CRISPR-based diagnostics use the CRISPR-Cas system trans-cleavage activity to identify specific target sequences. When activated, this activity cleaves surrounding reporter molecules, producing a detectable signal. This technique has great specificity, sensitivity, and rapid detection, making it an important molecular diagnostic tool for medical and infectious disease applications. Despite its potential, the present CRISPR/Cas system has challenges with its single-stranded DNA reporters, characterized by low stability and limited sensitivity, restricting effective application in complex biological settings. In this work, we investigate the trans-cleavage activity of CRISPR/Cas12a on substrates utilizing fluorescent polystyrene microspheres to detect tetracycline. This innovative discovery led to the development of microsphere probes addressing the stability and sensitivity issues associated with CRISPR/Cas biosensing. By attaching the ssDNA reporter to polystyrene microspheres, we discovered that the Cas12a system exhibits robust and sensitive trans-cleavage activity. Further work revealed that the trans-cleavage activity of Cas12a on the microsphere surface is significantly dependent on the concentration of the ssDNA reporters. Building on these intriguing discoveries, we developed microsphere-based fluorescent probes for CRISPR/Cas aptasensors, which showed stability and sensitivity in tetracycline biosensing. We demonstrated a highly sensitive detection of tetracycline with a detection limit of 0.1 μM. Finally, the practical use of a microsphere-based CRISPR/Cas aptasensor in spiked food samples was proven successful. These findings highlighted the remarkable potential of microsphere-based CRISPR/Cas aptasensors for biological research and medical diagnosis.
{"title":"Detection of Tetracycline with a CRISPR/Cas12a Aptasensor Using a Highly Efficient Fluorescent Polystyrene Microsphere Reporter System.","authors":"Bong Jing Yee, Siti Nurul Azian Zakaria, Rona Chandrawati, Minhaz Uddin Ahmed","doi":"10.1021/acssynbio.4c00200","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00200","url":null,"abstract":"<p><p>CRISPR-based diagnostics use the CRISPR-Cas system <i>trans</i>-cleavage activity to identify specific target sequences. When activated, this activity cleaves surrounding reporter molecules, producing a detectable signal. This technique has great specificity, sensitivity, and rapid detection, making it an important molecular diagnostic tool for medical and infectious disease applications. Despite its potential, the present CRISPR/Cas system has challenges with its single-stranded DNA reporters, characterized by low stability and limited sensitivity, restricting effective application in complex biological settings. In this work, we investigate the <i>trans</i>-cleavage activity of CRISPR/Cas12a on substrates utilizing fluorescent polystyrene microspheres to detect tetracycline. This innovative discovery led to the development of microsphere probes addressing the stability and sensitivity issues associated with CRISPR/Cas biosensing. By attaching the ssDNA reporter to polystyrene microspheres, we discovered that the Cas12a system exhibits robust and sensitive <i>trans</i>-cleavage activity. Further work revealed that the <i>trans</i>-cleavage activity of Cas12a on the microsphere surface is significantly dependent on the concentration of the ssDNA reporters. Building on these intriguing discoveries, we developed microsphere-based fluorescent probes for CRISPR/Cas aptasensors, which showed stability and sensitivity in tetracycline biosensing. We demonstrated a highly sensitive detection of tetracycline with a detection limit of 0.1 μM. Finally, the practical use of a microsphere-based CRISPR/Cas aptasensor in spiked food samples was proven successful. These findings highlighted the remarkable potential of microsphere-based CRISPR/Cas aptasensors for biological research and medical diagnosis.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141309475","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 : 2024-06-11DOI: 10.1021/acssynbio.4c00306
Qidou Gao, Yaqi Dong, Ying Huang, Sasa Liu, Xiaochun Zheng, Yiming Ma, Qingsheng Qi, Xue Wang, Zongbao Kent Zhao, Xiaobing Yang
Rhodotorula toruloides is a potential workhorse for production of various value-added chemicals including terpenoids, oleo-chemicals, and enzymes from low-cost feedstocks. However, the limited genetic toolbox is hindering its metabolic engineering. In the present study, four type I and one novel type II peroxisomal targeting signal (PTS1/PTS2) were characterized and employed for limonene production for the first time in R. toruloides. The implant of the biosynthesis pathway into the peroxisome led to 111.5 mg/L limonene in a shake flask culture. The limonene titer was further boosted to 1.05 g/L upon dual-metabolic regulation in the cytoplasm and peroxisome, which included employing the acetoacetyl-CoA synthase NphT7, adding an additional copy of native ATP-dependent citrate lyase, etc. The final yield was 0.053 g/g glucose, which was the highest ever reported. The newly characterized PTSs should contribute to the expansion of genetic toolboxes forR. toruloides. The results demonstrated that R. toruloides could be explored for efficient production of terpenoids.
红豆杉(Rhodotorula toruloides)是利用低成本原料生产各种高附加值化学品(包括萜类、油脂化学品和酶)的潜在主力军。然而,有限的遗传工具箱阻碍了它的代谢工程。在本研究中,首次对四种 I 型和一种新型 II 型过氧化物酶体靶向信号(PTS1/PTS2)进行了表征,并将其用于 R. toruloides 的柠檬烯生产。将生物合成途径植入过氧物酶体后,在摇瓶培养中可产生 111.5 mg/L 的柠檬烯。通过在细胞质和过氧物酶体中进行双代谢调节,包括使用乙酰乙酰-CoA 合成酶 NphT7、增加一个原生 ATP 依赖性柠檬酸裂解酶的拷贝等,柠檬烯的滴度进一步提高到 1.05 克/升。最终的产量为 0.053 克/克葡萄糖,这是迄今所报道的最高产量。新鉴定的 PTS 应有助于扩大 toruloides 的基因工具箱。研究结果表明,可以探索 R. toruloides 高效生产萜类化合物的途径。
{"title":"Dual-Regulation in Peroxisome and Cytoplasm toward Efficient Limonene Biosynthesis with <i>Rhodotorula toruloides</i>.","authors":"Qidou Gao, Yaqi Dong, Ying Huang, Sasa Liu, Xiaochun Zheng, Yiming Ma, Qingsheng Qi, Xue Wang, Zongbao Kent Zhao, Xiaobing Yang","doi":"10.1021/acssynbio.4c00306","DOIUrl":"10.1021/acssynbio.4c00306","url":null,"abstract":"<p><p><i>Rhodotorula toruloides</i> is a potential workhorse for production of various value-added chemicals including terpenoids, oleo-chemicals, and enzymes from low-cost feedstocks. However, the limited genetic toolbox is hindering its metabolic engineering. In the present study, four type I and one novel type II peroxisomal targeting signal (PTS1/PTS2) were characterized and employed for limonene production for the first time in <i>R. toruloides</i>. The implant of the biosynthesis pathway into the peroxisome led to 111.5 mg/L limonene in a shake flask culture. The limonene titer was further boosted to 1.05 g/L upon dual-metabolic regulation in the cytoplasm and peroxisome, which included employing the acetoacetyl-CoA synthase NphT7, adding an additional copy of native ATP-dependent citrate lyase, etc. The final yield was 0.053 g/g glucose, which was the highest ever reported. The newly characterized PTSs should contribute to the expansion of genetic toolboxes for<i>R. toruloides</i>. The results demonstrated that <i>R. toruloides</i> could be explored for efficient production of terpenoids.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141299339","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 : 2024-06-11DOI: 10.1021/acssynbio.4c00195
Jiawei Li, Lemin Wang, Nan Zhang, Si Cheng, Yi Wu* and Guang-Rong Zhao*,
Betanin is a water-soluble red-violet pigment belonging to the betacyanins family. It has become more and more attractive for its natural food colorant properties and health benefits. However, the commercial production of betanin, typically extracted from red beetroot, faces economic and sustainability challenges. Microbial heterologous production therefore offers a promising alternative. Here, we performed combinatorial engineering of plant P450 enzymes and precursor metabolisms to improve the de novo production of betanin in Saccharomyces cerevisiae. Semirational design by computer simulation and molecular docking was used to improve the catalytic activity of CYP76AD. Alanine substitution and site-directed saturation mutants were screened, with a combination mutant showing an approximately 7-fold increase in betanin titer compared to the wild type. Subsequently, betanin production was improved by enhancing the l-tyrosine pathway flux and UDP-glucose supply. Finally, after optimization of the fermentation process, the engineered strain BEW10 produced 134.1 mg/L of betanin from sucrose, achieving the highest reported titer of betanin in a shake flask by microbes. This work shows the P450 enzyme and metabolic engineering strategies for the efficient microbial production of natural complex products.
{"title":"Enzyme and Pathway Engineering for Improved Betanin Production in Saccharomyces cerevisiae","authors":"Jiawei Li, Lemin Wang, Nan Zhang, Si Cheng, Yi Wu* and Guang-Rong Zhao*, ","doi":"10.1021/acssynbio.4c00195","DOIUrl":"10.1021/acssynbio.4c00195","url":null,"abstract":"<p >Betanin is a water-soluble red-violet pigment belonging to the betacyanins family. It has become more and more attractive for its natural food colorant properties and health benefits. However, the commercial production of betanin, typically extracted from red beetroot, faces economic and sustainability challenges. Microbial heterologous production therefore offers a promising alternative. Here, we performed combinatorial engineering of plant P450 enzymes and precursor metabolisms to improve the <i>de novo</i> production of betanin in <i>Saccharomyces cerevisiae</i>. Semirational design by computer simulation and molecular docking was used to improve the catalytic activity of CYP76AD. Alanine substitution and site-directed saturation mutants were screened, with a combination mutant showing an approximately 7-fold increase in betanin titer compared to the wild type. Subsequently, betanin production was improved by enhancing the <span>l</span>-tyrosine pathway flux and UDP-glucose supply. Finally, after optimization of the fermentation process, the engineered strain BEW10 produced 134.1 mg/L of betanin from sucrose, achieving the highest reported titer of betanin in a shake flask by microbes. This work shows the P450 enzyme and metabolic engineering strategies for the efficient microbial production of natural complex products.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141304809","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 : 2024-06-11DOI: 10.1021/acssynbio.4c00161
Cong Wei, Xueying Lei and Songcheng Yu*,
A growing number of applications require simultaneous detection of multiplexed nucleic acid targets in a single reaction, which enables higher information density in combination with reduced assay time and cost. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-Cas system have broad applications for the detection of nucleic acids due to their strong specificity, high sensitivity, and excellent programmability. However, realizing multiplexed detection is still challenging for the CRISPR-Cas system due to the nonspecific collateral cleavage activity, limited signal reporting strategies, and possible cross-reactions. In this review, we summarize the principles, strategies, and features of multiplexed detection based on the CRISPR-Cas system and further discuss the challenges and perspective.
{"title":"Multiplexed Detection Strategies for Biosensors Based on the CRISPR-Cas System","authors":"Cong Wei, Xueying Lei and Songcheng Yu*, ","doi":"10.1021/acssynbio.4c00161","DOIUrl":"10.1021/acssynbio.4c00161","url":null,"abstract":"<p >A growing number of applications require simultaneous detection of multiplexed nucleic acid targets in a single reaction, which enables higher information density in combination with reduced assay time and cost. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-Cas system have broad applications for the detection of nucleic acids due to their strong specificity, high sensitivity, and excellent programmability. However, realizing multiplexed detection is still challenging for the CRISPR-Cas system due to the nonspecific collateral cleavage activity, limited signal reporting strategies, and possible cross-reactions. In this review, we summarize the principles, strategies, and features of multiplexed detection based on the CRISPR-Cas system and further discuss the challenges and perspective.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141299340","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 : 2024-06-11DOI: 10.1021/acssynbio.4c00297
Dong-Xun Li, Qi Guo*, Yu-Xin Yang, Shun-Jie Jiang, Xiao-Jun Ji, Chao Ye, Yue-Tong Wang and Tian-Qiong Shi*,
Monoterpenoids are an important subclass of terpenoids that play important roles in the energy, cosmetics, pharmaceuticals, and fragrances fields. With the development of biotechnology, microbial synthesis of monoterpenoids has received great attention. Yeasts such Saccharomyces cerevisiae and Yarrowia lipolytica are emerging as potential hosts for monoterpenoids production because of unique advantages including rapid growth cycles, mature gene editing tools, and clear genetic background. Recently, advancements in metabolic engineering and fermentation engineering have significantly enhanced the accumulation of monoterpenoids in cell factories. First, this review introduces the biosynthetic pathway of monoterpenoids and comprehensively summarizes the latest production strategies, which encompass enhancing precursor flux, modulating the expression of rate-limited enzymes, suppressing competitive pathway flux, mitigating cytotoxicity, optimizing substrate utilization, and refining the fermentation process. Subsequently, this review introduces four representative monoterpenoids. Finally, we outline the future prospects for efficient construction cell factories tailored for the production of monoterpenoids and other terpenoids.
{"title":"Recent Advances and Multiple Strategies of Monoterpenoid Overproduction in Saccharomyces cerevisiae and Yarrowia lipolytica","authors":"Dong-Xun Li, Qi Guo*, Yu-Xin Yang, Shun-Jie Jiang, Xiao-Jun Ji, Chao Ye, Yue-Tong Wang and Tian-Qiong Shi*, ","doi":"10.1021/acssynbio.4c00297","DOIUrl":"10.1021/acssynbio.4c00297","url":null,"abstract":"<p >Monoterpenoids are an important subclass of terpenoids that play important roles in the energy, cosmetics, pharmaceuticals, and fragrances fields. With the development of biotechnology, microbial synthesis of monoterpenoids has received great attention. Yeasts such <i>Saccharomyces cerevisiae</i> and <i>Yarrowia lipolytica</i> are emerging as potential hosts for monoterpenoids production because of unique advantages including rapid growth cycles, mature gene editing tools, and clear genetic background. Recently, advancements in metabolic engineering and fermentation engineering have significantly enhanced the accumulation of monoterpenoids in cell factories. First, this review introduces the biosynthetic pathway of monoterpenoids and comprehensively summarizes the latest production strategies, which encompass enhancing precursor flux, modulating the expression of rate-limited enzymes, suppressing competitive pathway flux, mitigating cytotoxicity, optimizing substrate utilization, and refining the fermentation process. Subsequently, this review introduces four representative monoterpenoids. Finally, we outline the future prospects for efficient construction cell factories tailored for the production of monoterpenoids and other terpenoids.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141299341","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 : 2024-06-11DOI: 10.1021/acssynbio.4c00112
Teng-Fei Long, Shi-Ying Zhou, Zi-Lei Huang, Gong Li, Qin Zhong, Xiao-Jing Zhang, Yuan-Yuan Li, Cai-Ping Chen, Li-Juan Xia, Ran Wei, Lei Wan, Ang Gao, Hao Ren, Xiao-Ping Liao, Ya-Hong Liu, Liang Chen and Jian Sun*,
Antimicrobial resistance poses a significant global challenge, demanding innovative approaches, such as the CRISPR-Cas-mediated resistance plasmid or gene-curing system, to effectively combat this urgent crisis. To enable successful curing of antimicrobial genes or plasmids through CRISPR-Cas technology, the development of an efficient broad-host-range delivery system is paramount. In this study, we have successfully designed and constructed a novel functional gene delivery plasmid, pQ-mini, utilizing the backbone of a broad-host-range Inc.Q plasmid. Moreover, we have integrated the CRISPR-Cas12f system into the pQ-mini plasmid to enable gene-curing in broad-host of bacteria. Our findings demonstrate that pQ-mini facilitates the highly efficient transfer of genetic elements to diverse bacteria, particularly in various species in the order of Enterobacterales, exhibiting a broader host range and superior conjugation efficiency compared to the commonly used pMB1-like plasmid. Notably, pQ-mini effectively delivers the CRISPR-Cas12f system to antimicrobial-resistant strains, resulting in remarkable curing efficiencies for plasmid-borne mcr-1 or blaKPC genes that are comparable to those achieved by the previously reported pCasCure system. In conclusion, our study successfully establishes and optimizes pQ-mini as a broad-host-range functional gene delivery vector. Furthermore, in combination with the CRISPR-Cas system, pQ-mini demonstrates its potential for broad-host delivery, highlighting its promising role as a novel antimicrobial tool against the growing threat of antimicrobial resistance.
{"title":"Innovative Delivery System Combining CRISPR-Cas12f for Combatting Antimicrobial Resistance in Gram-Negative Bacteria","authors":"Teng-Fei Long, Shi-Ying Zhou, Zi-Lei Huang, Gong Li, Qin Zhong, Xiao-Jing Zhang, Yuan-Yuan Li, Cai-Ping Chen, Li-Juan Xia, Ran Wei, Lei Wan, Ang Gao, Hao Ren, Xiao-Ping Liao, Ya-Hong Liu, Liang Chen and Jian Sun*, ","doi":"10.1021/acssynbio.4c00112","DOIUrl":"10.1021/acssynbio.4c00112","url":null,"abstract":"<p >Antimicrobial resistance poses a significant global challenge, demanding innovative approaches, such as the CRISPR-Cas-mediated resistance plasmid or gene-curing system, to effectively combat this urgent crisis. To enable successful curing of antimicrobial genes or plasmids through CRISPR-Cas technology, the development of an efficient broad-host-range delivery system is paramount. In this study, we have successfully designed and constructed a novel functional gene delivery plasmid, pQ-mini, utilizing the backbone of a broad-host-range Inc.Q plasmid. Moreover, we have integrated the CRISPR-Cas12f system into the pQ-mini plasmid to enable gene-curing in broad-host of bacteria. Our findings demonstrate that pQ-mini facilitates the highly efficient transfer of genetic elements to diverse bacteria, particularly in various species in the order of Enterobacterales, exhibiting a broader host range and superior conjugation efficiency compared to the commonly used pMB1-like plasmid. Notably, pQ-mini effectively delivers the CRISPR-Cas12f system to antimicrobial-resistant strains, resulting in remarkable curing efficiencies for plasmid-borne <i>mcr-1</i> or <i>bla</i><sub>KPC</sub> genes that are comparable to those achieved by the previously reported pCasCure system. In conclusion, our study successfully establishes and optimizes pQ-mini as a broad-host-range functional gene delivery vector. Furthermore, in combination with the CRISPR-Cas system, pQ-mini demonstrates its potential for broad-host delivery, highlighting its promising role as a novel antimicrobial tool against the growing threat of antimicrobial resistance.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141304810","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}
Escherichia coli, one of the most efficient expression hosts for recombinant proteins, is widely used in chemical, medical, food, and other industries. De novo engineering of gene regulation circuits and cell density-controlled E. coli cell lysis are promising directions for the release of intracellular bioproducts. Here, we developed an E. coli autolytic system, named the quorum sensing-mediated bacterial autolytic (QS-BA) system, by incorporating an acyl-homoserine lactone (AHL)-based YasI/YasR-type quorum sensing circuit from Pseudoalteromonas into E. coli cells. The results showed that the E. coli QS-BA system can release the intracellular bioproducts into the cell culture medium in terms of E. coli cell density, which offers an environmentally-friendly, economical, efficient, and flexible E. coli lysis platform for production of recombinant proteins. The QS-BA system has the potential to serve as an integrated system for the large-scale production of target products in E. coli for medical and industrial applications.
{"title":"Development of a Quorum Sensing-Mediated Bacterial Autolytic System in Escherichia coli for Automatic Release of Intracellular Products","authors":"Xiaofei Song, Yifan Zhao, Yixuan Ren, Ruoyu Liu, Mengting Zhang, Zhikai Zhang, Qiu Meng, Tingheng Zhu, Jianhua Yin and Zhiliang Yu*, ","doi":"10.1021/acssynbio.4c00084","DOIUrl":"10.1021/acssynbio.4c00084","url":null,"abstract":"<p ><i>Escherichia coli</i>, one of the most efficient expression hosts for recombinant proteins, is widely used in chemical, medical, food, and other industries. <i>De novo</i> engineering of gene regulation circuits and cell density-controlled <i>E. coli</i> cell lysis are promising directions for the release of intracellular bioproducts. Here, we developed an <i>E. coli</i> autolytic system, named the quorum sensing-mediated bacterial autolytic (QS-BA) system, by incorporating an acyl-homoserine lactone (AHL)-based YasI/YasR-type quorum sensing circuit from <i>Pseudoalteromonas</i> into <i>E. coli</i> cells. The results showed that the <i>E. coli</i> QS-BA system can release the intracellular bioproducts into the cell culture medium in terms of <i>E. coli</i> cell density, which offers an environmentally-friendly, economical, efficient, and flexible <i>E. coli</i> lysis platform for production of recombinant proteins. The QS-BA system has the potential to serve as an integrated system for the large-scale production of target products in <i>E. coli</i> for medical and industrial applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141299322","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}
Aromatic d-amino acids (d-AAs) play a pivotal role as important chiral building blocks and key intermediates in fine chemical and drug synthesis. Meso-diaminopimelate dehydrogenase (DAPDH) serves as an excellent biocatalyst in the synthesis of d-AAs and their derivatives. However, its strict substrate specificity and the lack of efficient engineering methods have hindered its widespread application. Therefore, this study aims to elucidate the catalytic mechanism underlying DAPDH from Proteus vulgaris (PvDAPDH) through the examination of its crystallographic structure, computational simulations of potential energies and molecular dynamics simulations, and site-directed mutagenesis. Mechanism-guided computational design showed that the optimal mutant PvDAPDH-M3 increased specific activity and catalytic efficiency (kcat/Km) for aromatic keto acids up to 124-fold and 92.4-fold, respectively, compared to that of the wild type. Additionally, it expanded the substrate scope to 10 aromatic keto acid substrates. Finally, six high-value-added aromatic d-AAs and their derivatives were synthesized using a one-pot three-enzyme cascade reaction, exhibiting a good conversion rate ranging from 32 to 84% and excellent stereoselectivity (enantiomeric excess >99%). These findings provide a potential synthetic pathway for the green industrial production of aromatic d-AAs.
{"title":"Mechanism-Guided Computational Design Drives meso-Diaminopimelate Dehydrogenase to Efficient Synthesis of Aromatic d-amino Acids","authors":"Tianfu Wu, Yihan Chen, Wanqing Wei, Wei Song, Jing Wu, Jian Wen, Guipeng Hu, Xiaomin Li, Cong Gao, Xiulai Chen and Liming Liu*, ","doi":"10.1021/acssynbio.4c00176","DOIUrl":"10.1021/acssynbio.4c00176","url":null,"abstract":"<p >Aromatic <span>d</span>-amino acids (<span>d</span>-AAs) play a pivotal role as important chiral building blocks and key intermediates in fine chemical and drug synthesis. <i>Meso</i>-diaminopimelate dehydrogenase (DAPDH) serves as an excellent biocatalyst in the synthesis of <span>d</span>-AAs and their derivatives. However, its strict substrate specificity and the lack of efficient engineering methods have hindered its widespread application. Therefore, this study aims to elucidate the catalytic mechanism underlying DAPDH from <i>Proteus vulgaris</i> (<i>Pv</i>DAPDH) through the examination of its crystallographic structure, computational simulations of potential energies and molecular dynamics simulations, and site-directed mutagenesis. Mechanism-guided computational design showed that the optimal mutant <i>Pv</i>DAPDH-M3 increased specific activity and catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) for aromatic keto acids up to 124-fold and 92.4-fold, respectively, compared to that of the wild type. Additionally, it expanded the substrate scope to 10 aromatic keto acid substrates. Finally, six high-value-added aromatic <span>d</span>-AAs and their derivatives were synthesized using a one-pot three-enzyme cascade reaction, exhibiting a good conversion rate ranging from 32 to 84% and excellent stereoselectivity (enantiomeric excess >99%). These findings provide a potential synthetic pathway for the green industrial production of aromatic <span>d</span>-AAs.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141282244","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 : 2024-06-06DOI: 10.1021/acssynbio.4c00070
Bakr Ahmed Taha*, Naser M. Ahmed, Rishi Kumar Talreja, Adawiya J. Haider, Yousif Al Mashhadany, Qussay Al-Jubouri, Aqilah Baseri Huddin, Mohd Hadri Hafiz Mokhtar, Sarvesh Rustagi, Ajeet Kaushik, Vishal Chaudhary* and Norhana Arsad*,
Antimicrobial resistance (AMR) poses a critical global One Health concern, ensuing from unintentional and continuous exposure to antibiotics, as well as challenges in accurate contagion diagnostics. Addressing AMR requires a strategic approach that emphasizes early stage prevention through screening in clinical, environmental, farming, and livestock settings to identify nonvulnerable antimicrobial agents and the associated genes. Conventional AMR diagnostics, like antibiotic susceptibility testing, possess drawbacks, including high costs, time-consuming processes, and significant manpower requirements, underscoring the need for intelligent, prompt, and on-site diagnostic techniques. Nanoenabled artificial intelligence (AI)-supported smart optical biosensors present a potential solution by facilitating rapid point-of-care AMR detection with real-time, sensitive, and portable capabilities. This Review comprehensively explores various types of optical nanobiosensors, such as surface plasmon resonance sensors, whispering-gallery mode sensors, optical coherence tomography, interference reflection imaging sensors, surface-enhanced Raman spectroscopy, fluorescence spectroscopy, microring resonance sensors, and optical tweezer biosensors, for AMR diagnostics. By harnessing the unique advantages of these nanoenabled smart biosensors, a revolutionary paradigm shift in AMR diagnostics can be achieved, characterized by rapid results, high sensitivity, portability, and integration with Internet-of-Things (IoT) technologies. Moreover, nanoenabled optical biosensors enable personalized monitoring and on-site detection, significantly reducing turnaround time and eliminating the human resources needed for sample preservation and transportation. Their potential for holistic environmental surveillance further enhances monitoring capabilities in diverse settings, leading to improved modern-age healthcare practices and more effective management of antimicrobial treatments. Embracing these advanced diagnostic tools promises to bolster global healthcare capacity to combat AMR and safeguard One Health.
抗菌素耐药性(AMR)是全球 "一体健康 "领域的一个重大问题,它源于对抗生素的无意和持续接触,以及准确传染诊断方面的挑战。解决 AMR 问题需要采取一种战略方法,强调通过在临床、环境、农业和畜牧业环境中进行筛查来确定非易感性抗菌剂和相关基因,从而进行早期预防。传统的 AMR 诊断方法(如抗生素药敏试验)具有成本高、过程耗时、需要大量人力等缺点,因此需要智能、快速和现场诊断技术。由纳米人工智能(AI)支持的智能光学生物传感器提供了一种潜在的解决方案,它具有实时、灵敏和便携的特点,可促进快速的护理点 AMR 检测。本综述全面探讨了用于 AMR 诊断的各种类型的光学纳米生物传感器,如表面等离子体共振传感器、耳语-画廊模式传感器、光学相干断层扫描、干涉反射成像传感器、表面增强拉曼光谱、荧光光谱、微孔共振传感器和光镊生物传感器。利用这些纳米智能生物传感器的独特优势,可以实现 AMR 诊断模式的革命性转变,其特点是结果快速、灵敏度高、便携,并可与物联网(IoT)技术集成。此外,纳米光学生物传感器还能进行个性化监测和现场检测,大大缩短了周转时间,并省去了样本保存和运输所需的人力资源。它们在整体环境监测方面的潜力进一步增强了在不同环境中的监测能力,从而改善了现代医疗保健实践,并更有效地管理抗菌治疗。采用这些先进的诊断工具有望增强全球医疗保健能力,以对抗 AMR 并保障 "全民健康"。
{"title":"Synergizing Nanomaterials and Artificial Intelligence in Advanced Optical Biosensors for Precision Antimicrobial Resistance Diagnosis","authors":"Bakr Ahmed Taha*, Naser M. Ahmed, Rishi Kumar Talreja, Adawiya J. Haider, Yousif Al Mashhadany, Qussay Al-Jubouri, Aqilah Baseri Huddin, Mohd Hadri Hafiz Mokhtar, Sarvesh Rustagi, Ajeet Kaushik, Vishal Chaudhary* and Norhana Arsad*, ","doi":"10.1021/acssynbio.4c00070","DOIUrl":"10.1021/acssynbio.4c00070","url":null,"abstract":"<p >Antimicrobial resistance (AMR) poses a critical global One Health concern, ensuing from unintentional and continuous exposure to antibiotics, as well as challenges in accurate contagion diagnostics. Addressing AMR requires a strategic approach that emphasizes early stage prevention through screening in clinical, environmental, farming, and livestock settings to identify nonvulnerable antimicrobial agents and the associated genes. Conventional AMR diagnostics, like antibiotic susceptibility testing, possess drawbacks, including high costs, time-consuming processes, and significant manpower requirements, underscoring the need for intelligent, prompt, and on-site diagnostic techniques. Nanoenabled artificial intelligence (AI)-supported smart optical biosensors present a potential solution by facilitating rapid point-of-care AMR detection with real-time, sensitive, and portable capabilities. This Review comprehensively explores various types of optical nanobiosensors, such as surface plasmon resonance sensors, whispering-gallery mode sensors, optical coherence tomography, interference reflection imaging sensors, surface-enhanced Raman spectroscopy, fluorescence spectroscopy, microring resonance sensors, and optical tweezer biosensors, for AMR diagnostics. By harnessing the unique advantages of these nanoenabled smart biosensors, a revolutionary paradigm shift in AMR diagnostics can be achieved, characterized by rapid results, high sensitivity, portability, and integration with Internet-of-Things (IoT) technologies. Moreover, nanoenabled optical biosensors enable personalized monitoring and on-site detection, significantly reducing turnaround time and eliminating the human resources needed for sample preservation and transportation. Their potential for holistic environmental surveillance further enhances monitoring capabilities in diverse settings, leading to improved modern-age healthcare practices and more effective management of antimicrobial treatments. Embracing these advanced diagnostic tools promises to bolster global healthcare capacity to combat AMR and safeguard One Health.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141260149","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}
Genome editing is the basis for the modification of engineered microbes. In the process of genome editing, the design of editing sequences, such as primers and sgRNA, is very important for the accurate positioning of editing sites and efficient sequence editing. The whole process of genome editing involves multiple rounds and types of editing sequence design, while the development of related whole-workflow design tools for high-throughput experimental requirements lags. Here, we propose AutoESDCas, an online tool for the end-to-end editing sequence design for microbial genome editing based on the CRISPR/Cas system. This tool facilitates all types of genetic manipulation covering diverse experimental requirements and design scenarios, enables biologists to quickly and efficiently obtain all editing sequences needed for the entire genome editing process, and empowers high-throughput strain modification. Notably, with its off-target risk assessment function for editing sequences, the usability of the design results is significantly improved. AutoESDCas is freely available at https://autoesdcas.biodesign.ac.cn/with the source code at https://github.com/tibbdc/AutoESDCas/.
{"title":"AutoESDCas: A Web-Based Tool for the Whole-Workflow Editing Sequence Design for Microbial Genome Editing Based on the CRISPR/Cas System","authors":"Chunhe Yang, Yi Yang, Guangyun Chu, Ruoyu Wang, Haoran Li, Yufeng Mao, Meng Wang, Jian Zhang, Xiaoping Liao* and Hongwu Ma*, ","doi":"10.1021/acssynbio.4c00063","DOIUrl":"10.1021/acssynbio.4c00063","url":null,"abstract":"<p >Genome editing is the basis for the modification of engineered microbes. In the process of genome editing, the design of editing sequences, such as primers and sgRNA, is very important for the accurate positioning of editing sites and efficient sequence editing. The whole process of genome editing involves multiple rounds and types of editing sequence design, while the development of related whole-workflow design tools for high-throughput experimental requirements lags. Here, we propose AutoESDCas, an online tool for the end-to-end editing sequence design for microbial genome editing based on the CRISPR/Cas system. This tool facilitates all types of genetic manipulation covering diverse experimental requirements and design scenarios, enables biologists to quickly and efficiently obtain all editing sequences needed for the entire genome editing process, and empowers high-throughput strain modification. Notably, with its off-target risk assessment function for editing sequences, the usability of the design results is significantly improved. AutoESDCas is freely available at https://autoesdcas.biodesign.ac.cn/with the source code at https://github.com/tibbdc/AutoESDCas/.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141282243","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}