspergillus oryzae</em> and <em>Aspergillus niger</em> serve as valuable microbial cell factories with diverse applications. However, genetic manipulation in these fungi is often hindered by the lack of efficient resistance-based transformation systems. To address this challenge, we developed a novel transformation system for both <em>A. oryzae</em> and <em>A. niger</em> based on zhongshengmycin, a cost-effective streptothricin-class antibiotic, used in combination with the resistance gene encoding nourseothricin acetyltransferase (NAT). Our tests revealed that sensitization with SDS and EDTA-Na₂ disrupts cell membrane integrity, thereby enhancing the susceptibility of <em>Aspergillus</em> to zhongshengmycin. Through an attB/attP-mediated integration system, we achieved heterologous expression of β-glucuronidase in <em>A. oryzae</em> with a selection efficiency of 85.71 % (6/7). Additionally, recombination of <em>megfp-sed5</em> in <em>A. niger</em> achieved a transformation efficiency of 83.33 % (10/12), comparable to that of the PyrG system, thus establishing a versatile platform for fungal biotechnology. This system enables precise genomic integration for metabolic rewiring, offering new strategies for the synthetic biology-driven improvement of industrial filamentous fungi.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 205-215"},"PeriodicalIF":4.4,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145220361","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":"A smart gene circuit for precise regulation of tumor cell behavior","authors":"Hao Fu ,&nbsp;Zhiwei Zhou ,&nbsp;Lan Fang ,&nbsp;Qing Wang ,&nbsp;Xin Tang ,&nbsp;Wei Su ,&nbsp;Xuancai Chen ,&nbsp;Yachun Tang ,&nbsp;Qun Zhou","doi":"10.1016/j.synbio.2025.09.016","DOIUrl":"10.1016/j.synbio.2025.09.016","url":null,"abstract":"<div><div>Personalized gene circuit is a robust mode of cellular regulation that can manipulate intracellular gene expression to achieve desired functional regulation. However, the construction of logic circuits that automatically sense the characteristics of a particular environment within a cell is often difficult and lacking in sensitivity. Here, we synthesize from scratch specific promoters capable of sensing in cells, and use the combination of different types of promoters to construct smart gene circuits that can regulate gene expression in specific cell types sensitively. In detail, the tumour-specific promoter and the prostate tissue-specific promoter were constructed to be combined together into generating an artificial AND-gate gene circuit using the CRISPR-Cas9 system which could identify prostate cancer selectively. We then utilized this artificial gene circuit to drive targeted genes expression, such as P21, E-cadherin and Bax, to inhibit multifunctional prostate cancer cells but not other cells. Moreover, we applied gene circuits to redirect endogenous genes within cells and significantly and specifically suppressed the tumor growth of prostate cancer in vivo. Overall, these results highlight the clinical potential of these gene circuits as specific tools for prostate cancer detection and treatment, which is a new method for specifically reprogramming prostate cancer cells <em>in vivo</em> and may serve as a promising treatment strategy.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 237-246"},"PeriodicalIF":4.4,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145220420","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":"Efficient trans-aconitic acid production using systematically metabolic engineered Escherichia coli","authors":"Leilei Guo ,&nbsp;Yi Cheng ,&nbsp;Xiaoxu Tan ,&nbsp;Hongxu Zhang ,&nbsp;Hairong Yu ,&nbsp;Wenjia Tian ,&nbsp;Mingyuan Liu ,&nbsp;Weikang Sun ,&nbsp;Kaiyu Gao ,&nbsp;Tianyi Jiang ,&nbsp;Qianjin Kang ,&nbsp;Wensi Meng ,&nbsp;Yidong Liu ,&nbsp;Zhaoqi Kang ,&nbsp;Chuanjuan Lü ,&nbsp;Chao Gao ,&nbsp;Ping Xu ,&nbsp;Cuiqing Ma","doi":"10.1016/j.synbio.2025.09.009","DOIUrl":"10.1016/j.synbio.2025.09.009","url":null,"abstract":"<div><div><em>trans</em>-Aconitic acid (TAA) is a versatile platform biochemical exhibiting extensive applications. In this work, <em>Escherichia coli</em> W3110 (DE3) was metabolic modified for the de novo biosynthesis of TAA. Firstly, a pyruvate accumulation chassis strain <em>E. coli</em> W3110-P6 was constructed through deletion of six byproducts generation genes. Secondly, the aconitate isomerase was screened from four candidates and co-overexpressed with TAA transporter to construct heterologous TAA biosynthetic pathway in <em>E. coli</em> W3110-P6. Thirdly, the genes <em>pyc</em>^P458S encoding a feedback-insensitive pyruvate carboxylase and <em>gltA</em> encoding an allosterically unaffected citrate synthase were overexpressed, and <em>aceA</em> encoding the isocitrate lyase was deleted, to increase precursor supply and TAA generation of the recombinant <em>E. coli</em>. Finally, the fermentation condition of the obtained strain <em>E. coli</em> W3110-TAA08 was optimized. TAA at a concentration of 37.32 g/L was generated within 40 h, with a yield of 0.76 g/g glucose and a productivity of 0.93 g/L/h.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 110-116"},"PeriodicalIF":4.4,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145096564","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/Cas12a genome editing toolbox in Kluyveromyces marxianus and its application in succinic acid biosynthesis","authors":"Hao Zha ,&nbsp;Yanjie Li ,&nbsp;Zhongmei Hu,&nbsp;Jiacheng Li,&nbsp;Yujie Xie,&nbsp;Mingtao Zhao,&nbsp;Lili Ren,&nbsp;Biao Zhang","doi":"10.1016/j.synbio.2025.09.015","DOIUrl":"10.1016/j.synbio.2025.09.015","url":null,"abstract":"<div><div><em>Kluyveromyces marxianus</em> is a promising thermotolerant yeast for industrial biotechnology, but lacks efficient genome engineering tools. A CRISPR/Cas12a genome editing toolbox for <em>K</em>. <em>marxianus</em> was developed for the first time in this study. A plasmid-free transient system achieved single-gene knockout efficiencies of about 50 %–100 % in <em>Δku70</em> strain. Even with homology arms as short as 35 bp, the knockout efficiency remained 66.67 %. Chromosomal integration of Cas12a enabled single-to-triple fragment knock-ins efficiency of 82.93–85.70 % and 94.50 % for large fragment (&gt;5 kb) integrations. Applying this system, the roles of succinate dehydrogenase (<em>SDH</em>) genes <em>SDH1-SDH5</em> were elucidated. Combinatorial <em>SDH</em> genes knockouts redirected carbon flux toward succinic acid (SA), but increased glycerol/acetate byproducts. Subsequent <em>GPD1</em>/<em>ACH1/ADH2A</em> co-knockout in a <em>Δsdh1,3,5,4A,2</em> strain with <em>NDE1</em> overexpression (YZH43) yielded a chassis producing 32.38 g/L SA from glucose at 37 °C, which is the highest reported titer in <em>K. marxianus</em>, while reducing ethanol, acetate, and glycerol by 60.79 %, 89.24 %, and 67.5 %, respectively. At 46 °C, YZH43 produced 20.51 g/L SA through simultaneous saccharification and fermentation (SSF) using cellulose as substrate. This work provides a high-efficiency CRISPR/Cas12a platform for <em>K. marxianus</em>, enabling rapid metabolic engineering for value-added chemical production, and demonstrates its utility in developing thermotolerant SA-overproducing strains.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 193-204"},"PeriodicalIF":4.4,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145158419","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}