Pub Date : 2024-11-01DOI: 10.1016/j.ymben.2024.10.008
Victor Forman , Dan Luo , Sotirios C. Kampranis , Dan Stærk , Birger Lindberg Møller , Irini Pateraki
Cytochrome B5s, or CytB5s, are small heme-binding proteins, ubiquitous across all kingdoms of life that serve mainly as electron donors to enzymes engaged in oxidative reactions. They often function as redox partners of the cytochrome P450s (CYPs), a superfamily of enzymes participating in multiple biochemical processes. In plants, CYPs catalyze key reactions in the biosynthesis of plant specialized metabolites with their activity dependent on electron donation often from cytochrome P450 oxidoreductases (CPRs or PORs). In eukaryotic microsomal CYPs, CytB5s frequently participate in the electron transfer process although their exact role remains understudied, especially in plant systems. In this study, we assess the role of CytB5s in the heterologous biotechnological production of plant specialized metabolites in yeast. For this, we used as a case-study the biosynthesis of forskolin - a bioactive diterpenoid produced exclusively from the plant Coleus forskohlii. The complete biosynthetic pathway for forskolin is known and includes three CYP enzymes. We reconstructed the entire forskolin pathway in the yeast Saccharomyces cerevisiae, and upon co-expression of the three CytB5s - identified in C. forskohlii transcriptomes - alleviation of a CYP-related bottleneck step was noticed only when a specific CytB5, CfCytB5A, was used. Co-expression of CfCytB5A in yeast, in combination with forskolin pathway engineering, resulted in forskolin production at titers of 1.81 g/L in a bioreactor. Our findings demonstrate that CytB5s not only play an important role in plant specialized metabolism but also, they can interact with precision with specific CYPs, indicating that the properties of CytB5s are far from understood. Moreover, our work highlights how CytB5s may act as indispensable components in the sustainable microbial production of plant metabolites, when their biosynthetic pathways involve CYP enzymes.
{"title":"Not all cytochrome b5s are created equal: How a specific CytB5 boosts forskolin biosynthesis in Saccharomyces cerevisiae","authors":"Victor Forman , Dan Luo , Sotirios C. Kampranis , Dan Stærk , Birger Lindberg Møller , Irini Pateraki","doi":"10.1016/j.ymben.2024.10.008","DOIUrl":"10.1016/j.ymben.2024.10.008","url":null,"abstract":"<div><div>Cytochrome B5s, or CytB5s, are small heme-binding proteins, ubiquitous across all kingdoms of life that serve mainly as electron donors to enzymes engaged in oxidative reactions. They often function as redox partners of the cytochrome P450s (CYPs), a superfamily of enzymes participating in multiple biochemical processes. In plants, CYPs catalyze key reactions in the biosynthesis of plant specialized metabolites with their activity dependent on electron donation often from cytochrome P450 oxidoreductases (CPRs or PORs). In eukaryotic microsomal CYPs, CytB5s frequently participate in the electron transfer process although their exact role remains understudied, especially in plant systems. In this study, we assess the role of CytB5s in the heterologous biotechnological production of plant specialized metabolites in yeast. For this, we used as a case-study the biosynthesis of forskolin - a bioactive diterpenoid produced exclusively from the plant <em>Coleus forskohlii</em>. The complete biosynthetic pathway for forskolin is known and includes three CYP enzymes. We reconstructed the entire forskolin pathway in the yeast <em>Saccharomyces cerevisiae,</em> and upon co-expression of the three CytB5s - identified in <em>C. forskohlii</em> transcriptomes - alleviation of a CYP-related bottleneck step was noticed only when a specific CytB5, <em>Cf</em>CytB5A, was used. Co-expression of <em>Cf</em>CytB5A in yeast, in combination with forskolin pathway engineering, resulted in forskolin production at titers of 1.81 g/L in a bioreactor. Our findings demonstrate that CytB5s not only play an important role in plant specialized metabolism but also, they can interact with precision with specific CYPs, indicating that the properties of CytB5s are far from understood. Moreover, our work highlights how CytB5s may act as indispensable components in the sustainable microbial production of plant metabolites, when their biosynthetic pathways involve CYP enzymes.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 288-299"},"PeriodicalIF":6.8,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142503498","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-01DOI: 10.1016/j.ymben.2024.10.011
Eric C. Holmes, Alissa C. Bleem, Christopher W. Johnson, Gregg T. Beckham
Bioconversion of high-volume waste streams into value-added products will be an integral component of the growing bioeconomy. Volatile fatty acids (VFAs) (e.g., butyrate, valerate, and hexanoate) are an emerging and promising waste-derived feedstock for microbial carbon upcycling. Cupriavidus necator H16 is a favorable host for conversion of VFAs into various bioproducts due to its diverse carbon metabolism, ease of metabolic engineering, and use at industrial scales. Here, we report that a common strategy to improve product titers in C. necator, deletion of the polyhydroxybutyrate (PHB) biosynthetic operon, results in a significant growth defect on VFA substrates. Using adaptive laboratory evolution, we identify mutations to the regulator gene phaR, the two-component response regulator-histidine kinase pair encoded by H16_A1372/H16_A1373, and the tripartite transporter assembly encoded by H16_A2296-A2298 as causative for improved growth on VFA substrates. Deletion of phaR and H16_A1373 led to significantly reduced NADH abundance accompanied by large changes to expression of genes involved in carbon metabolism, balance of electron carriers, and oxidative stress tolerance that may be responsible for improved growth of these engineered strains. These results provide insight into the role of PHB biosynthesis in carbon and energy metabolism and highlight a key role for the regulator PhaR in global regulatory networks. By combining mutations, we generated platform strains with significant growth improvements on VFAs, which can enable improved conversion of waste-derived VFA substrates to target bioproducts.
{"title":"Adaptive laboratory evolution and metabolic engineering of Cupriavidus necator for improved catabolism of volatile fatty acids","authors":"Eric C. Holmes, Alissa C. Bleem, Christopher W. Johnson, Gregg T. Beckham","doi":"10.1016/j.ymben.2024.10.011","DOIUrl":"10.1016/j.ymben.2024.10.011","url":null,"abstract":"<div><div>Bioconversion of high-volume waste streams into value-added products will be an integral component of the growing bioeconomy. Volatile fatty acids (VFAs) (e.g., butyrate, valerate, and hexanoate) are an emerging and promising waste-derived feedstock for microbial carbon upcycling. <em>Cupriavidus necator</em> H16 is a favorable host for conversion of VFAs into various bioproducts due to its diverse carbon metabolism, ease of metabolic engineering, and use at industrial scales. Here, we report that a common strategy to improve product titers in <em>C. necator</em>, deletion of the polyhydroxybutyrate (PHB) biosynthetic operon, results in a significant growth defect on VFA substrates. Using adaptive laboratory evolution, we identify mutations to the regulator gene <em>phaR</em>, the two-component response regulator-histidine kinase pair encoded by <em>H16_A1372</em>/<em>H16_A1373</em>, and the tripartite transporter assembly encoded by <em>H16_A2296</em>-<em>A2298</em> as causative for improved growth on VFA substrates. Deletion of <em>phaR</em> and <em>H16_A1373</em> led to significantly reduced NADH abundance accompanied by large changes to expression of genes involved in carbon metabolism, balance of electron carriers, and oxidative stress tolerance that may be responsible for improved growth of these engineered strains. These results provide insight into the role of PHB biosynthesis in carbon and energy metabolism and highlight a key role for the regulator PhaR in global regulatory networks. By combining mutations, we generated platform strains with significant growth improvements on VFAs, which can enable improved conversion of waste-derived VFA substrates to target bioproducts.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 262-273"},"PeriodicalIF":6.8,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142553087","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-23DOI: 10.1016/j.ymben.2024.10.010
Soo Young Moon , Nan Yeong An , Seung Soo Oh , Ju Young Lee
Maintaining a delicate balance of adenosine-5′-triphosphate (ATP) is crucial not only for optimal cellular functions but also for improved metabolite production, indicating the need for careful regulation of ATP demands in metabolic engineering. This study explored the modification of ATP metabolism to enhance adipic acid production in Escherichia coli, focusing on the reverse adipate degradation pathway (RADP), and ATP-consuming cycles were fine-tuned by controlling the overexpression of genes (panK and acs) to balance ATP consumption and adipic acid production. As a result, we successfully achieved a significant increase (19.5-fold) in adipic acid production, reaching 1093.11 mg/L in a shake flask, compared to that in the control strain (wild-type E. coli harboring the RADP). Our transcriptomic analysis indicated that modulation of ATP metabolism, along with a balanced supply of pathway precursors, affects metabolic fluxes, enhancing adipic acid biosynthesis in E. coli. This study suggests the potential of metabolic reprogramming of ATP to meet biosynthetic demands, which may improve the production of adipic acid and other ATP-derived chemicals.
保持腺苷-5'-三磷酸（ATP）的微妙平衡不仅对优化细胞功能至关重要，而且对提高代谢产物的产量也至关重要，这表明在代谢工程中需要仔细调节 ATP 需求。本研究以反向己二酸降解途径（RADP）为重点，探索了如何改变 ATP 代谢以提高大肠杆菌的己二酸产量，并通过控制基因（panK 和 acs）的过量表达对 ATP 消耗循环进行了微调，以平衡 ATP 消耗和己二酸产量。结果，与对照菌株（携带 RADP 的野生型大肠杆菌）相比，我们成功地使己二酸产量大幅增加（19.5 倍），在摇瓶中达到 1,093.11 mg/L。我们的转录组分析表明，调节 ATP 代谢以及途径前体的平衡供应会影响代谢通量，从而增强大肠杆菌的己二酸生物合成。这项研究表明，ATP 的代谢重编程有可能满足生物合成的需求，从而提高己二酸和其他 ATP 衍生化学物质的产量。
{"title":"Coordinated reprogramming of ATP metabolism strongly enhances adipic acid production in Escherichia coli","authors":"Soo Young Moon , Nan Yeong An , Seung Soo Oh , Ju Young Lee","doi":"10.1016/j.ymben.2024.10.010","DOIUrl":"10.1016/j.ymben.2024.10.010","url":null,"abstract":"<div><div>Maintaining a delicate balance of adenosine-5′-triphosphate (ATP) is crucial not only for optimal cellular functions but also for improved metabolite production, indicating the need for careful regulation of ATP demands in metabolic engineering. This study explored the modification of ATP metabolism to enhance adipic acid production in <em>Escherichia coli</em>, focusing on the reverse adipate degradation pathway (RADP), and ATP-consuming cycles were fine-tuned by controlling the overexpression of genes (<em>panK</em> and <em>acs</em>) to balance ATP consumption and adipic acid production. As a result, we successfully achieved a significant increase (19.5-fold) in adipic acid production, reaching 1093.11 mg/L in a shake flask, compared to that in the control strain (wild-type <em>E. coli</em> harboring the RADP). Our transcriptomic analysis indicated that modulation of ATP metabolism, along with a balanced supply of pathway precursors, affects metabolic fluxes, enhancing adipic acid biosynthesis in <em>E. coli</em>. This study suggests the potential of metabolic reprogramming of ATP to meet biosynthetic demands, which may improve the production of adipic acid and other ATP-derived chemicals.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 234-241"},"PeriodicalIF":6.8,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142503497","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-22DOI: 10.1016/j.ymben.2024.10.007
Sebastian Wenk , Vittorio Rainaldi , Karin Schann , Hai He , Madeleine Bouzon , Volker Döring , Steffen N. Lindner , Arren Bar-Even
Atmospheric CO2 poses a major threat to life on Earth by causing global warming and climate change. On the other hand, it can be considered as a resource that is scalable enough to establish a circular carbon economy. Accordingly, technologies to capture and convert CO2 into reduced one-carbon (C1) compounds (e.g. formic acid) are developing and improving fast. Driven by the idea of creating sustainable bioproduction platforms, natural and synthetic C1-utilization pathways are engineered into industrially relevant microbes. The realization of synthetic C1-assimilation cycles in living organisms is a promising but challenging endeavour. Here, we engineer the Serine Threonine Cycle, a synthetic C1-assimilation cycle in Escherichia coli to achieve growth on formic acid. Our stepwise engineering approach in tailored selection strains combined with adaptive laboratory evolution experiments enabled formatotrophic growth of the organism. Whole genome sequencing and reverse engineering allowed us to determine the key mutations linked to pathway activity. The Serine Threonine Cycle strains created in this work use formic acid as sole carbon and energy source and can grow at ambient CO2 cultivation conditions. This work sets an example for the engineering of complex C1-assimilation cycles in heterotrophic microbes.
{"title":"Evolution-assisted engineering of E. coli enables growth on formic acid at ambient CO2 via the Serine Threonine Cycle","authors":"Sebastian Wenk , Vittorio Rainaldi , Karin Schann , Hai He , Madeleine Bouzon , Volker Döring , Steffen N. Lindner , Arren Bar-Even","doi":"10.1016/j.ymben.2024.10.007","DOIUrl":"10.1016/j.ymben.2024.10.007","url":null,"abstract":"<div><div>Atmospheric CO<sub>2</sub> poses a major threat to life on Earth by causing global warming and climate change. On the other hand, it can be considered as a resource that is scalable enough to establish a circular carbon economy. Accordingly, technologies to capture and convert CO<sub>2</sub> into reduced one-carbon (C<sub>1</sub>) compounds (e.g. formic acid) are developing and improving fast. Driven by the idea of creating sustainable bioproduction platforms, natural and synthetic C<sub>1</sub>-utilization pathways are engineered into industrially relevant microbes. The realization of synthetic C<sub>1</sub>-assimilation cycles in living organisms is a promising but challenging endeavour. Here, we engineer the Serine Threonine Cycle, a synthetic C<sub>1</sub>-assimilation cycle in <em>Escherichia coli</em> to achieve growth on formic acid. Our stepwise engineering approach in tailored selection strains combined with adaptive laboratory evolution experiments enabled formatotrophic growth of the organism. Whole genome sequencing and reverse engineering allowed us to determine the key mutations linked to pathway activity. The Serine Threonine Cycle strains created in this work use formic acid as sole carbon and energy source and can grow at ambient CO<sub>2</sub> cultivation conditions. This work sets an example for the engineering of complex C<sub>1</sub>-assimilation cycles in heterotrophic microbes.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 14-24"},"PeriodicalIF":6.8,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142490609","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Geobacillus thermoglucosidasius NCIMB 11955 possesses advantages, such as high-temperature tolerance, rapid growth rate, and low contamination risk. Additionally, it features efficient gene editing tools, making it one of the most promising next-generation cell factories. However, as a non-model microorganism, a lack of metabolic information significantly hampers the construction of high-precision metabolic flux models. Here, we propose a BioIntelliModel (BIM) strategy based on artificial intelligence technology for the automated construction of enzyme-constrained models. 1) . BIM utilises the Contrastive Learning Enabled Enzyme Annotation (CLEAN) prediction tool to analyse the entire genome sequence of G. thermoglucosidasius NCIMB 11955, uncovering potential functional proteins in non-model strains. 2). The MetaPatchM module of BIM automates the repair of the metabolic network model. 3). The Tianjin University of Science and Technology-kcat (TUST-kcat) module predicts the kcat values of enzymes within the model. 4). The Enzyme-insert procedure constructs an enzyme-constrained model and performs a global scan to address overconstraint issues. Enzymatic data were automatically integrated into the metabolic flux model, creating an enzyme-constrained model, ec_G-ther11955. To validate model accuracy, we used both the p-thermo and ec_G-ther11955 models to predict riboflavin production strategies. The ec_G-ther11955 model demonstrated significantly higher accuracy. To further verify its efficacy, we employed ec_G-ther11955 to guide the rational design of L-valine-producing strains. Using the Optimisation Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions (OptForce), Predictive Knockout Targeting (PKT), and Flux Scanning based on Enforced Objective Flux (FSEOF) algorithms, we identified 24 knockout and overexpression targets, achieving an accuracy rate of 87.5%. Ultimately, this led to an increase of 664.04% in L-valine titre. This study provides a novel strategy for rapidly constructing non-model strain models and demonstrates the tremendous potential of artificial intelligence in metabolic engineering.
{"title":"AI-based automated construction of high-precision Geobacillus thermoglucosidasius enzyme constraint model.","authors":"Minghao Zhang,Haijiao Shi,Xiaohong Wang,Yanan Zhu,Zilong Li,Linna Tu,Yu Zheng,Menglei Xia,Weishan Wang,Min Wang","doi":"10.1016/j.ymben.2024.10.006","DOIUrl":"https://doi.org/10.1016/j.ymben.2024.10.006","url":null,"abstract":"Geobacillus thermoglucosidasius NCIMB 11955 possesses advantages, such as high-temperature tolerance, rapid growth rate, and low contamination risk. Additionally, it features efficient gene editing tools, making it one of the most promising next-generation cell factories. However, as a non-model microorganism, a lack of metabolic information significantly hampers the construction of high-precision metabolic flux models. Here, we propose a BioIntelliModel (BIM) strategy based on artificial intelligence technology for the automated construction of enzyme-constrained models. 1) . BIM utilises the Contrastive Learning Enabled Enzyme Annotation (CLEAN) prediction tool to analyse the entire genome sequence of G. thermoglucosidasius NCIMB 11955, uncovering potential functional proteins in non-model strains. 2). The MetaPatchM module of BIM automates the repair of the metabolic network model. 3). The Tianjin University of Science and Technology-kcat (TUST-kcat) module predicts the kcat values of enzymes within the model. 4). The Enzyme-insert procedure constructs an enzyme-constrained model and performs a global scan to address overconstraint issues. Enzymatic data were automatically integrated into the metabolic flux model, creating an enzyme-constrained model, ec_G-ther11955. To validate model accuracy, we used both the p-thermo and ec_G-ther11955 models to predict riboflavin production strategies. The ec_G-ther11955 model demonstrated significantly higher accuracy. To further verify its efficacy, we employed ec_G-ther11955 to guide the rational design of L-valine-producing strains. Using the Optimisation Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions (OptForce), Predictive Knockout Targeting (PKT), and Flux Scanning based on Enforced Objective Flux (FSEOF) algorithms, we identified 24 knockout and overexpression targets, achieving an accuracy rate of 87.5%. Ultimately, this led to an increase of 664.04% in L-valine titre. This study provides a novel strategy for rapidly constructing non-model strain models and demonstrates the tremendous potential of artificial intelligence in metabolic engineering.","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"33 1","pages":""},"PeriodicalIF":8.4,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142486335","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-18DOI: 10.1016/j.ymben.2024.10.006
Minghao Zhang , Haijiao Shi , Xiaohong Wang , Yanan Zhu , Zilong Li , Linna Tu , Yu Zheng , Menglei Xia , Weishan Wang , Min Wang
Geobacillus thermoglucosidasius NCIMB 11955 possesses advantages, such as high-temperature tolerance, rapid growth rate, and low contamination risk. Additionally, it features efficient gene editing tools, making it one of the most promising next-generation cell factories. However, as a non-model microorganism, a lack of metabolic information significantly hampers the construction of high-precision metabolic flux models. Here, we propose a BioIntelliModel (BIM) strategy based on artificial intelligence technology for the automated construction of enzyme-constrained models. 1). BIM utilises the Contrastive Learning Enabled Enzyme Annotation (CLEAN) prediction tool to analyse the entire genome sequence of G. thermoglucosidasius NCIMB 11955, uncovering potential functional proteins in non-model strains. 2). The MetaPatchM module of BIM automates the repair of the metabolic network model. 3). The Tianjin University of Science and Technology-kcat (TUST-kcat) module predicts the kcat values of enzymes within the model. 4). The Enzyme-insert procedure constructs an enzyme-constrained model and performs a global scan to address overconstraint issues. Enzymatic data were automatically integrated into the metabolic flux model, creating an enzyme-constrained model, ec_G-ther11955. To validate model accuracy, we used both the p-thermo and ec_G-ther11955 models to predict riboflavin production strategies. The ec_G-ther11955 model demonstrated significantly higher accuracy. To further verify its efficacy, we employed ec_G-ther11955 to guide the rational design of L-valine-producing strains. Using the Optimisation Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions (OptForce), Predictive Knockout Targeting (PKT), and Flux Scanning based on Enforced Objective Flux (FSEOF) algorithms, we identified 24 knockout and overexpression targets, achieving an accuracy rate of 87.5%. Ultimately, this led to an increase of 664.04% in L-valine titre. This study provides a novel strategy for rapidly constructing non-model strain models and demonstrates the tremendous potential of artificial intelligence in metabolic engineering.
{"title":"AI-based automated construction of high-precision Geobacillus thermoglucosidasius enzyme constraint model","authors":"Minghao Zhang , Haijiao Shi , Xiaohong Wang , Yanan Zhu , Zilong Li , Linna Tu , Yu Zheng , Menglei Xia , Weishan Wang , Min Wang","doi":"10.1016/j.ymben.2024.10.006","DOIUrl":"10.1016/j.ymben.2024.10.006","url":null,"abstract":"<div><div><em>Geobacillus thermoglucosidasius</em> NCIMB 11955 possesses advantages, such as high-temperature tolerance, rapid growth rate, and low contamination risk. Additionally, it features efficient gene editing tools, making it one of the most promising next-generation cell factories. However, as a non-model microorganism, a lack of metabolic information significantly hampers the construction of high-precision metabolic flux models. Here, we propose a BioIntelliModel (BIM) strategy based on artificial intelligence technology for the automated construction of enzyme-constrained models. 1). BIM utilises the Contrastive Learning Enabled Enzyme Annotation (CLEAN) prediction tool to analyse the entire genome sequence of <em>G. thermoglucosidasius</em> NCIMB 11955, uncovering potential functional proteins in non-model strains. 2). The MetaPatchM module of BIM automates the repair of the metabolic network model. 3). The Tianjin University of Science and Technology-<em>k</em><sub>cat</sub> (TUST-<em>k</em><sub>cat</sub>) module predicts the <em>k</em><sub>cat</sub> values of enzymes within the model. 4). The Enzyme-insert procedure constructs an enzyme-constrained model and performs a global scan to address overconstraint issues. Enzymatic data were automatically integrated into the metabolic flux model, creating an enzyme-constrained model, ec_G-ther11955. To validate model accuracy, we used both the <em>p-thermo</em> and ec_G-ther11955 models to predict riboflavin production strategies. The ec_G-ther11955 model demonstrated significantly higher accuracy. To further verify its efficacy, we employed ec_G-ther11955 to guide the rational design of L-valine-producing strains. Using the Optimisation Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions (OptForce), Predictive Knockout Targeting (PKT), and Flux Scanning based on Enforced Objective Flux (FSEOF) algorithms, we identified 24 knockout and overexpression targets, achieving an accuracy rate of 87.5%. Ultimately, this led to an increase of 664.04% in L-valine titre. This study provides a novel strategy for rapidly constructing non-model strain models and demonstrates the tremendous potential of artificial intelligence in metabolic engineering.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 208-233"},"PeriodicalIF":6.8,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142469629","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-15DOI: 10.1016/j.ymben.2024.10.005
Hyeongmin Seo, Sofia H. Capece, John D. Hill, Jonathan K. Otten, Eleftherios T. Papoutsakis
The butyrate biosynthetic pathway not only contributes to electron management and energy generation in butyrate forming bacteria, but also confers evolutionary advantages to the host by inhibiting the growth of surrounding butyrate-sensitive microbes. While high butyrate levels induce toxic stress, effects of non-toxic levels on cell growth, health, metabolism, and sporulation remain unclear. Here, we show that butyrate stimulates cellular processes of Clostridium acetobutylicum, a model butyrate forming Firmicute. First, we deleted the 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from the C. acetobutylicum chromosome to eliminate the butyrate synthetic pathway and thus butyrate formation. A xylose inducible Cas9 cassette was chromosomally integrated and utilized for the one-step markerless gene deletions. Non-toxic butyrate levels significantly affected growth, health, and sporulation of C. acetobutylicum. After deleting spo0A, the gene encoding the master regulator of sporulation, Spo0A, and conducting butyrate addition experiments, we conclude that butyrate affects cellular metabolism through both Spo0A-dependent and independent mechanisms. We also deleted the hbd gene from the chromosome of the asporogenous C. acetobutylicum M5 strain lacking the pSOL1 plasmid to examine the potential involvement of pSOL1 genes on the observed butyrate effects. Addition of crotonate, the precursor of butyrate biosynthesis, to the hbd deficient M5 strain was used to probe the role of butyrate biosynthesis pathway in electron and metabolic fluxes. Finally, we found that butyrate addition can enhance the growth of the non-butyrate forming Clostridium saccharolyticum. Our data suggest that butyrate functions as a stimulator of cellular processes, like a growth factor, in C. acetobutylicum and potentially evolutionarily related Clostridium organisms.
{"title":"Butyrate as a growth factor of Clostridium acetobutylicum","authors":"Hyeongmin Seo, Sofia H. Capece, John D. Hill, Jonathan K. Otten, Eleftherios T. Papoutsakis","doi":"10.1016/j.ymben.2024.10.005","DOIUrl":"10.1016/j.ymben.2024.10.005","url":null,"abstract":"<div><div>The butyrate biosynthetic pathway not only contributes to electron management and energy generation in butyrate forming bacteria, but also confers evolutionary advantages to the host by inhibiting the growth of surrounding butyrate-sensitive microbes. While high butyrate levels induce toxic stress, effects of non-toxic levels on cell growth, health, metabolism, and sporulation remain unclear. Here, we show that butyrate stimulates cellular processes of <em>Clostridium acetobutylicum</em>, a model butyrate forming Firmicute. First, we deleted the 3-hydroxybutyryl-CoA dehydrogenase gene (<em>hbd</em>) from the <em>C. acetobutylicum</em> chromosome to eliminate the butyrate synthetic pathway and thus butyrate formation. A xylose inducible Cas9 cassette was chromosomally integrated and utilized for the one-step markerless gene deletions. Non-toxic butyrate levels significantly affected growth, health, and sporulation of <em>C. acetobutylicum</em>. After deleting <em>spo0A</em>, the gene encoding the master regulator of sporulation, Spo0A, and conducting butyrate addition experiments, we conclude that butyrate affects cellular metabolism through both Spo0A-dependent and independent mechanisms. We also deleted the <em>hbd</em> gene from the chromosome of the asporogenous <em>C. acetobutylicum</em> M5 strain lacking the pSOL1 plasmid to examine the potential involvement of pSOL1 genes on the observed butyrate effects. Addition of crotonate, the precursor of butyrate biosynthesis, to the <em>hbd</em> deficient M5 strain was used to probe the role of butyrate biosynthesis pathway in electron and metabolic fluxes. Finally, we found that butyrate addition can enhance the growth of the non-butyrate forming <em>Clostridium saccharolyticum</em>. Our data suggest that butyrate functions as a stimulator of cellular processes, like a growth factor, in <em>C. acetobutylicum</em> and potentially evolutionarily related <em>Clostridium</em> organisms.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 194-207"},"PeriodicalIF":6.8,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142469630","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-14DOI: 10.1016/j.ymben.2024.10.004
Nan Lu , Minhua Wei , Xuejing Yang , Yingzi Li , Hao Sun , Qianyu Yan , Haibin Zhang , Jilong He , Jie Ma , Menglei Xia , Chenglin Zhang
L-isoleucine, an essential amino acid, is widely used in the pharmaceutical and food industries. However, the current production efficiency is insufficient to meet the increasing demands. In this study, we aimed to develop an efficient L-isoleucine-producing strain of Escherichia coli. First, accumulation of L-isoleucine was achieved by employing feedback-resistant enzymes. Next, a growth-coupled L-isoleucine synthetic pathway was established by introducing the metA-metB-based α-ketobutyrate-generating bypass, which significantly increased L-isoleucine production to 7.4 g/L. Upon employing an activity-improved cystathionine γ-synthase mutant obtained from adaptive laboratory evolution, L-isoleucine production further increased to 8.5 g/L. Subsequently, the redox flux was improved by bypassing the NADPH-dependent aspartate aminotransferase pathway and employing the NADH-dependent pathway and transhydrogenase. Finally, L-isoleucine efflux was enhanced by modifying the transport system. After fed-batch fermentation for 48 h, the resultant strain, ISO-12, reached an L-isoleucine production titer of 51.5 g/L and yield of 0.29 g/g glucose. The strains developed in this study achieved a higher L-isoleucine production efficiency than those reported previously. These strategies will aid in the development of cell factories that produce L-isoleucine and related products.
{"title":"Growth-coupled production of L-isoleucine in Escherichia coli via metabolic engineering","authors":"Nan Lu , Minhua Wei , Xuejing Yang , Yingzi Li , Hao Sun , Qianyu Yan , Haibin Zhang , Jilong He , Jie Ma , Menglei Xia , Chenglin Zhang","doi":"10.1016/j.ymben.2024.10.004","DOIUrl":"10.1016/j.ymben.2024.10.004","url":null,"abstract":"<div><div>L-isoleucine, an essential amino acid, is widely used in the pharmaceutical and food industries. However, the current production efficiency is insufficient to meet the increasing demands. In this study, we aimed to develop an efficient L-isoleucine-producing strain of <em>Escherichia coli</em>. First, accumulation of L-isoleucine was achieved by employing feedback-resistant enzymes. Next, a growth-coupled L-isoleucine synthetic pathway was established by introducing the <em>metA</em>-<em>metB</em>-based α-ketobutyrate-generating bypass, which significantly increased L-isoleucine production to 7.4 g/L. Upon employing an activity-improved cystathionine γ-synthase mutant obtained from adaptive laboratory evolution, L-isoleucine production further increased to 8.5 g/L. Subsequently, the redox flux was improved by bypassing the NADPH-dependent aspartate aminotransferase pathway and employing the NADH-dependent pathway and transhydrogenase. Finally, L-isoleucine efflux was enhanced by modifying the transport system. After fed-batch fermentation for 48 h, the resultant strain, ISO-12, reached an L-isoleucine production titer of 51.5 g/L and yield of 0.29 g/g glucose. The strains developed in this study achieved a higher L-isoleucine production efficiency than those reported previously. These strategies will aid in the development of cell factories that produce L-isoleucine and related products.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 181-193"},"PeriodicalIF":6.8,"publicationDate":"2024-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142447953","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-09DOI: 10.1016/j.ymben.2024.10.002
Ruxue Sun , Peijun Yu , Liying Guo , Yufei Huang , Yanhong Nie , Yunpeng Yang
Escherichia coli Nissle 1917 (EcN), the probiotic featured with well-established safety in different host, is emerging as a favored chassis for the construction of engineered probiotics for disease treatment. However, limited by the low intestinal colonization ability of EcN, repeated administration is required to maximize the health benefits of the EcN-derived engineered probiotics. Here, using fecal metabolites as “metabolites pool”, we developed a metabolomic strategy to characterize the comprehensive metabolic profile of EcN. Compared with Prevotella copri DSM 18205 (P. copri), one of the dominant microbes in gut flora, EcN exhibited minor growth advantage under the fecal metabolites-containing condition for its lower metabolic capability towards fecal metabolites. Further study indicated that EcN lacked the ability to import the oligopeptides containing more than two amino acids. The shortage of oligopeptides-derived amino acids might limit the growth of EcN by restricting its purine metabolism. Assisted with the bioinformatic and qRT-PCR analyses, we identified a tripeptides-specific importer Pc-OPT in P. copri, which was mainly distributed in genera Prevotella and Bacteroides. Overexpression of Pc-OPT improved the tripeptides importation of EcN and promoted its growth and intestinal colonization. Notably, 16S rRNA gene amplicon sequencing results indicated that strengthening the oligopeptides importation ability of EcN might promote its intestinal colonization by adjusting the gut microbial composition. Our study reveals that the growth and intestinal colonization of EcN is limited by its insufficient oligopeptides importation and paves road for promoting the efficacy of the EcN-derived synthetic probiotics by improving their intestinal colonization ability.
{"title":"Improving the growth and intestinal colonization of Escherichia coli Nissle 1917 by strengthening its oligopeptides importation ability","authors":"Ruxue Sun , Peijun Yu , Liying Guo , Yufei Huang , Yanhong Nie , Yunpeng Yang","doi":"10.1016/j.ymben.2024.10.002","DOIUrl":"10.1016/j.ymben.2024.10.002","url":null,"abstract":"<div><div><em>Escherichia coli</em> Nissle 1917 (EcN), the probiotic featured with well-established safety in different host, is emerging as a favored chassis for the construction of engineered probiotics for disease treatment. However, limited by the low intestinal colonization ability of EcN, repeated administration is required to maximize the health benefits of the EcN-derived engineered probiotics. Here, using fecal metabolites as “metabolites pool”, we developed a metabolomic strategy to characterize the comprehensive metabolic profile of EcN. Compared with <em>Prevotella copri</em> DSM 18205 (<em>P. copri</em>), one of the dominant microbes in gut flora, EcN exhibited minor growth advantage under the fecal metabolites-containing condition for its lower metabolic capability towards fecal metabolites. Further study indicated that EcN lacked the ability to import the oligopeptides containing more than two amino acids. The shortage of oligopeptides-derived amino acids might limit the growth of EcN by restricting its purine metabolism. Assisted with the bioinformatic and qRT-PCR analyses, we identified a tripeptides-specific importer Pc-OPT in <em>P. copri</em>, which was mainly distributed in genera <em>Prevotella</em> and <em>Bacteroides</em>. Overexpression of Pc-OPT improved the tripeptides importation of EcN and promoted its growth and intestinal colonization. Notably, 16S rRNA gene amplicon sequencing results indicated that strengthening the oligopeptides importation ability of EcN might promote its intestinal colonization by adjusting the gut microbial composition. Our study reveals that the growth and intestinal colonization of EcN is limited by its insufficient oligopeptides importation and paves road for promoting the efficacy of the EcN-derived synthetic probiotics by improving their intestinal colonization ability.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 157-171"},"PeriodicalIF":6.8,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Salicin is a natural glycoside compound widely used to treat fever, inflammation, and analgesia. Currently, salicin is primarily extracted from willow bark, which is not only cumbersome in terms of extraction and separate steps, but also subject to seasonal and geographic limitations. In this study, a highly efficient biosynthetic pathway for salicin synthesis was designed and constructed in E. coli. The most important precursor in the synthetic pathway of salicin designed in this study is salicyl alcohol. Building on a previously constructed biosynthetic salicylic acid metabolic pathway, the production of salicyl alcohol in shake flask fermentation reached 1.7 g/L by increasing the supply of shikimic acid pathway precursor PEP and salicyl alcohol precursor chorismate. According to the principle of substrate similarity, this study identified the key enzyme OsSGT1 from Oryza sativa, which uses E. coli endogenous UDP-glucose as a glycosyl donor to glycosylate salicyl alcohol into salicin. By redefining the optimal substrate of OsSGT1, and balancing metabolic flux along with increasing the supply of UDP-glucose, salicin production in shake flasks reached 4 g/L. Finally, culturing the high-yield strain in a 3-L fermenter resulted in the synthesis of 14.62 g/L of salicin. To the best of our knowledge, this achievement marks the highest salicin production through microbial fermentation to date.
{"title":"Engineering a novel pathway for efficient biosynthesis of salicin in Escherichia coli","authors":"Jingyan Wang, Qianjing Zhao, Xin Chen, Yichen Lu, Xinxiao Sun, Qipeng Yuan, Jia Wang, Xiaolin Shen","doi":"10.1016/j.ymben.2024.10.003","DOIUrl":"10.1016/j.ymben.2024.10.003","url":null,"abstract":"<div><div>Salicin is a natural glycoside compound widely used to treat fever, inflammation, and analgesia. Currently, salicin is primarily extracted from willow bark, which is not only cumbersome in terms of extraction and separate steps, but also subject to seasonal and geographic limitations. In this study, a highly efficient biosynthetic pathway for salicin synthesis was designed and constructed in <em>E. coli</em>. The most important precursor in the synthetic pathway of salicin designed in this study is salicyl alcohol. Building on a previously constructed biosynthetic salicylic acid metabolic pathway, the production of salicyl alcohol in shake flask fermentation reached 1.7 g/L by increasing the supply of shikimic acid pathway precursor PEP and salicyl alcohol precursor chorismate. According to the principle of substrate similarity, this study identified the key enzyme OsSGT1 from <em>Oryza sativa</em>, which uses <em>E. coli</em> endogenous UDP-glucose as a glycosyl donor to glycosylate salicyl alcohol into salicin. By redefining the optimal substrate of OsSGT1, and balancing metabolic flux along with increasing the supply of UDP-glucose, salicin production in shake flasks reached 4 g/L. Finally, culturing the high-yield strain in a 3-L fermenter resulted in the synthesis of 14.62 g/L of salicin. To the best of our knowledge, this achievement marks the highest salicin production through microbial fermentation to date.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 172-180"},"PeriodicalIF":6.8,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400707","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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