Synthetic and Systems Biotechnology最新文献

{"title":"Predicting effective drug combinations for cancer treatment using a graph-based approach","authors":"Qi Wang ,&nbsp;Xiya Liu ,&nbsp;Guiying Yan","doi":"10.1016/j.synbio.2024.09.003","DOIUrl":"10.1016/j.synbio.2024.09.003","url":null,"abstract":"<div><div>Drug combination therapy, involving the use of two or more drugs, has been widely employed to treat complex diseases such as cancer. It enhances therapeutic efficacy, reduces drug resistance, and minimizes side effects. However, traditional methods to identify effective drug combinations are time-consuming, costly, and less efficient than computational methods. Therefore, developing computational approaches to predict drug combinations has become increasingly important.</div><div>In this paper, we developed the Random Walk with Restart for Drug Combination (RWRDC) model to predict effective drug combinations for cancer therapy. The RWRDC model offers a quantitative mathematical method for predicting the potential effective drug combinations. Cross-validation results indicate that the RWRDC model outperforms other predictive models, particularly in breast, colorectal, and lung cancer predictions across various performance metrics. We have theoretically proven the convergence of its algorithm and provided an explanation for the algorithm's rationality. A targeted case study on breast cancer further highlights the capability of RWRDC to identify effective drug combinations. These findings highlight our model as a novel and effective tool for discovering potential effective drug combinations, offering new possibilities in therapy. Additionally, the graph-based framework of RWRDC holds potential for predicting drug combinations in other complex diseases, expanding its utility in the medical field.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 148-155"},"PeriodicalIF":4.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142445486","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessing the physiological properties of baker's yeast based on single-cell Raman spectrum technology","authors":"Xi Sun ,&nbsp;Xin Zhou ,&nbsp;Ran Yu ,&nbsp;Xiaofang Zhou ,&nbsp;Jun Zhang ,&nbsp;Teng Xu ,&nbsp;Jianmei Wang ,&nbsp;Mengqi Li ,&nbsp;Xiaoting Li ,&nbsp;Min Zhang ,&nbsp;Jian Xu ,&nbsp;Jia Zhang","doi":"10.1016/j.synbio.2024.09.004","DOIUrl":"10.1016/j.synbio.2024.09.004","url":null,"abstract":"<div><div>With rapid progress in the yeast fermentation industry, a comprehensive commercial yeast quality assessment approach integrating efficiency, accuracy, sensitivity, and cost-effectiveness is required. In this study, a new yeast quality assessment method based on single-cell Raman technology was developed and contrasted with traditional methods. The findings demonstrated significant associations (Pearson correlation coefficient of 0.933 on average) between the two methods in measuring physiological indicators, including cell viability and intracellular trehalose content, demonstrating the credibility of the Raman method compared to the traditional method. Furthermore, the sensitivity of the Raman method in viable but non-culturable cells was higher in measuring yeast cell viability (17.9 % more sensitive). According to the accurate quantitative analysis of metabolic activity level (MAL) of yeast cells, the cell vitality was accurately quantified at population and single-cell levels, offering a more comprehensive assessment of yeast fermentation performance. Overall, the single-cell Raman method integrates credibility, feasibility, accuracy, and sensitivity in yeast quality assessment, offering a new technological framework for quality assessments of live-cell yeast products.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 110-118"},"PeriodicalIF":4.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142441270","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineered probiotic-mediated intratumoral delivery and controlled release of bacterial collagenase for cancer therapy","authors":"Hong-Rui Li,&nbsp;Bang-Ce Ye","doi":"10.1016/j.synbio.2024.09.001","DOIUrl":"10.1016/j.synbio.2024.09.001","url":null,"abstract":"<div><div>Elevated collagen levels within breast tumors are strongly associated with tumor progression and present a barrier to effective therapeutic agent penetration within the tumor microenvironment (TME), leading to poor clinical outcomes. To address this challenge, we engineered a probiotic strain to degrade collagen within the TME by selectively colonizing in tumors and releasing bacterial collagenase in a lysis-dependent manner. Initially, we constructed a therapeutic bacterial strain designed to lyse within the TME and release an encoded immunotoxin comprising a nanobody targeting CD47 (CD47nb) and a modified <em>Pseudomonas</em> exotoxin A (PE38KDEL). The introduction of collagenase-expressing bacteria, in conjunction with therapeutic immunotoxin, reduced collagen fiber levels within the TME, resulting in inhibited tumor growth and prolonged survival in a murine model of breast cancer. Furthermore, we investigated the broader applicability of the collagenase-expressing bacterial strain in combination with chemotherapeutic drugs, such as doxorubicin. Remarkably, synergistic antitumor effects were observed in mice treated with this combination therapy. In conclusion, our study demonstrates that probiotic delivery of bacterial collagenase offers a promising adjuvant treatment strategy for selectively degrading intratumoral collagen, thereby improving the efficacy of anticancer therapies in breast cancer.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 226-236"},"PeriodicalIF":4.4,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142659617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Tissue chips as headway model and incitement technology","authors":"Prerna Suchitan Modi ,&nbsp;Abhishek Singh ,&nbsp;Awyang Chaturvedi ,&nbsp;Shailly Agarwal ,&nbsp;Raghav Dutta ,&nbsp;Ranu Nayak ,&nbsp;Alok Kumar Singh","doi":"10.1016/j.synbio.2024.08.007","DOIUrl":"10.1016/j.synbio.2024.08.007","url":null,"abstract":"<div><p>Tissue on a chip or organ-on-chip (OOC) is a technology that's dignified to form a transformation in drug discovery through the use of advanced platforms. These are 3D in<em>-vitro</em> cell culture models that mimic micro-environment of human organs or tissues on artificial microstructures built on a portable microfluidic chip without involving sacrificial humans or animals.</p><p>This review article aims to offer readers a thorough and insightful understanding of technology. It begins with an in-depth understanding of chip design and instrumentation, underlining its pivotal role and the imperative need for its development in the modern scientific landscape. The review article explores into the myriad applications of OOC technology, showcasing its transformative impact on fields such as radiobiology, drug discovery and screening, and its pioneering use in space research. In addition to highlighting these diverse applications, the article provides a critical analysis of the current challenges that OOC technology faces. It examines both the biological and technical limitations that hinder its progress and efficacy and discusses the potential advancements and innovations that could drive the OOC technology forward. Through this comprehensive review, readers will gain a deep appreciation of the significance, capabilities, and evolving landscape of OOC technology.</p></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 86-101"},"PeriodicalIF":4.4,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405805X24001182/pdfft?md5=fdd969b5051ee124e51a362370907009&pid=1-s2.0-S2405805X24001182-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142096001","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering the TetR-family transcriptional regulator XNR_0706 to enhance heterologous spinosad production in Streptomyces albus B4 chassis","authors":"Xingjun Cui ,&nbsp;Hao Tang ,&nbsp;Wenzong Wang ,&nbsp;Wenping Wei ,&nbsp;Jing Wu ,&nbsp;Bang-Ce Ye","doi":"10.1016/j.synbio.2024.08.008","DOIUrl":"10.1016/j.synbio.2024.08.008","url":null,"abstract":"<div><div>The TetR family of regulators are an important group of transcription regulators that regulate diverse cellular processes in prokaryotes. In this study, we found that XNR_0706, a TetR family regulator, controlled the expression of <em>XNR_0345</em>, <em>XNR_0454</em>, <em>XNR_0513</em> and <em>XNR_1438</em> putatively involved in fatty acid β-oxidation by interacting with the promoter regions in <em>Streptomyces albus</em> B4. The transcription level of these four genes was downregulated in <em>XNR_0706</em> deletion strain (ΔXNR_0706) and restored by <em>XNR_0706</em> complementation in Δ0706/pIB-<em>0706</em>, demonstrating that XNR_0706 was a positive transcriptional regulator of the genes. With toxic long-chain fatty acids addition in TSB media, deletion of <em>XNR_0706</em> caused significantly poor growth, whereas <em>XNR_0706</em> complementation increased the utilization of additional fatty acids, resulting in restored growth. Fatty acid β-oxidation is one source of acetyl- and malonyl-CoA precursors for polyketides biosynthesis in actinobacteria. Overexpression of <em>XNR_0706</em> in B4/spnNEW, a spinosad heterologous expression strain derived from <em>S. albus</em> B4, increased spinosad yield by 20.6 %. Additionally, supplement of 0.3 g/L fatty acids resulted in a further 42.4 % increase in spinosad yield. Our study reveals a regulatory mechanism in long-chain fatty acids metabolism in <em>S. albus</em> and these insights into the molecular regulation of β-oxidation by XNR_0706 are instrumental for increasing secondary metabolites in actinobacteria.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 218-225"},"PeriodicalIF":4.4,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142659616","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme","authors":"Yongjian Qiao,&nbsp;Junbo Wang,&nbsp;Dashan Zhang,&nbsp;Xiaoqing Zheng,&nbsp;Baixin Lin,&nbsp;Yongkang Huang,&nbsp;Yulin Liao,&nbsp;Zixin Deng,&nbsp;Lingxin Kong,&nbsp;Delin You","doi":"10.1016/j.synbio.2024.08.006","DOIUrl":"10.1016/j.synbio.2024.08.006","url":null,"abstract":"<div><p>Anisomycin (compound <strong>1</strong>), a multifunctional pyrrolidine antibiotic, primarily inhibits protein biosynthesis by binding to the ribosome. Upon binding to the ribosome, the para-phenol moiety of anisomycin inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incoming aminoacyl-tRNAs, disrupting peptide bond formation. Hence, the para-methoxyphenyl group serves as a starting point for developing novel anisomycin analogs with potent antifungal and insecticidal properties. However, the activation and condensation mechanism of phenylpyruvic acid has not yet been elucidated. In this study, genetic manipulations of <em>aniP</em> and its homologue <em>siAniP</em> confirmed their indispensable role in <strong>1</strong> biosynthesis. Bioinformatics analysis suggested that AniP and siAniP function as transketolase. siAniP was found to catalyzed condensation between 4-hydroxyphenylpyruvic acid (<strong>3</strong>) and glyceraldehyde (GA), initiating pyrrolidine synthesis. siAniP was specific for aromatic keto acids and tolerant of aliphatic and aromatic aldehydes, and was able to catalyze the asymmetric intermolecular condensation of two keto acids, leading to the formation of 24 α-hydroxy ketone. To the best of our knowledge, siAniP is the first TK that catalyzes the transfer of a C2 ketol and symmetrical intermolecular coupling using aromatic keto acids as donor substrates. Structural analysis, docking model construction, and site-directed mutagenesis identified that I220, H275, R322 and W391 were crucial for substrate binding. Moreover, sequence similarity network (SSN)-based genome neighborhood network (GNN) analyses of AniP suggested the widespread occurrence of the AniP-like-mediated reaction in the biosynthesis of <strong>1</strong> and its analogs, particularly in the assembly of benzylpyrrolidine. These findings not only expand the repertoire of TKs but also provide a potent biocatalyst that could be used for the structural innovation of <strong>1</strong> and its derivatives.</p></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 76-85"},"PeriodicalIF":4.4,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405805X24001170/pdfft?md5=a3b2902611aa71c849c7eb7f73b3c4a3&pid=1-s2.0-S2405805X24001170-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142050440","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhancing levan biosynthesis by destroying the strongly acidic environment caused by membrane-bound glucose dehydrogenase (mGDH) in Gluconobacter sp. MP2116","authors":"Junjie Tian ,&nbsp;Shumin Wei ,&nbsp;Wenxing Liang ,&nbsp;Guangyuan Wang","doi":"10.1016/j.synbio.2024.08.005","DOIUrl":"10.1016/j.synbio.2024.08.005","url":null,"abstract":"<div><p>Levan produced by <em>Gluconobacter</em> spp. has great potential in biotechnological applications. However, <em>Gluconobacter</em> spp. can synthesize organic acids during fermentation, resulting in environmental acidification. Few studies have focused on the effects of environmental acidification on levan synthesis. This study revealed that the organic acids, mainly gluconic acid (GA) and 2-keto-gluconic acid (2KGA) secreted by <em>Gluconobacter</em> sp. MP2116 created a highly acidic environment (pH &lt; 3) that inhibited levan biosynthesis. The levansucrase derived from strain MP2116 had high enzyme activity at pH 4.0 ∼ pH 6.5. When the ambient pH was less than 3, the enzyme activity decreased by 67 %. Knocking out the <em>mgdh</em> gene of membrane-bound glucose dehydrogenase (mGDH) in the GA and 2KGA synthesis pathway in strain MP2116 eliminated the inhibitory effect of high acid levels on levansucrase function. As a result, the levan yield increased from 7.4 g/l (wild-type) to 18.8 g/l (Δ<em>mgdh</em>) during fermentation without pH control. This study provides a new strategy for improving levan production by preventing the inhibition of polysaccharide synthesis by environmental acidification.</p></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 68-75"},"PeriodicalIF":4.4,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405805X24001169/pdfft?md5=d2bc19bfe837c1f30763278c26cd8634&pid=1-s2.0-S2405805X24001169-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142039739","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Systematic metabolic engineering enables highly efficient production of vitamin A in Saccharomyces cerevisiae","authors":"Yi Shi ,&nbsp;Shuhuan Lu ,&nbsp;Xiao Zhou ,&nbsp;Xinhui Wang ,&nbsp;Chenglong Zhang ,&nbsp;Nan Wu ,&nbsp;Tianyu Dong ,&nbsp;Shilong Xing ,&nbsp;Ying Wang ,&nbsp;Wenhai Xiao ,&nbsp;Mingdong Yao","doi":"10.1016/j.synbio.2024.08.004","DOIUrl":"10.1016/j.synbio.2024.08.004","url":null,"abstract":"<div><p>Vitamin A is a micronutrient critical for versatile biological functions and has been widely used in the food, cosmetics, pharmaceutical, and nutraceutical industries. Synthetic biology and metabolic engineering enable microbes, especially the model organism <em>Saccharomyces cerevisiae</em> (generally recognised as safe) to possess great potential for the production of vitamin A. Herein, we first generated a vitamin A-producing strain by mining β-carotene 15,15′-mono(di)oxygenase from different sources and identified two isoenzymes <em>Mbblh</em> and <em>Ssbco</em> with comparable catalytic properties but different catalytic mechanisms. Combinational expression of isoenzymes increased the flux from β-carotene to vitamin A metabolism. To modulate the vitamin A components, retinol dehydrogenase 12 from <em>Homo sapiens</em> was introduced to achieve more than 90 % retinol purity using shake flask fermentation. Overexpressing <em>POS5Δ17</em> enhanced the reduced nicotinamide adenine dinucleotide phosphate pool, and the titer of vitamin A was elevated by almost 46 %. Multi-copy integration of the key rate-limiting step gene <em>Mbblh</em> further improved the synthesis of vitamin A. Consequently, the titer of vitamin A in the strain harbouring the Ura3 marker was increased to 588 mg/L at the shake-flask level. Eventually, the highest reported titer of 5.21 g/L vitamin A in <em>S. cerevisiae</em> was achieved in a 1-L bioreactor. This study unlocked the potential of <em>S. cerevisiae</em> for synthesising vitamin A in a sustainable and economical way, laying the foundation for the commercial-scale production of bio-based vitamin A.</p></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 58-67"},"PeriodicalIF":4.4,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405805X24001157/pdfft?md5=e084c2a4b3b96f9c53f2c9bc1be6d9ea&pid=1-s2.0-S2405805X24001157-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142012685","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering artificial cross-species promoters with different transcriptional strengths","authors":"Wenjie Zuo ,&nbsp;Guobin Yin ,&nbsp;Luyao Zhang ,&nbsp;Weijiao Zhang ,&nbsp;Ruirui Xu ,&nbsp;Yang Wang ,&nbsp;Jianghua Li ,&nbsp;Zhen Kang","doi":"10.1016/j.synbio.2024.08.003","DOIUrl":"10.1016/j.synbio.2024.08.003","url":null,"abstract":"<div><p>As a fundamental tool in synthetic biology, promoters are pivotal in regulating gene expression, enabling precise genetic control and spurring innovation across diverse biotechnological applications. However, most advances in engineered genetic systems rely on host-specific regulation of the genetic portion. With the burgeoning diversity of synthetic biology chassis cells, there emerges a pressing necessity to broaden the universal promoter toolkit spectrum, ensuring adaptability across various microbial chassis cells for enhanced applicability and customization in the evolving landscape of synthetic biology. In this study, we analyzed and validated the primary structures of natural endogenous promoters from <em>Escherichia coli</em>, <em>Bacillus subtilis</em>, <em>Corynebacterium glutamicum</em>, <em>Saccharomyces cerevisiae</em>, and <em>Pichia pastoris</em>, and through strategic integration and rational modification of promoter motifs, we developed a series of cross-species promoters (P_sh) with transcriptional activity in five strains (prokaryotic and eukaryotic). This series of cross species promoters can significantly expand the synthetic biology promoter toolkit while providing a foundation and inspiration for standardized development of universal components The combinatorial use of key elements from prokaryotic and eukaryotic promoters presented in this study represents a novel strategy that may offer new insights and methods for future advancements in promoter engineering.</p></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 49-57"},"PeriodicalIF":4.4,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405805X24001145/pdfft?md5=0d60323195a11533b64443b42b3f90d5&pid=1-s2.0-S2405805X24001145-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141953883","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishment of the CRISPR-Cpf1 gene editing system in Bacillus licheniformis and multiplexed gene knockout","authors":"Suxin Liu ,&nbsp;Fengxu Xiao ,&nbsp;Youran Li ,&nbsp;Yupeng Zhang ,&nbsp;Yanling Wang ,&nbsp;Guiyang Shi","doi":"10.1016/j.synbio.2024.08.002","DOIUrl":"10.1016/j.synbio.2024.08.002","url":null,"abstract":"<div><p><em>Bacillus licheniformis</em> is a significant industrial microorganism. Traditional gene editing techniques relying on homologous recombination often exhibit low efficiency due to their reliance on resistance genes. Additionally, the established CRISPR gene editing technology, utilizing Cas9 endonuclease, faces challenges in achieving simultaneous knockout of multiple genes. To address this limitation, the CRISPR-Cpf1 system has been developed, enabling multiplexed gene editing across various microorganisms. Key to the efficient gene editing capability of this system is the rigorous screening of highly effective expression elements to achieve conditional expression of protein Cpf1. In this study, we employed mCherry as a reporter gene and harnessed P_<em>mal</em> for regulating the expression of Cpf1 to establish the CRISPR-Cpf1 gene editing system in <em>Bacillus licheniformis</em>. Our system achieved a 100 % knockout efficiency for the single gene <em>vpr</em> and up to 80 % for simultaneous knockout of the double genes <em>epr</em> and <em>mpr</em>. Furthermore, the culture of a series of protease-deficient strains revealed that the protease encoded by <em>aprE</em> contributed significantly to extracellular enzyme activity (approximately 80 %), whereas proteases encoded by <em>vpr</em>, <em>epr</em>, and <em>mpr</em> genes contributed to a smaller proportion of extracellular enzyme activity. These findings provide support for effective molecular modification and metabolic regulation in industrial organisms.</p></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 39-48"},"PeriodicalIF":4.4,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405805X24001133/pdfft?md5=f69f2f7760a9aa1a358ca6c2a316639b&pid=1-s2.0-S2405805X24001133-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141964151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}