Pub Date : 2024-07-02DOI: 10.1021/acssynbio.4c00318
Sefli Sri Wahyu Effendi, I-Son Ng
Carbon dioxide emission and acidification during chemical biosynthesis are critical challenges toward microbial cell factories' sustainability and efficiency. Due to its acidophilic traits among workhorse lineages, the probiotic Escherichia coli Nissle (EcN) has emerged as a promising chemical bioproducer. However, EcN lacks a CO2-fixing system. Herein, EcN was equipped with a simultaneous CO2 fixation system and subsequently utilized to produce low-emission 5-aminolevulinic acid (5-ALA). Two different artificial CO2-assimilating pathways were reconstructed: the novel ribose-1,5-bisphosphate (R15P) route and the conventional ribulose-5-phosphate (Ru5P) route. CRISPRi was employed to target the pfkAB and zwf genes in order to redirect the carbon flux. As expected, the CRISPRi design successfully strengthened the CO2 fixation. The CO2-fixing route via R15P resulted in high biomass, while the engineered Ru5P route acquired the highest 5-ALA and suppressed the CO2 release by 77%. CO2 fixation during 5-ALA production in EcN was successfully synchronized through fine-tuning the non-native pathways with CRISPRi.
{"title":"Non-native Pathway Engineering with CRISPRi for Carbon Dioxide Assimilation and Valued 5-Aminolevulinic Acid Synthesis in <i>Escherichia coli</i> Nissle.","authors":"Sefli Sri Wahyu Effendi, I-Son Ng","doi":"10.1021/acssynbio.4c00318","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00318","url":null,"abstract":"<p><p>Carbon dioxide emission and acidification during chemical biosynthesis are critical challenges toward microbial cell factories' sustainability and efficiency. Due to its acidophilic traits among workhorse lineages, the probiotic <i>Escherichia coli</i> Nissle (EcN) has emerged as a promising chemical bioproducer. However, EcN lacks a CO<sub>2</sub>-fixing system. Herein, EcN was equipped with a simultaneous CO<sub>2</sub> fixation system and subsequently utilized to produce low-emission 5-aminolevulinic acid (5-ALA). Two different artificial CO<sub>2</sub>-assimilating pathways were reconstructed: the novel ribose-1,5-bisphosphate (R15P) route and the conventional ribulose-5-phosphate (Ru5P) route. CRISPRi was employed to target the <i>pfk</i>AB and <i>zwf</i> genes in order to redirect the carbon flux. As expected, the CRISPRi design successfully strengthened the CO<sub>2</sub> fixation. The CO<sub>2</sub>-fixing route via R15P resulted in high biomass, while the engineered Ru5P route acquired the highest 5-ALA and suppressed the CO<sub>2</sub> release by 77%. CO<sub>2</sub> fixation during 5-ALA production in EcN was successfully synchronized through fine-tuning the non-native pathways with CRISPRi.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141489923","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}
l-Valine, an essential amino acid, serves as a valuable compound in various industries. However, engineering strains with both high yield and purity are yet to be delivered for microbial l-valine production. We engineered a Corynebacterium glutamicum strain capable of highly efficient production of l-valine. We initially introduced an acetohydroxy acid synthase mutant from an industrial l-valine producer and optimized a cofactor-balanced pathway, followed by the activation of the nonphosphoenolpyruvate-dependent carbohydrate phosphotransferase system and the introduction of an exogenous Entner-Doudoroff pathway. Subsequently, we weakened anaplerotic pathways, and attenuated the tricarboxylic acid cycle via start codon substitution in icd, encoding isocitrate dehydrogenase. Finally, to balance bacterial growth and l-valine production, an l-valine biosensor-dependent genetic circuit was established to dynamically repress citrate synthase expression. The engineered strain Val19 produced 103 g/L of l-valine with a high yield of 0.35 g/g glucose and a productivity of 2.67 g/L/h. This represents the highest reported l-valine production in C. glutamicum via direct fermentation and exhibits potential for its industrial-scale production, leveraging the advantages of C. glutamicum over other microbes.
{"title":"Metabolic Engineering of <i>Corynebacterium glutamicum</i> for the High-Level Production of l-Valine under Aerobic Conditions.","authors":"Feiao Wang, Ningyun Cai, Yanlin Leng, Chen Wu, Yanan Wang, Siyu Tian, Chenglin Zhang, Qingyang Xu, Huadong Peng, Ning Chen, Yanjun Li","doi":"10.1021/acssynbio.4c00278","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00278","url":null,"abstract":"<p><p>l-Valine, an essential amino acid, serves as a valuable compound in various industries. However, engineering strains with both high yield and purity are yet to be delivered for microbial l-valine production. We engineered a <i>Corynebacterium glutamicum</i> strain capable of highly efficient production of l-valine. We initially introduced an acetohydroxy acid synthase mutant from an industrial l-valine producer and optimized a cofactor-balanced pathway, followed by the activation of the nonphosphoenolpyruvate-dependent carbohydrate phosphotransferase system and the introduction of an exogenous Entner-Doudoroff pathway. Subsequently, we weakened anaplerotic pathways, and attenuated the tricarboxylic acid cycle via start codon substitution in <i>icd</i>, encoding isocitrate dehydrogenase. Finally, to balance bacterial growth and l-valine production, an l-valine biosensor-dependent genetic circuit was established to dynamically repress citrate synthase expression. The engineered strain Val19 produced 103 g/L of l-valine with a high yield of 0.35 g/g glucose and a productivity of 2.67 g/L/h. This represents the highest reported l-valine production in <i>C. glutamicum</i> via direct fermentation and exhibits potential for its industrial-scale production, leveraging the advantages of <i>C. glutamicum</i> over other microbes.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141464233","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 : 2024-06-28DOI: 10.1021/acssynbio.4c00154
Yannan Wang, Yujue Wang, Na Tang, Zhipeng Wang, Deng Pan, Quanjiang Ji
Cas12f nucleases are one of the most compact genome editors, exhibiting promising potential for in vivo therapeutic applications. However, the availability of active Cas12f genome editors remains relatively limited in the field. Here, we report the characterization and engineering of a novel miniature Cas12f endonuclease from Eubacterium siraeum (EsCas12f1, 433 amino acids). We elucidate the specific Protospacer Adjacent Motifs preference and the detailed biochemical properties for DNA targeting and cleavage. By employing rational design strategies, we systematically optimize the guide RNA of EsCas12f1, converting the initially ineffective CRISPR-EsCas12f1 system into an efficient bacterial genome editor. Furthermore, we demonstrate the capacity of EsCas12f1 for in vitro nucleic-acid diagnostics. In summary, our results enrich the miniature CRISPR-Cas toolbox and pave the way for the application of EsCas12f1 for both genome editing and in vitro diagnostics.
{"title":"Characterization and Engineering of a Novel Miniature <i>Eubacterium siraeum</i> CRISPR-Cas12f System.","authors":"Yannan Wang, Yujue Wang, Na Tang, Zhipeng Wang, Deng Pan, Quanjiang Ji","doi":"10.1021/acssynbio.4c00154","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00154","url":null,"abstract":"<p><p>Cas12f nucleases are one of the most compact genome editors, exhibiting promising potential for <i>in vivo</i> therapeutic applications. However, the availability of active Cas12f genome editors remains relatively limited in the field. Here, we report the characterization and engineering of a novel miniature Cas12f endonuclease from <i>Eubacterium siraeum</i> (EsCas12f1, 433 amino acids). We elucidate the specific Protospacer Adjacent Motifs preference and the detailed biochemical properties for DNA targeting and cleavage. By employing rational design strategies, we systematically optimize the guide RNA of EsCas12f1, converting the initially ineffective CRISPR-EsCas12f1 system into an efficient bacterial genome editor. Furthermore, we demonstrate the capacity of EsCas12f1 for <i>in vitro</i> nucleic-acid diagnostics. In summary, our results enrich the miniature CRISPR-Cas toolbox and pave the way for the application of EsCas12f1 for both genome editing and <i>in vitro</i> diagnostics.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141464232","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 : 2024-06-27DOI: 10.1021/acssynbio.3c00572
P V Phaneuf, S H Kim, K Rychel, C Rode, F Beulig, B O Palsson, L Yang
As the availability of data sets increases, meta-analysis leveraging aggregated and interoperable data types is proving valuable. This study leveraged a meta-analysis workflow to identify mutations that could improve robustness to reactive oxygen species (ROS) stresses using an industrially important melatonin production strain as an example. ROS stresses often occur during cultivation and negatively affect strain performance. Cellular response to ROS is also linked to the SOS response and resistance to pH fluctuations, which is important to strain robustness in large-scale biomanufacturing. This work integrated more than 7000 E. coli adaptive laboratory evolution (ALE) mutations across 59 experiments to statistically associate mutated genes to 2 ROS tolerance ALE conditions from 72 unique conditions. Mutant oxyR, fur, iscR, and ygfZ were significantly associated and hypothesized to contribute fitness in ROS stress. Across these genes, 259 total mutations were inspected in conjunction with transcriptomics from 46 iModulon experiments. Ten mutations were chosen for reintroduction based on mutation clustering and coinciding transcriptional changes as evidence of fitness impact. Strains with mutations reintroduced into oxyR, fur, iscR, and ygfZ exhibited increased tolerance to H2O2 and acid stress and reduced SOS response, all of which are related to ROS. Additionally, new evidence was generated toward understanding the function of ygfZ, an uncharacterized gene. This meta-analysis approach utilized aggregated and interoperable multiomics data sets to identify mutations conferring industrially relevant phenotypes with the least drawbacks, describing an approach for data-driven strain engineering to optimize microbial cell factories.
{"title":"Meta-analysis Driven Strain Design for Mitigating Oxidative Stresses Important in Biomanufacturing.","authors":"P V Phaneuf, S H Kim, K Rychel, C Rode, F Beulig, B O Palsson, L Yang","doi":"10.1021/acssynbio.3c00572","DOIUrl":"https://doi.org/10.1021/acssynbio.3c00572","url":null,"abstract":"<p><p>As the availability of data sets increases, meta-analysis leveraging aggregated and interoperable data types is proving valuable. This study leveraged a meta-analysis workflow to identify mutations that could improve robustness to reactive oxygen species (ROS) stresses using an industrially important melatonin production strain as an example. ROS stresses often occur during cultivation and negatively affect strain performance. Cellular response to ROS is also linked to the SOS response and resistance to pH fluctuations, which is important to strain robustness in large-scale biomanufacturing. This work integrated more than 7000 <i>E. coli</i> adaptive laboratory evolution (ALE) mutations across 59 experiments to statistically associate mutated genes to 2 ROS tolerance ALE conditions from 72 unique conditions. Mutant <i>oxyR</i>, <i>fur</i>, <i>iscR</i>, and <i>ygfZ</i> were significantly associated and hypothesized to contribute fitness in ROS stress. Across these genes, 259 total mutations were inspected in conjunction with transcriptomics from 46 iModulon experiments. Ten mutations were chosen for reintroduction based on mutation clustering and coinciding transcriptional changes as evidence of fitness impact. Strains with mutations reintroduced into <i>oxyR</i>, <i>fur</i>, <i>iscR</i>, and <i>ygfZ</i> exhibited increased tolerance to H<sub>2</sub>O<sub>2</sub> and acid stress and reduced SOS response, all of which are related to ROS. Additionally, new evidence was generated toward understanding the function of <i>ygfZ</i>, an uncharacterized gene. This meta-analysis approach utilized aggregated and interoperable multiomics data sets to identify mutations conferring industrially relevant phenotypes with the least drawbacks, describing an approach for data-driven strain engineering to optimize microbial cell factories.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141453682","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 : 2024-06-26DOI: 10.1021/acssynbio.4c00178
Sara M Eslami, Chandrashekhar Padhi, Imran R Rahman, Wilfred A van der Donk
Cyclic peptides, such as most ribosomally synthesized and post-translationally modified peptides (RiPPs), represent a burgeoning area of interest in therapeutic and biotechnological research because of their conformational constraints and reduced susceptibility to proteolytic degradation compared to their linear counterparts. Herein, an expression system is reported that enables the production of structurally diverse lanthipeptides and derivatives in mammalian cells. Successful targeting of lanthipeptides to the nucleus, the endoplasmic reticulum, and the plasma membrane is demonstrated. In vivo expression and targeting of such peptides in mammalian cells may allow for screening of lanthipeptide-based cyclic peptide inhibitors of native, organelle-specific protein-protein interactions in mammalian systems.
{"title":"Expression and Subcellular Localization of Lanthipeptides in Human Cells.","authors":"Sara M Eslami, Chandrashekhar Padhi, Imran R Rahman, Wilfred A van der Donk","doi":"10.1021/acssynbio.4c00178","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00178","url":null,"abstract":"<p><p>Cyclic peptides, such as most ribosomally synthesized and post-translationally modified peptides (RiPPs), represent a burgeoning area of interest in therapeutic and biotechnological research because of their conformational constraints and reduced susceptibility to proteolytic degradation compared to their linear counterparts. Herein, an expression system is reported that enables the production of structurally diverse lanthipeptides and derivatives in mammalian cells. Successful targeting of lanthipeptides to the nucleus, the endoplasmic reticulum, and the plasma membrane is demonstrated. In vivo expression and targeting of such peptides in mammalian cells may allow for screening of lanthipeptide-based cyclic peptide inhibitors of native, organelle-specific protein-protein interactions in mammalian systems.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141453681","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 : 2024-06-25DOI: 10.1021/acssynbio.4c00056
Daniel Stukenberg, Anna Faber, Anke Becker
In recent years, the fast-growing bacterium Vibrio natriegens has gained increasing attention as it has the potential to become a next-generation chassis for synthetic biology. A wide range of genetic parts and genome engineering methods have already been developed. However, there is still a need for a well-characterized tool to effectively and gradually reduce the expression levels of native genes. To bridge this gap, we created graded-CRISPRi, a system utilizing gRNA variants that lead to varying levels of repression strength. By incorporating multiple gRNA sequences into our design, we successfully extended this concept to simultaneously repress four distinct reporter genes. Furthermore, we demonstrated the capability of using graded-CRISPRi to target native genes, thereby examining the effect of various knockdown levels on growth.
{"title":"Graded-CRISPRi, a Tool for Tuning the Strengths of CRISPRi-Mediated Knockdowns in <i>Vibrio natriegens</i> Using gRNA Libraries.","authors":"Daniel Stukenberg, Anna Faber, Anke Becker","doi":"10.1021/acssynbio.4c00056","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00056","url":null,"abstract":"<p><p>In recent years, the fast-growing bacterium <i>Vibrio natriegens</i> has gained increasing attention as it has the potential to become a next-generation chassis for synthetic biology. A wide range of genetic parts and genome engineering methods have already been developed. However, there is still a need for a well-characterized tool to effectively and gradually reduce the expression levels of native genes. To bridge this gap, we created graded-CRISPRi, a system utilizing gRNA variants that lead to varying levels of repression strength. By incorporating multiple gRNA sequences into our design, we successfully extended this concept to simultaneously repress four distinct reporter genes. Furthermore, we demonstrated the capability of using graded-CRISPRi to target native genes, thereby examining the effect of various knockdown levels on growth.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141445530","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 : 2024-06-25DOI: 10.1021/acssynbio.4c00077
Vikram Mubayi, Colleen B Ahern, Magdalena Calusinska, Michelle A O'Malley
Polymer production is rapidly increasing, but there are no large-scale technologies available to effectively mitigate the massive accumulation of these recalcitrant materials. One potential solution is the development of a carbon-neutral polymer life cycle, where microorganisms convert plant biomass to chemicals, which are used to synthesize biodegradable materials that ultimately contribute to the growth of new plants. Realizing a circular carbon life cycle requires the integration of knowledge across microbiology, bioengineering, materials science, and organic chemistry, which itself has hindered large-scale industrial advances. This review addresses the biodegradation status of common synthetic polymers, identifying novel microbes and enzymes capable of metabolizing these recalcitrant materials and engineering approaches to enhance their biodegradation pathways. Design considerations for the next generation of biodegradable polymers are also reviewed, and finally, opportunities to apply findings from lignocellulosic biodegradation to the design and biodegradation of similarly recalcitrant synthetic polymers are discussed.
{"title":"Toward a Circular Bioeconomy: Designing Microbes and Polymers for Biodegradation.","authors":"Vikram Mubayi, Colleen B Ahern, Magdalena Calusinska, Michelle A O'Malley","doi":"10.1021/acssynbio.4c00077","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00077","url":null,"abstract":"<p><p>Polymer production is rapidly increasing, but there are no large-scale technologies available to effectively mitigate the massive accumulation of these recalcitrant materials. One potential solution is the development of a carbon-neutral polymer life cycle, where microorganisms convert plant biomass to chemicals, which are used to synthesize biodegradable materials that ultimately contribute to the growth of new plants. Realizing a circular carbon life cycle requires the integration of knowledge across microbiology, bioengineering, materials science, and organic chemistry, which itself has hindered large-scale industrial advances. This review addresses the biodegradation status of common synthetic polymers, identifying novel microbes and enzymes capable of metabolizing these recalcitrant materials and engineering approaches to enhance their biodegradation pathways. Design considerations for the next generation of biodegradable polymers are also reviewed, and finally, opportunities to apply findings from lignocellulosic biodegradation to the design and biodegradation of similarly recalcitrant synthetic polymers are discussed.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141449034","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 : 2024-06-24DOI: 10.1021/acssynbio.4c00273
Ye Chan Kim, Hee-Wang Yoo, Beom Gi Park, Sharad Sarak, Ji-Sook Hahn, Byung-Gee Kim, Hyungdon Yun
Metabolically engineered microbial consortia can contribute as a promising production platform for the supply of polyamide monomers. To date, the biosynthesis of long-chain α,ω-diamines from n-alkanes is challenging because of the inert nature of n-alkanes and the complexity of the overall synthesis pathway. We combined an engineered Yarrowia lipolytica module with Escherichia coli modules to obtain a mixed strain microbial consortium that could catalyze an efficient biotransformation of n-alkanes into corresponding α,ω-diamines. The engineered Y. lipolytica strain was constructed (YALI10) wherein the two genes responsible for β-oxidation and the five genes responsible for the overoxidation of fatty aldehydes were deleted. This newly constructed YALI10 strain expressing transaminase (TA) could produce 0.2 mM 1,12-dodecanediamine (40.1 mg/L) from 10 mM n-dodecane. The microbial consortia comprising engineered Y. lipolytica strains for the oxidation of n-alkanes (OM) and an E. coli amination module (AM) expressing an aldehyde reductase (AHR) and transaminase (TA) improved the production of 1,12-diamine up to 1.95 mM (391 mg/L) from 10 mM n-dodecane. Finally, combining the E. coli reduction module (RM) expressing a carboxylic acid reductase (CAR) and an sfp phosphopantetheinyl transferase with OM and AM further improved the production of 1,12-diamine by catalyzing the reduction of undesired 1,12-diacids into 1,12-diols, which further undergo amination to give 1,12-diamine as the target product. This newly constructed mixed strain consortium comprising three modules in one pot gave 4.1 mM (41%; 816 mg/L) 1,12-diaminododecane from 10 mM n-dodecane. The whole-cell consortia reported herein present an elegant "greener" alternative for the biosynthesis of various α,ω-diamines (C8, C10, C12, and C14) from corresponding n-alkanes.
经过代谢工程改造的微生物联合体可作为一个前景广阔的生产平台,供应聚酰胺单体。迄今为止,由于正构烷烃的惰性和整个合成途径的复杂性,从正构烷烃中生物合成长链 α、ω-二胺具有挑战性。我们将工程化的脂肪溶解蓍草菌(Yarrowia lipolytica)模块与大肠杆菌(Escherichia coli)模块相结合,获得了一个混合菌株微生物联合体,该联合体可催化正构烷烃向相应的α,ω-二胺的高效生物转化。在构建的脂肪溶解酵母工程菌株(YALI10)中,负责β氧化的两个基因和负责脂肪醛过氧化的五个基因被删除。这种新构建的表达转氨酶(TA)的 YALI10 菌株可从 10 mM 正十二烷中产生 0.2 mM 1,12-十二烷二胺(40.1 mg/L)。由用于氧化正构烷烃(OM)的脂肪溶解酵母菌株和表达醛还原酶(AHR)和转氨酶(TA)的大肠杆菌氨化模块(AM)组成的微生物联合体可将 10 mM 正十二烷中 1,12-二胺的产量提高到 1.95 mM(391 mg/L)。最后,将表达羧酸还原酶(CAR)和 sfp 磷酸泛硫乙烯基转移酶的大肠杆菌还原模块(RM)与 OM 和 AM 结合使用,通过催化将不需要的 1,12-二酸还原成 1,12-二醇,再经过氨化反应生成目标产物 1,12-二胺,从而进一步提高了 1,12-二胺的产量。这种新构建的混合菌株联合体由一个罐中的三个模块组成,能从 10 mM 正十二烷中产生 4.1 mM(41%;816 mg/L)的 1,12-二氨基十二烷。本文报告的全细胞联合菌株为从相应的正烷烃中生物合成各种α,ω-二胺(C8、C10、C12 和 C14)提供了一种优雅的 "绿色 "替代方法。
{"title":"One-Pot Biocatalytic Route from Alkanes to α,ω-Diamines by Whole-Cell Consortia of Engineered <i>Yarrowia lipolytica</i> and <i>Escherichia coli</i>.","authors":"Ye Chan Kim, Hee-Wang Yoo, Beom Gi Park, Sharad Sarak, Ji-Sook Hahn, Byung-Gee Kim, Hyungdon Yun","doi":"10.1021/acssynbio.4c00273","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00273","url":null,"abstract":"<p><p>Metabolically engineered microbial consortia can contribute as a promising production platform for the supply of polyamide monomers. To date, the biosynthesis of long-chain α,ω-diamines from <i>n</i>-alkanes is challenging because of the inert nature of <i>n</i>-alkanes and the complexity of the overall synthesis pathway. We combined an engineered <i>Yarrowia lipolytica</i> module with <i>Escherichia coli</i> modules to obtain a mixed strain microbial consortium that could catalyze an efficient biotransformation of <i>n</i>-alkanes into corresponding α,ω-diamines. The engineered <i>Y. lipolytica</i> strain was constructed (YALI10) wherein the two genes responsible for β-oxidation and the five genes responsible for the overoxidation of fatty aldehydes were deleted. This newly constructed YALI10 strain expressing transaminase (TA) could produce 0.2 mM 1,12-dodecanediamine (40.1 mg/L) from 10 mM <i>n</i>-dodecane. The microbial consortia comprising engineered <i>Y. lipolytica</i> strains for the oxidation of <i>n</i>-alkanes (O<sup>M</sup>) and an <i>E. coli</i> amination module (A<sup>M</sup>) expressing an aldehyde reductase (AHR) and transaminase (TA) improved the production of 1,12-diamine up to 1.95 mM (391 mg/L) from 10 mM <i>n</i>-dodecane. Finally, combining the <i>E. coli</i> reduction module (R<sup>M</sup>) expressing a carboxylic acid reductase (CAR) and an sfp phosphopantetheinyl transferase with O<sup>M</sup> and A<sup>M</sup> further improved the production of 1,12-diamine by catalyzing the reduction of undesired 1,12-diacids into 1,12-diols, which further undergo amination to give 1,12-diamine as the target product. This newly constructed mixed strain consortium comprising three modules in one pot gave 4.1 mM (41%; 816 mg/L) 1,12-diaminododecane from 10 mM <i>n</i>-dodecane. The whole-cell consortia reported herein present an elegant \"greener\" alternative for the biosynthesis of various α,ω-diamines (C8, C10, C12, and C14) from corresponding <i>n</i>-alkanes.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141441756","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}
Artificial riboswitches responsive to user-defined analytes can be constructed by successfully inserting in vitro selected aptamers, which bind to the analytes, into untranslated regions of mRNA. Among them, eukaryotic riboswitches are more promising as biosensors than bacterial ones because they function well at ambient temperature. In addition, cell-free expression systems allow the broader use of these riboswitches as cell-free biosensors in an environmentally friendly manner without cellular limitations. The current best cell-free eukaryotic riboswitch regulates eukaryotic canonical translation initiation through self-cleavage mediated by an implanted analyte-responsive ribozyme (i.e., an aptazyme, an aptamer-ribozyme fusion). However, it has critical flaws as a sensor: due to the less-active ribozyme used, self-cleavage and translation reactions must be conducted separately and sequentially, and a different aptazyme has to be selected to change the analyte specificity, even if an aptamer for the next analyte is available. We here stepwise engineered novel types of cell-free eukaryotic riboswitches that harness highly active self-cleavage and thus require no reaction partitioning. Despite the single-step and one-pot reaction, these riboswitches showed higher analyte dose dependency and sensitivities than the current best cell-free eukaryotic riboswitch requiring multistep reactions. In addition, the analyte specificity can be changed in an extremely facile way, simply by aptamer substitution (and the subsequent simple fine-tuning for giant aptamers). Given that cell-free systems can be lyophilized for storage and transport, the present one-pot and thus easy-to-handle cell-free biosensors utilizing eukaryotic riboswitches are expected to be widely used for on-the-spot sensing of analytes at ambient temperature.
{"title":"Cell-Free Biosensors Based on Modular Eukaryotic Riboswitches That Function in One Pot at Ambient Temperature.","authors":"Atsushi Ogawa, Masahiro Fujikawa, Kazuki Onishi, Hajime Takahashi","doi":"10.1021/acssynbio.4c00341","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00341","url":null,"abstract":"<p><p>Artificial riboswitches responsive to user-defined analytes can be constructed by successfully inserting <i>in vitro</i> selected aptamers, which bind to the analytes, into untranslated regions of mRNA. Among them, eukaryotic riboswitches are more promising as biosensors than bacterial ones because they function well at ambient temperature. In addition, cell-free expression systems allow the broader use of these riboswitches as cell-free biosensors in an environmentally friendly manner without cellular limitations. The current best cell-free eukaryotic riboswitch regulates eukaryotic canonical translation initiation through self-cleavage mediated by an implanted analyte-responsive ribozyme (i.e., an aptazyme, an aptamer-ribozyme fusion). However, it has critical flaws as a sensor: due to the less-active ribozyme used, self-cleavage and translation reactions must be conducted separately and sequentially, and a different aptazyme has to be selected to change the analyte specificity, even if an aptamer for the next analyte is available. We here stepwise engineered novel types of cell-free eukaryotic riboswitches that harness highly active self-cleavage and thus require no reaction partitioning. Despite the single-step and one-pot reaction, these riboswitches showed higher analyte dose dependency and sensitivities than the current best cell-free eukaryotic riboswitch requiring multistep reactions. In addition, the analyte specificity can be changed in an extremely facile way, simply by aptamer substitution (and the subsequent simple fine-tuning for giant aptamers). Given that cell-free systems can be lyophilized for storage and transport, the present one-pot and thus easy-to-handle cell-free biosensors utilizing eukaryotic riboswitches are expected to be widely used for on-the-spot sensing of analytes at ambient temperature.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141441755","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}