Pub Date : 2025-11-17DOI: 10.1016/j.ymben.2025.11.014
Linh Khanh Nong, Chandran Sathesh-Prabu, Sung Kuk Lee, Donghyuk Kim
Pseudomonas putida strains are prized biocatalysts, renowned for their versatility in degrading diverse chemicals, tolerating organic solvents, and withstanding environmental stressors. Central to their adaptive success is the precise regulation of primary carbon metabolism, with HexR emerging as a key regulator. While previous research has explored HexR binding through in vitro assays and comparative transcriptomics, the in vivo binding sites and genome-scale regulon remain uncharted. This study presents a comparative analysis of P. putida KT2440, comparing expression profiles of wild-type and hexR deletion mutant strains across distinct growth substrates: glucose (glycolytic), acetate, succinate (gluconeogenic), and glycerol (inducing both metabolic responses). Our findings revealed an extensive regulatory role of HexR in acetate metabolism, simultaneously suppressing the glycolytic pathway while enhancing pyruvate metabolism, glyoxylate shunt, and gluconeogenesis to support growth. Integration of ChIP-exo data identified 29 HexR binding locations in the KT2440 strain grown on acetate, directly regulating 75 genes. Complementing these findings, model-based in silico simulations provided contextual insight into metabolic flux states, deepening our understanding of carbon metabolism orchestrated by this transcription factor. This study thus offers a holistic view of the HexR regulatory landscape, highlighting its relevance in P. putida KT2440 metabolism and laying the groundwork for future metabolic engineering efforts in this versatile organism.
{"title":"Redefining HexR regulatory landscape in Pseudomonas putida KT2440 through integrative systems biology","authors":"Linh Khanh Nong, Chandran Sathesh-Prabu, Sung Kuk Lee, Donghyuk Kim","doi":"10.1016/j.ymben.2025.11.014","DOIUrl":"10.1016/j.ymben.2025.11.014","url":null,"abstract":"<div><div><em>Pseudomonas putida</em> strains are prized biocatalysts, renowned for their versatility in degrading diverse chemicals, tolerating organic solvents, and withstanding environmental stressors. Central to their adaptive success is the precise regulation of primary carbon metabolism, with HexR emerging as a key regulator. While previous research has explored HexR binding through <em>in vitro</em> assays and comparative transcriptomics, the <em>in vivo</em> binding sites and genome-scale regulon remain uncharted. This study presents a comparative analysis of <em>P. putida</em> KT2440, comparing expression profiles of wild-type and <em>hexR</em> deletion mutant strains across distinct growth substrates: glucose (glycolytic), acetate, succinate (gluconeogenic), and glycerol (inducing both metabolic responses). Our findings revealed an extensive regulatory role of HexR in acetate metabolism, simultaneously suppressing the glycolytic pathway while enhancing pyruvate metabolism, glyoxylate shunt, and gluconeogenesis to support growth. Integration of ChIP-exo data identified 29 HexR binding locations in the KT2440 strain grown on acetate, directly regulating 75 genes. Complementing these findings, model-based <em>in silico</em> simulations provided contextual insight into metabolic flux states, deepening our understanding of carbon metabolism orchestrated by this transcription factor. This study thus offers a holistic view of the HexR regulatory landscape, highlighting its relevance in <em>P. putida</em> KT2440 metabolism and laying the groundwork for future metabolic engineering efforts in this versatile organism.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 77-89"},"PeriodicalIF":6.8,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145536219","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 : 2025-11-10DOI: 10.1016/j.ymben.2025.11.009
Sami Kazaz , Yu-Ton Chen , Senri Yamamoto , Yang Tian , Chien-Yuan Lin , Dylan Chin , İrem Pamukçu , Ibraheem Mohammed Al Shammaa , Yusuf Selman Akbas , Monikaben Nimavat , Emine Akyuz Turumtay , Edward E.K. Baidoo , Albert P. Kausch , Yuki Tobimatsu , Aymerick Eudes
The shikimate pathway is a crucial metabolic route for the biosynthesis of numerous valuable chemicals. In this study, we engineered the shikimate pathway in plants via expression of microbial enzymes to produce the two important antioxidants gallate and arbutin. The engineered pathways utilize the aromatics protocatechuate and 4-hydroxybenzoate as metabolic intermediates. Through transient expression in Nicotiana benthamiana leaves, we first identified biosynthetic routes for the production of gallate from either chorismate or 3-dehydroshikimate. Gallate production was then achieved in Arabidopsis using a genetic background that overproduces protocatechuate and via expression of a mutated version of the 4-hydroxybenzoate hydroxylase PobA from Pseudomonas sp. Arbutin production was obtained in Arabidopsis using a genetic background that overproduces 4-hydroxybenzoate and via expression of the monooxygenase MNX1 from Candida parapsilosis. The best Arabidopsis transgenic lines accumulated gallate and arbutin in the range of 0.25 and 0.93 dry weight % (dwt%), respectively. Using sorghum for large-scale in planta production, the titers of gallate and arbutin produced from the intermediate 4-hydroxybenzoate reached 0.58 dwt% and 0.50 dwt%, respectively, in mature transgenic plants, surpassing levels typically observed in plants that naturally produce these compounds. Gallate and arbutin were readily extracted from plant tissues using methanol solvent. Analysis of extractive-free biomass showed only trace amounts of gallate and its precursors 4-hydroxybenzoate and protocatechuate crosslinked to cell walls, suggesting that they mainly occur as soluble conjugated forms stored in the vacuole. This study presents alternative synthesis routes using plant hosts for the eco-friendly production of gallate and arbutin.
{"title":"Engineered plants for the production of the antioxidants arbutin and gallate","authors":"Sami Kazaz , Yu-Ton Chen , Senri Yamamoto , Yang Tian , Chien-Yuan Lin , Dylan Chin , İrem Pamukçu , Ibraheem Mohammed Al Shammaa , Yusuf Selman Akbas , Monikaben Nimavat , Emine Akyuz Turumtay , Edward E.K. Baidoo , Albert P. Kausch , Yuki Tobimatsu , Aymerick Eudes","doi":"10.1016/j.ymben.2025.11.009","DOIUrl":"10.1016/j.ymben.2025.11.009","url":null,"abstract":"<div><div>The shikimate pathway is a crucial metabolic route for the biosynthesis of numerous valuable chemicals. In this study, we engineered the shikimate pathway in plants via expression of microbial enzymes to produce the two important antioxidants gallate and arbutin. The engineered pathways utilize the aromatics protocatechuate and 4-hydroxybenzoate as metabolic intermediates. Through transient expression in <em>Nicotiana benthamiana</em> leaves, we first identified biosynthetic routes for the production of gallate from either chorismate or 3-dehydroshikimate. Gallate production was then achieved in Arabidopsis using a genetic background that overproduces protocatechuate and via expression of a mutated version of the 4-hydroxybenzoate hydroxylase PobA from <em>Pseudomonas</em> sp. Arbutin production was obtained in Arabidopsis using a genetic background that overproduces 4-hydroxybenzoate and via expression of the monooxygenase MNX1 from <em>Candida parapsilosis</em>. The best Arabidopsis transgenic lines accumulated gallate and arbutin in the range of 0.25 and 0.93 dry weight % (dwt%), respectively. Using sorghum for large-scale <em>in planta</em> production, the titers of gallate and arbutin produced from the intermediate 4-hydroxybenzoate reached 0.58 dwt% and 0.50 dwt%, respectively, in mature transgenic plants, surpassing levels typically observed in plants that naturally produce these compounds. Gallate and arbutin were readily extracted from plant tissues using methanol solvent. Analysis of extractive-free biomass showed only trace amounts of gallate and its precursors 4-hydroxybenzoate and protocatechuate crosslinked to cell walls, suggesting that they mainly occur as soluble conjugated forms stored in the vacuole. This study presents alternative synthesis routes using plant hosts for the eco-friendly production of gallate and arbutin.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 57-66"},"PeriodicalIF":6.8,"publicationDate":"2025-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145492048","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 : 2025-11-08DOI: 10.1016/j.ymben.2025.11.006
Razieh Rafieenia , Jing Fu , Piotr Hapeta , Marko Storch , Rodrigo Ledesma-Amaro
The oleaginous yeast, Yarrowia lipolytica has gained interest as a biotechnological chassis to produce foods, chemicals, pharmaceuticals, and biofuels. To reduce production costs and sustainability, inexpensive and abundant feedstocks such as lignocellulose must be used for bioproduction. Since lignocellulosic biomass contains components that cannot be utilised by Y. lipolytica, it is important to use engineering biology to enable their utilisation. L-arabinose is the second most abundant pentose in lignocellulose after xylose. However, it has received much less attention than xylose as a bioresource. In the present study, we first engineered Y. lipolytica to grow on L-arabinose as the sole carbon source. We used several wild-type and engineered strains to express the multigene arabinose cassette. Second, we used adaptive laboratory evolution to improve the utilisation of arabinose by the engineered strains. Third, we enabled the production of β-carotene from arabinose by expressing a β-carotene cassette in the evolved strain. Using minimal YNB medium supplemented with 20 g/l of arabinose as the sole carbon source resulted in the complete utilisation of L-arabinose within 120 h. In bioreactors, a β-carotene production of 418.89 mg/l was achieved with the complete utilisation of 60 g/l of L-arabinose. This study is the first to engineer L-arabinose utilisation in Y. lipolytica, opening new avenues for biomanufacturing using alternative carbon sources.
{"title":"Advancing arabinose-based bioproduction in Yarrowia lipolytica by integrating metabolic engineering and adaptive laboratory evolution","authors":"Razieh Rafieenia , Jing Fu , Piotr Hapeta , Marko Storch , Rodrigo Ledesma-Amaro","doi":"10.1016/j.ymben.2025.11.006","DOIUrl":"10.1016/j.ymben.2025.11.006","url":null,"abstract":"<div><div>The oleaginous yeast, <em>Yarrowia lipolytica</em> has gained interest as a biotechnological chassis to produce foods, chemicals, pharmaceuticals, and biofuels. To reduce production costs and sustainability, inexpensive and abundant feedstocks such as lignocellulose must be used for bioproduction. Since lignocellulosic biomass contains components that cannot be utilised by <em>Y. lipolytica</em>, it is important to use engineering biology to enable their utilisation. L-arabinose is the second most abundant pentose in lignocellulose after xylose. However, it has received much less attention than xylose as a bioresource. In the present study, we first engineered <em>Y. lipolytica</em> to grow on L-arabinose as the sole carbon source. We used several wild-type and engineered strains to express the multigene arabinose cassette. Second, we used adaptive laboratory evolution to improve the utilisation of arabinose by the engineered strains. Third, we enabled the production of β-carotene from arabinose by expressing a β-carotene cassette in the evolved strain. Using minimal YNB medium supplemented with 20 g/l of arabinose as the sole carbon source resulted in the complete utilisation of L-arabinose within 120 h. In bioreactors, a β-carotene production of 418.89 mg/l was achieved with the complete utilisation of 60 g/l of L-arabinose. This study is the first to engineer L-arabinose utilisation in <em>Y. lipolytica</em>, opening new avenues for biomanufacturing using alternative carbon sources.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 15-23"},"PeriodicalIF":6.8,"publicationDate":"2025-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145461945","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}
The reductive tricarboxylic acid (rTCA) cycle is a crucial metabolic pathway employed in the microbial production of C4-dicarboxylic acids, especially succinic acid (SA). However, the inherent redox constraints associated with this cycle pose significant limitations on the yields of SA. Here, we address this critical bottleneck by engineering a non-canonical reductive TCA (Nc-rTCA) pathway in oleaginous yeast Yarrowia lipolytica. Our approach substitutes the NADH-dependent conversion of oxaloacetate to fumarate in the native rTCA cycle with an engineered cascade utilizing aspartate aminotransferase, aspartate ammonia-lyase, and glutamate dehydrogenase, effectively decoupling SA synthesis from NADH limitations. This NADPH-dependent module resulted in a remarkable 1.28-fold increase in fumarate titer. Further metabolic optimization in the engineered strain Ncr12 minimized malate byproduct formation, achieving an SA titer of 98.16 g/L with a high yield of 0.91 g/g glucose in 5-L bioreactors. Importantly, the Nc-rTCA pathway demonstrated potential for industrial application, yielding 74.34 g/L SA at 0.98 g/g glucose from lignocellulosic hydrolysate and 117.74 g/L SA at 0.74 g/g from glycerol. Our findings address the longstanding redox imbalance issues that have challenged rTCA-based engineering and establish a scalable platform for bio-based C4-dicarboxylic acid production.
{"title":"Engineered non-canonical reductive TCA pathway drives high-yield succinic acid biosynthesis in Yarrowia lipolytica","authors":"Huilin Tao, Aomei Hao, Xiaoyue Pan, Yutao Zhong, Zhiyong Cui, Qingsheng Qi","doi":"10.1016/j.ymben.2025.11.003","DOIUrl":"10.1016/j.ymben.2025.11.003","url":null,"abstract":"<div><div>The reductive tricarboxylic acid (rTCA) cycle is a crucial metabolic pathway employed in the microbial production of C4-dicarboxylic acids, especially succinic acid (SA). However, the inherent redox constraints associated with this cycle pose significant limitations on the yields of SA. Here, we address this critical bottleneck by engineering a non-canonical reductive TCA (Nc-rTCA) pathway in oleaginous yeast <em>Yarrowia lipolytica</em>. Our approach substitutes the NADH-dependent conversion of oxaloacetate to fumarate in the native rTCA cycle with an engineered cascade utilizing aspartate aminotransferase, aspartate ammonia-lyase, and glutamate dehydrogenase, effectively decoupling SA synthesis from NADH limitations. This NADPH-dependent module resulted in a remarkable 1.28-fold increase in fumarate titer. Further metabolic optimization in the engineered strain Ncr12 minimized malate byproduct formation, achieving an SA titer of 98.16 g/L with a high yield of 0.91 g/g glucose in 5-L bioreactors. Importantly, the Nc-rTCA pathway demonstrated potential for industrial application, yielding 74.34 g/L SA at 0.98 g/g glucose from lignocellulosic hydrolysate and 117.74 g/L SA at 0.74 g/g from glycerol. Our findings address the longstanding redox imbalance issues that have challenged rTCA-based engineering and establish a scalable platform for bio-based C4-dicarboxylic acid production.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 36-44"},"PeriodicalIF":6.8,"publicationDate":"2025-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145461946","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 : 2025-11-06DOI: 10.1016/j.ymben.2025.11.007
Ian S. Yunus , David N. Carruthers , Yan Chen , Jennifer W. Gin , Edward E.K. Baidoo , Christopher J. Petzold , Hector Garcia Martin , Paul D. Adams , Aindrila Mukhopadhyay , Taek Soon Lee
CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, its application is often constrained by two challenges: (i) rationally identifying effective gene targets for downregulation and (ii) efficiently constructing multiplexed CRISPRi systems. In this study, we address both challenges by integrating a computational prioritization tool with a versatile assembly method for building multiplexed CRISPRi systems. FluxRETAP (Flux-Reaction Target Prioritization) accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers in Pseudomonas putida KT2440, outperforming a conventional non-computational, pathway-guided target selection. The highest isoprenol titer of nearly 1.5 g/L was achieved by knocking down PP_4118 (a gene encoding α-ketoglutarate dehydrogenase). The use of VAMMPIRE (Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation) enabled accurate assembly of CRISPRi constructs containing up to five sgRNA arrays, reducing context dependency and achieving uniform, position-independent gene downregulation. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.
{"title":"Predictive CRISPR-mediated gene downregulation for enhanced production of sustainable aviation fuel precursor in Pseudomonas putida","authors":"Ian S. Yunus , David N. Carruthers , Yan Chen , Jennifer W. Gin , Edward E.K. Baidoo , Christopher J. Petzold , Hector Garcia Martin , Paul D. Adams , Aindrila Mukhopadhyay , Taek Soon Lee","doi":"10.1016/j.ymben.2025.11.007","DOIUrl":"10.1016/j.ymben.2025.11.007","url":null,"abstract":"<div><div>CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, its application is often constrained by two challenges: (i) rationally identifying effective gene targets for downregulation and (ii) efficiently constructing multiplexed CRISPRi systems. In this study, we address both challenges by integrating a computational prioritization tool with a versatile assembly method for building multiplexed CRISPRi systems. FluxRETAP (Flux-Reaction Target Prioritization) accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers in <em>Pseudomonas putida</em> KT2440, outperforming a conventional non-computational, pathway-guided target selection. The highest isoprenol titer of nearly 1.5 g/L was achieved by knocking down PP_4118 (a gene encoding α-ketoglutarate dehydrogenase). The use of VAMMPIRE (Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation) enabled accurate assembly of CRISPRi constructs containing up to five sgRNA arrays, reducing context dependency and achieving uniform, position-independent gene downregulation. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 67-76"},"PeriodicalIF":6.8,"publicationDate":"2025-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145447343","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 : 2025-11-05DOI: 10.1016/j.ymben.2025.11.004
Juthamas Jaroensuk , Joshua P. Abraham , Baltazar E. Zuniga , Hawkins S. Shepard , Michael Wei , Russell Williams , Stewart A. Morley , Maneesh Lingwan , Jiahong Zhou , Michael A. Jindra , Poonam Jyoti , Bo Wang , Jody C. May , John A. McLean , Jamey D. Young , Brian F. Pfleger , Doug K. Allen
Free fatty acid (FFA) production in bacteria is a key target for metabolic engineering. The knockout of the acyl-ACP synthetase (AAS) prevents reincorporation of FFA into the fatty acid biosynthetic cycle and is widely used to enhance their secretion. However, the role of AAS in membrane lipid remodeling under environmental stress, such as altered temperature, remains poorly understood. In cyanobacteria, temperature shifts are known to affect fatty acid desaturation and membrane fluidity, yet it is unclear whether AAS contributes to these adaptive responses through re-esterification of membrane-released acyl chains. We elucidated unique aspects of fatty acid metabolism in response to temperature changes in biotechnologically relevant microbes with the development of an efficient method for quantifying acyl-ACP intermediates using anion exchange chromatography (AEX). In Escherichia coli, which performs desaturation during fatty acid biosynthesis, we detected saturated and unsaturated acyl-ACPs that confirm biosynthetic pathway operation. In the cyanobacteria, Picosynechococcus sp. PCC 7002 and the Δaas strain, changes between two temperatures were interpreted with support from proteomic and lipidomic analyses and indicated that the AAS is tied to membrane lipid remodeling. Further, polyunsaturated acyl-ACPs were detected in the Δaas strain, which was unexpected because fatty acid synthesis does not produce polyunsaturates in cyanobacteria, suggesting the presence of alternative acyl-activating enzymes or unknown acyl-ACP desaturases. This study highlights the possible link between acyl chain recycling and lipid remodeling in cyanobacteria and demonstrates the utility of AEX-based acyl-ACP profiling in dissecting fatty acid metabolism.
{"title":"Disruption of acyl-acyl carrier protein (acyl-ACP) synthetase in cyanobacteria impairs lipid remodeling as revealed by acyl-ACP measurements","authors":"Juthamas Jaroensuk , Joshua P. Abraham , Baltazar E. Zuniga , Hawkins S. Shepard , Michael Wei , Russell Williams , Stewart A. Morley , Maneesh Lingwan , Jiahong Zhou , Michael A. Jindra , Poonam Jyoti , Bo Wang , Jody C. May , John A. McLean , Jamey D. Young , Brian F. Pfleger , Doug K. Allen","doi":"10.1016/j.ymben.2025.11.004","DOIUrl":"10.1016/j.ymben.2025.11.004","url":null,"abstract":"<div><div>Free fatty acid (FFA) production in bacteria is a key target for metabolic engineering. The knockout of the acyl-ACP synthetase (AAS) prevents reincorporation of FFA into the fatty acid biosynthetic cycle and is widely used to enhance their secretion. However, the role of AAS in membrane lipid remodeling under environmental stress, such as altered temperature, remains poorly understood. In cyanobacteria, temperature shifts are known to affect fatty acid desaturation and membrane fluidity, yet it is unclear whether AAS contributes to these adaptive responses through re-esterification of membrane-released acyl chains. We elucidated unique aspects of fatty acid metabolism in response to temperature changes in biotechnologically relevant microbes with the development of an efficient method for quantifying acyl-ACP intermediates using anion exchange chromatography (AEX). In <em>Escherichia coli,</em> which performs desaturation during fatty acid biosynthesis, we detected saturated and unsaturated acyl-ACPs that confirm biosynthetic pathway operation. In the cyanobacteria, <em>Picosynechococcus</em> sp. PCC 7002 and the Δ<em>aas</em> strain, changes between two temperatures were interpreted with support from proteomic and lipidomic analyses and indicated that the AAS is tied to membrane lipid remodeling. Further, polyunsaturated acyl-ACPs were detected in the Δ<em>aas</em> strain, which was unexpected because fatty acid synthesis does not produce polyunsaturates in cyanobacteria, suggesting the presence of alternative acyl-activating enzymes or unknown acyl-ACP desaturases. This study highlights the possible link between acyl chain recycling and lipid remodeling in cyanobacteria and demonstrates the utility of AEX-based acyl-ACP profiling in dissecting fatty acid metabolism.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 45-56"},"PeriodicalIF":6.8,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145447241","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 : 2025-11-05DOI: 10.1016/j.ymben.2025.11.002
Cui Guo , Nguyen N.T. Luu , Maryem M. Adwer , Hemen Hosseinzadeh , Venkatesh Balan , Yajun Yan , Yuheng Lin
Psychedelic-assisted therapy is emerging as a highly promising approach for treating depression, with psilocybin, a psychoactive compound in magic mushrooms, gaining the most recognition for its efficacy in treating post-traumatic stress disorder and treatment-resistant depression. However, its low natural abundance makes extraction costly, necessitating alternative production methods. While engineered microbial production has been explored, dependence on the CYP450 hydroxylase (PsiH) in the natural biosynthetic pathway remains a major bottleneck, limiting production efficiency. Here, we report the design, validation, and optimization of artificial biosynthetic pathways in Escherichia coli that bypass PsiH, enabling efficient psilocybin and psilocin production. De novo biosynthesis of psilocybin achieved record titers of 557.91 mg/L in shake flasks and 2.00 g/L in a bioreactor, outperforming previous microbial engineering efforts. This work demonstrates the great commercial potential of microbial psilocybin production via combinatorial metabolic engineering and synthetic biology approaches.
{"title":"Engineering artificial biosynthetic pathways for efficient microbial production of psilocybin and psilocin","authors":"Cui Guo , Nguyen N.T. Luu , Maryem M. Adwer , Hemen Hosseinzadeh , Venkatesh Balan , Yajun Yan , Yuheng Lin","doi":"10.1016/j.ymben.2025.11.002","DOIUrl":"10.1016/j.ymben.2025.11.002","url":null,"abstract":"<div><div>Psychedelic-assisted therapy is emerging as a highly promising approach for treating depression, with psilocybin, a psychoactive compound in magic mushrooms, gaining the most recognition for its efficacy in treating post-traumatic stress disorder and treatment-resistant depression. However, its low natural abundance makes extraction costly, necessitating alternative production methods. While engineered microbial production has been explored, dependence on the CYP450 hydroxylase (PsiH) in the natural biosynthetic pathway remains a major bottleneck, limiting production efficiency. Here, we report the design, validation, and optimization of artificial biosynthetic pathways in <em>Escherichia coli</em> that bypass PsiH, enabling efficient psilocybin and psilocin production. <em>De novo</em> biosynthesis of psilocybin achieved record titers of 557.91 mg/L in shake flasks and 2.00 g/L in a bioreactor, outperforming previous microbial engineering efforts. This work demonstrates the great commercial potential of microbial psilocybin production via combinatorial metabolic engineering and synthetic biology approaches.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 24-34"},"PeriodicalIF":6.8,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145447345","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 : 2025-11-05DOI: 10.1016/j.ymben.2025.11.005
Chenyang Zhang , Xuan Zhou , Wei Wei , Jiahui Yu , Yaokang Wu , Yanfeng Liu , Jianghua Li , Guocheng Du , Jian Chen , Tongcheng Xu , Xueqin Lv , Xianhao Xu , Long Liu
Human milk fats (HMFs) could facilitate nutrient absorption in the infant gut, with 1,3-olein-2-palmitin (OPO) and 1-olein-2-palmitin-3-linolein (OPL) being the most abundant components. The construction of microbial cell factories has garnered significant interest due to their potential to synthesize HMFs from cheap raw materials. However, the substrate preference of endogenous triglyceride (TAG) synthases and the complex fatty acid (FA) composition limit OPO and OPL synthesis. This study developed a microbial cell factory for OPO and OPL production by reconstructing and fine-tuning the lipid metabolic network in Saccharomyces cerevisiae. First, the TAG biosynthesis pathway of S. cerevisiae was reconstructed, resulting in more than 70 % of palmitic acid (C16:0) in TAG being esterified to the sn-2 position, while simultaneously achieving de novo OPO synthesis. Further optimization of intracellular FA composition improved the OPO proportion in TAG to 26.59 %. De novo synthesis of OPL was achieved by introducing a heterologous synthesis pathway of linoleic acid (C18:2). A push-pull strategy was employed to promote FA and TAG synthesis, resulting in a 3.86-fold increase in TAG production and reaching 81.2 mg/g dry cell weight in shake flask. In a 3-L bioreactor, the engineered strain HF-35 achieved OPO and OPL titers of 85.68 mg/L and 162.30 mg/L, respectively, representing the highest reported titers of OPO and OPL using glucose as the substrate to date. This study demonstrated that regulating lipid metabolism is an effective strategy for specific TAG synthesis and lays the foundation for large-scale production of OPO and OPL.
{"title":"De novo production of 1,3-olein-2-palmitin (OPO) and 1-olein-2-palmitin-3-linolein (OPL) by multiplexed reconstruction of lipid metabolism in yeasts","authors":"Chenyang Zhang , Xuan Zhou , Wei Wei , Jiahui Yu , Yaokang Wu , Yanfeng Liu , Jianghua Li , Guocheng Du , Jian Chen , Tongcheng Xu , Xueqin Lv , Xianhao Xu , Long Liu","doi":"10.1016/j.ymben.2025.11.005","DOIUrl":"10.1016/j.ymben.2025.11.005","url":null,"abstract":"<div><div>Human milk fats (HMFs) could facilitate nutrient absorption in the infant gut, with 1,3-olein-2-palmitin (OPO) and 1-olein-2-palmitin-3-linolein (OPL) being the most abundant components. The construction of microbial cell factories has garnered significant interest due to their potential to synthesize HMFs from cheap raw materials. However, the substrate preference of endogenous triglyceride (TAG) synthases and the complex fatty acid (FA) composition limit OPO and OPL synthesis. This study developed a microbial cell factory for OPO and OPL production by reconstructing and fine-tuning the lipid metabolic network in <em>Saccharomyces cerevisiae</em>. First, the TAG biosynthesis pathway of <em>S</em>. <em>cerevisiae</em> was reconstructed, resulting in more than 70 % of palmitic acid (C16:0) in TAG being esterified to the <em>sn</em>-2 position, while simultaneously achieving <em>de novo</em> OPO synthesis. Further optimization of intracellular FA composition improved the OPO proportion in TAG to 26.59 %. <em>De novo</em> synthesis of OPL was achieved by introducing a heterologous synthesis pathway of linoleic acid (C18:2). A push-pull strategy was employed to promote FA and TAG synthesis, resulting in a 3.86-fold increase in TAG production and reaching 81.2 mg/g dry cell weight in shake flask. In a 3-L bioreactor, the engineered strain HF-35 achieved OPO and OPL titers of 85.68 mg/L and 162.30 mg/L, respectively, representing the highest reported titers of OPO and OPL using glucose as the substrate to date. This study demonstrated that regulating lipid metabolism is an effective strategy for specific TAG synthesis and lays the foundation for large-scale production of OPO and OPL.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 1-14"},"PeriodicalIF":6.8,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145447230","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 : 2025-11-04DOI: 10.1016/j.ymben.2025.11.001
Tingfeng Cheng , Suihao Yan , Min Xu , Lei Zhao
Microbial natural products (NPs) are a pivotal reservoir for drugs used in human health and agriculture. Andrimid, a polyketide-non-ribosomal peptide hybrid antibiotic inhibiting bacterial acetyl-CoA carboxylase, shows enormous potential in antibiotic drug development to mitigate antimicrobial resistance. However, industrial-scale manufacturing and downstream development of andrimid are largely prohibited due to its milligram level production in microorganisms. Herein, using an integrative multi-omics approach, we improved the yield of andrimid remarkably from milligram to gram level in a non-model environmental soil bacterium, Erwinia persicina BST187, isolated from the rhizosphere of tomato. Systematic reprogramming of the pathways for carbon source uptake, competing metabolites biosynthesis, supply of essential building blocks including phenylalanine, glycine, valine and malonyl-CoA and cofactor biosynthesis using CRIPSR/Cas9 based gene editing tools, coupled with fine-tuning the transcription of the biosynthetic genes of andrimid, resulted in the generation of the optimal producer, G17. Combined with fermentation optimization, andrimid was produced to a highest level of 1099.42 mg/L with a productivity of 15.3 mg/L/h using a 5 L bioreactor, representing a 628-fold increase compared to the parental strain. This study showcases the genome wide engineering of non-model bacteria and generates a plasmid- and inducer-free E. persicina strain for high-level andrimid production, providing a blueprint for systems-driven metabolic engineering of complex bioactive NPs for biomanufacturing.
{"title":"From soil to biomanufacturing: Systems-driven metabolic pathway rewiring in non-model bacteria for gram-scale antibiotic production","authors":"Tingfeng Cheng , Suihao Yan , Min Xu , Lei Zhao","doi":"10.1016/j.ymben.2025.11.001","DOIUrl":"10.1016/j.ymben.2025.11.001","url":null,"abstract":"<div><div>Microbial natural products (NPs) are a pivotal reservoir for drugs used in human health and agriculture. Andrimid, a polyketide-non-ribosomal peptide hybrid antibiotic inhibiting bacterial acetyl-CoA carboxylase, shows enormous potential in antibiotic drug development to mitigate antimicrobial resistance. However, industrial-scale manufacturing and downstream development of andrimid are largely prohibited due to its milligram level production in microorganisms. Herein, using an integrative multi-omics approach, we improved the yield of andrimid remarkably from milligram to gram level in a non-model environmental soil bacterium, <em>Erwinia persicina</em> BST187, isolated from the rhizosphere of tomato. Systematic reprogramming of the pathways for carbon source uptake, competing metabolites biosynthesis, supply of essential building blocks including phenylalanine, glycine, valine and malonyl-CoA and cofactor biosynthesis using CRIPSR/Cas9 based gene editing tools, coupled with fine-tuning the transcription of the biosynthetic genes of andrimid, resulted in the generation of the optimal producer, G17. Combined with fermentation optimization, andrimid was produced to a highest level of 1099.42 mg/L with a productivity of 15.3 mg/L/h using a 5 L bioreactor, representing a 628-fold increase compared to the parental strain. This study showcases the genome wide engineering of non-model bacteria and generates a plasmid- and inducer-free <em>E. persicina</em> strain for high-level andrimid production, providing a blueprint for systems-driven metabolic engineering of complex bioactive NPs for biomanufacturing.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"93 ","pages":"Pages 271-285"},"PeriodicalIF":6.8,"publicationDate":"2025-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145442020","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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