Pub Date : 2025-12-11DOI: 10.1016/j.ymben.2025.12.001
Dileep Sai Kumar Palur , Shannon R. Pressley , Alex McGill , Yuanyuan Bai , Hai Yu , Xi Chen , Shota Atsumi
Human milk oligosaccharides (HMOs), such as lacto-N-tetraose (LNT), play critical roles in infant health by shaping gut microbiota and modulating immune function. While LNT is already produced at industrial scales, efficient microbial routes to more complex HMOs derived from LNT remain limited. Here, we established a simplified microbial platform in Escherichia coli that produces LNT directly from lactose as the sole carbon and precursor source. A key innovation was construction of a strain library with tunable β-galactosidase (LacZ) activity, enabling controlled lactose hydrolysis to generate glucose and galactose for UDP-sugar biosynthesis while preserving sufficient intact lactose as the scaffold for LNT assembly. Quantitative profiling of intracellular UDP-sugars further guided identification of metabolic bottlenecks. The optimized strain achieved co-production of 2.4 g/L LNT and 2.0 g/L lacto-N-triose II (LNT II) from 10 g/L lactose. This streamlined strategy demonstrates the feasibility of producing LNT from a single substrate and provides a versatile foundation for scalable microbial biosynthesis of more complex HMOs.
{"title":"Lacto-N-tetraose biosynthesis from lactose via metabolically rewired Escherichia coli","authors":"Dileep Sai Kumar Palur , Shannon R. Pressley , Alex McGill , Yuanyuan Bai , Hai Yu , Xi Chen , Shota Atsumi","doi":"10.1016/j.ymben.2025.12.001","DOIUrl":"10.1016/j.ymben.2025.12.001","url":null,"abstract":"<div><div>Human milk oligosaccharides (HMOs), such as lacto-<em>N</em>-tetraose (LNT), play critical roles in infant health by shaping gut microbiota and modulating immune function. While LNT is already produced at industrial scales, efficient microbial routes to more complex HMOs derived from LNT remain limited. Here, we established a simplified microbial platform in <em>Escherichia coli</em> that produces LNT directly from lactose as the sole carbon and precursor source. A key innovation was construction of a strain library with tunable β-galactosidase (LacZ) activity, enabling controlled lactose hydrolysis to generate glucose and galactose for UDP-sugar biosynthesis while preserving sufficient intact lactose as the scaffold for LNT assembly. Quantitative profiling of intracellular UDP-sugars further guided identification of metabolic bottlenecks. The optimized strain achieved co-production of 2.4 g/L LNT and 2.0 g/L lacto-<em>N</em>-triose II (LNT II) from 10 g/L lactose. This streamlined strategy demonstrates the feasibility of producing LNT from a single substrate and provides a versatile foundation for scalable microbial biosynthesis of more complex HMOs.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 182-191"},"PeriodicalIF":6.8,"publicationDate":"2025-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145731784","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 : 2025-12-11DOI: 10.1016/j.ymben.2025.12.003
Chao Wu , Jeffrey N. Law , Onyeka Onyenemezu , Jetendra K. Roy , Peter C. St. John , Robert L. Jernigan , Yannick J. Bomble , Laura Jarboe
Escherichia coli employs diverse strategies to adapt to acidic environments that disrupt enzyme activity and the thermodynamic feasibility of essential reactions. To understand the impact of pH stress on cell metabolism, we present the PET-FBA (pH-, Enzyme protein allocation-, and Thermodynamics-constrained Flux Balance Analysis) framework. PET-FBA extends genome-scale modeling by integrating enzyme protein costs and reaction Gibbs free energy changes. Additionally, by incorporating pH-dependent enzyme kinetics in response to intracellular acidification, this framework enables the simulation of E. coli's metabolic adjustments across varying external pH levels. The model's accuracy is validated by comparing in silico growth simulations with experimental measurements under both anaerobic and aerobic conditions, as well as in silico gene knockouts of essential genes. By explicitly incorporating pH effects, our model accurately replicates the metabolic shift towards lactate production as the primary fermentation product at low pH in anaerobic conditions. This shift is only predicted when enzyme kinetics are dynamically adjusted as a function of pH. Further analysis revealed that this shift can be attributed to the reduced protein efficiency of the acetyl-CoA branch compared to lactate dehydrogenase under acidic stress, which then becomes crucial for maintaining NAD regeneration and cell growth at low pH. Furthermore, we identified strategies for enhancing cell growth under acidic anaerobic conditions by improving the enzyme activity of lactate dehydrogenase and pyruvate formate lyase, which increases NAD production efficiency and reduces enzyme protein allocation costs. Designed as a lightweight yet versatile framework, PET-FBA enables efficient genome-scale metabolic analysis. Using E. coli as a model system, our framework provides a systematic approach to understanding metabolic responses to environmental stress, pinpointing key metabolic bottlenecks, and identifying potential targets for strain optimization. This work also highlights the critical role of intracellular acidification in shaping enzyme performance and microbial adaptation. The PET-FBA framework is implemented as a Python package at https://github.com/Chaowu88/etfba, with detailed documentation provided at https://etfba.readthedocs.io.
{"title":"PET-FBA: A lightweight enzyme allocation and thermodynamics-constrained flux analysis approach to explore Escherichia coli metabolic adaptation to intracellular acidification","authors":"Chao Wu , Jeffrey N. Law , Onyeka Onyenemezu , Jetendra K. Roy , Peter C. St. John , Robert L. Jernigan , Yannick J. Bomble , Laura Jarboe","doi":"10.1016/j.ymben.2025.12.003","DOIUrl":"10.1016/j.ymben.2025.12.003","url":null,"abstract":"<div><div><em>Escherichia coli</em> employs diverse strategies to adapt to acidic environments that disrupt enzyme activity and the thermodynamic feasibility of essential reactions. To understand the impact of pH stress on cell metabolism, we present the PET-FBA (pH-, Enzyme protein allocation-, and Thermodynamics-constrained Flux Balance Analysis) framework. PET-FBA extends genome-scale modeling by integrating enzyme protein costs and reaction Gibbs free energy changes. Additionally, by incorporating pH-dependent enzyme kinetics in response to intracellular acidification, this framework enables the simulation of <em>E. coli</em>'s metabolic adjustments across varying external pH levels. The model's accuracy is validated by comparing <em>in silico</em> growth simulations with experimental measurements under both anaerobic and aerobic conditions, as well as <em>in silico</em> gene knockouts of essential genes. By explicitly incorporating pH effects, our model accurately replicates the metabolic shift towards lactate production as the primary fermentation product at low pH in anaerobic conditions. This shift is only predicted when enzyme kinetics are dynamically adjusted as a function of pH. Further analysis revealed that this shift can be attributed to the reduced protein efficiency of the acetyl-CoA branch compared to lactate dehydrogenase under acidic stress, which then becomes crucial for maintaining NAD regeneration and cell growth at low pH. Furthermore, we identified strategies for enhancing cell growth under acidic anaerobic conditions by improving the enzyme activity of lactate dehydrogenase and pyruvate formate lyase, which increases NAD production efficiency and reduces enzyme protein allocation costs. Designed as a lightweight yet versatile framework, PET-FBA enables efficient genome-scale metabolic analysis. Using <em>E. coli</em> as a model system, our framework provides a systematic approach to understanding metabolic responses to environmental stress, pinpointing key metabolic bottlenecks, and identifying potential targets for strain optimization. This work also highlights the critical role of intracellular acidification in shaping enzyme performance and microbial adaptation. The PET-FBA framework is implemented as a Python package at <span><span>https://github.com/Chaowu88/etfba</span><svg><path></path></svg></span>, with detailed documentation provided at <span><span>https://etfba.readthedocs.io</span><svg><path></path></svg></span>.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 202-212"},"PeriodicalIF":6.8,"publicationDate":"2025-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145731099","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 : 2025-12-11DOI: 10.1016/j.ymben.2025.12.002
Weiming Tu , Jiabao Xu , Yongshuo Ma , Constantinos Katsimpouras , Gregory Stephanopoulos
Balancing metabolic pathways is critical for engineering microbial platforms to efficiently and robustly synthesize value-added bioproducts. In the oleaginous yeast Yarrowia lipolytica engineered for β-carotene production, lipid synthesis supports carotenoid storage but also competes with carotenoid synthesis for cellular resources, necessitating precise regulation for optimal resource allocation. In this study, we establish a machine learning framework that captures the complex interactions among three key metabolic modules for β-carotene synthesis: the mevalonate pathway (precursor supply for β-carotene), lipid synthesis (storage capacity), and the β-carotene synthetic cluster (product formation). This computational framework enables the prediction of β-carotene output based on gene combinations and guides iterative gene integration strategies across these interconnected pathways to optimize production. Using this approach, the best-performing strain YLT226 achieved a 7-fold increase in β-carotene titer compared to the initial strain YLT001 through nine rounds of guided gene integration. This work provides a promising strategy for understanding and engineering metabolic flux distributions.
{"title":"Machine learning-driven optimization of metabolic balance for β-carotene production","authors":"Weiming Tu , Jiabao Xu , Yongshuo Ma , Constantinos Katsimpouras , Gregory Stephanopoulos","doi":"10.1016/j.ymben.2025.12.002","DOIUrl":"10.1016/j.ymben.2025.12.002","url":null,"abstract":"<div><div>Balancing metabolic pathways is critical for engineering microbial platforms to efficiently and robustly synthesize value-added bioproducts. In the oleaginous yeast <em>Yarrowia lipolytica</em> engineered for β-carotene production, lipid synthesis supports carotenoid storage but also competes with carotenoid synthesis for cellular resources, necessitating precise regulation for optimal resource allocation. In this study, we establish a machine learning framework that captures the complex interactions among three key metabolic modules for β-carotene synthesis: the mevalonate pathway (precursor supply for β-carotene), lipid synthesis (storage capacity), and the β-carotene synthetic cluster (product formation). This computational framework enables the prediction of β-carotene output based on gene combinations and guides iterative gene integration strategies across these interconnected pathways to optimize production. Using this approach, the best-performing strain YLT226 achieved a 7-fold increase in β-carotene titer compared to the initial strain YLT001 through nine rounds of guided gene integration. This work provides a promising strategy for understanding and engineering metabolic flux distributions.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 192-201"},"PeriodicalIF":6.8,"publicationDate":"2025-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145731098","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 : 2025-12-01DOI: 10.1016/j.ymben.2025.11.012
Eric J. Mooney , Patrick F. Suthers , Wheaton L. Schroeder , Hoang V. Dinh , Xi Li , Yihui Shen , Tianxia Xiao , Catherine M. Call , Heide Baron , Arjuna M. Subramanian , Daniel R. Weilandt , Felix C. Keber , Martin Wühr , Joshua D. Rabinowitz , Costas D. Maranas
The yeast Rhodotorula toruloides is a promising bioproduction organism due to its high lipid yields and ability to grow on cheap and abundant substrates. Quantitative, systems-level assessment of its metabolic activity is accordingly merited. Resource-balance analysis (RBA) models capture not only reaction stoichiometry but also enzyme requirements for catalysis, providing valuable tools for understanding metabolic trade-offs and optimizing metabolic engineering strategies. Here, we present systems-level measurements of R. toruloides metabolic flux based on isotope tracing and metabolic flux analysis. In combination with new proteomic measurements, these flux data are used to parameterize a genome-scale resource balance model rtRBA. We find that S. cerevisiae and R. toruloides grow at nearly indistinguishable rates using similar biosynthetic but dramatically different central metabolic programs. R. toruloides consumes one-fifth as much glucose, which it metabolizes primarily via the pentose phosphate pathway and TCA cycle unlike primarily glycolysis in S. cerevisiae. Overall, across these two divergent yeasts, protein abundances aligned more closely than metabolic flux. Resource balance modeling of these metabolic programs predicts superior theoretical yields but lower productivities in R. toruloides than S. cerevisiae for industrial chemicals, highlighting the value of rapid glucose uptake for productivity but respiratory metabolism for yields.
{"title":"Metabolic flux and resource balance in the oleaginous yeast Rhodotorula toruloides","authors":"Eric J. Mooney , Patrick F. Suthers , Wheaton L. Schroeder , Hoang V. Dinh , Xi Li , Yihui Shen , Tianxia Xiao , Catherine M. Call , Heide Baron , Arjuna M. Subramanian , Daniel R. Weilandt , Felix C. Keber , Martin Wühr , Joshua D. Rabinowitz , Costas D. Maranas","doi":"10.1016/j.ymben.2025.11.012","DOIUrl":"10.1016/j.ymben.2025.11.012","url":null,"abstract":"<div><div>The yeast <em>Rhodotorula toruloides</em> is a promising bioproduction organism due to its high lipid yields and ability to grow on cheap and abundant substrates. Quantitative, systems-level assessment of its metabolic activity is accordingly merited. Resource-balance analysis (RBA) models capture not only reaction stoichiometry but also enzyme requirements for catalysis, providing valuable tools for understanding metabolic trade-offs and optimizing metabolic engineering strategies. Here, we present systems-level measurements of <em>R. toruloides</em> metabolic flux based on isotope tracing and metabolic flux analysis. In combination with new proteomic measurements, these flux data are used to parameterize a genome-scale resource balance model rtRBA. We find that <em>S. cerevisiae</em> and <em>R. toruloides</em> grow at nearly indistinguishable rates using similar biosynthetic but dramatically different central metabolic programs. <em>R. toruloides</em> consumes one-fifth as much glucose, which it metabolizes primarily via the pentose phosphate pathway and TCA cycle unlike primarily glycolysis in <em>S. cerevisiae.</em> Overall, across these two divergent yeasts, protein abundances aligned more closely than metabolic flux. Resource balance modeling of these metabolic programs predicts superior theoretical yields but lower productivities in <em>R. toruloides</em> than <em>S. cerevisiae</em> for industrial chemicals, highlighting the value of rapid glucose uptake for productivity but respiratory metabolism for yields.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 169-181"},"PeriodicalIF":6.8,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145657141","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 : 2025-11-30DOI: 10.1016/j.ymben.2025.11.017
Rou Wen , Yiling Chen , Jiale Wang , Weinan Yang , Fang Yang , Qiong Wu , Fuqing Wu , Xu Yan , Guo-Qiang Chen
Poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P (3HB-co-4HB) or P34HB) is a promising biopolyester for applications in food packaging, medical sutures, drug delivery, and tissue engineering due to its tunable thermomechanical properties. However, industrial-scale production of P34HB from glucose remains challenging. In this study, scalable P34HB production by engineered Halomonas bluephagenesis was developed. A de novo 4HB synthesis pathway was introduced into an endogenous toxin-antitoxin plasmid, enabling stable expression in the absence of antibiotics. Promoter engineering and pathway optimization fine-tuned 4HB molar ratio in P34HB from 18 to 39 mol%. The engineered H. bluephagenesis WR3 demonstrated successful scale-up from 7-L to 100-L and 5000-L bioreactors, achieving a maximum cell dry weight (CDW) of 72 g/L and 84% P (3HB-co-30 mol% 4HB) from the cultures in 7-L bioreactor. In further scale-up studies, H. bluephagenesis WR3 maintained comparable 4HB ratios, producing 69 g/L and 71 g/L CDW containing 74% and 61% P34HB copolymer in 100-L and 5000-L scale bioreactors, respectively. The amorphous P (3HB-co-30 mol% 4HB) exhibited high ductility, with an elongation at break of over 800% and a Young's modulus of 164 MPa. Additionally, morphology engineering and a controllable cell lysis were applied to enhance downstream processing efficiency. The optimized H. bluephagenesis WR25 produced 97 g/L CDW containing 83% P (3HB-co-20 mol% 4HB) in 7-L bioreactor, and 83 g/L CDW with 80% P34HB in 100-L bioreactor, while maintaining a consistently high glucose to P34HB conversion efficiency of 37%. This study provides a robust and cost-effective platform for industrial P34HB production from glucose harboring the toxin-antitoxin stable plasmid encoded with the 4HB pathway.
{"title":"Pilot production of P(3HB-co-4HB) by engineered Halomonas bluephagenesis harboring an endogenous plasmid grown on glucose","authors":"Rou Wen , Yiling Chen , Jiale Wang , Weinan Yang , Fang Yang , Qiong Wu , Fuqing Wu , Xu Yan , Guo-Qiang Chen","doi":"10.1016/j.ymben.2025.11.017","DOIUrl":"10.1016/j.ymben.2025.11.017","url":null,"abstract":"<div><div>Poly (3-hydroxybutyrate<em>-co</em>-4-hydroxybutyrate) (P (3HB-<em>co</em>-4HB) or P34HB) is a promising biopolyester for applications in food packaging, medical sutures, drug delivery, and tissue engineering due to its tunable thermomechanical properties. However, industrial-scale production of P34HB from glucose remains challenging. In this study, scalable P34HB production by engineered <em>Halomonas bluephagenesis</em> was developed. A <em>de novo</em> 4HB synthesis pathway was introduced into an endogenous toxin-antitoxin plasmid, enabling stable expression in the absence of antibiotics. Promoter engineering and pathway optimization fine-tuned 4HB molar ratio in P34HB from 18 to 39 mol%. The engineered <em>H. bluephagenesis</em> WR3 demonstrated successful scale-up from 7-L to 100-L and 5000-L bioreactors, achieving a maximum cell dry weight (CDW) of 72 g/L and 84% P (3HB-<em>co</em>-30 mol% 4HB) from the cultures in 7-L bioreactor. In further scale-up studies, <em>H. bluephagenesis</em> WR3 maintained comparable 4HB ratios, producing 69 g/L and 71 g/L CDW containing 74% and 61% P34HB copolymer in 100-L and 5000-L scale bioreactors, respectively. The amorphous P (3HB-<em>co</em>-30 mol% 4HB) exhibited high ductility, with an elongation at break of over 800% and a Young's modulus of 164 MPa. Additionally, morphology engineering and a controllable cell lysis were applied to enhance downstream processing efficiency. The optimized <em>H. bluephagenesis</em> WR25 produced 97 g/L CDW containing 83% P (3HB-<em>co</em>-20 mol% 4HB) in 7-L bioreactor, and 83 g/L CDW with 80% P34HB in 100-L bioreactor, while maintaining a consistently high glucose to P34HB conversion efficiency of 37%. This study provides a robust and cost-effective platform for industrial P34HB production from glucose harboring the toxin-antitoxin stable plasmid encoded with the 4HB pathway.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 153-168"},"PeriodicalIF":6.8,"publicationDate":"2025-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145619676","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}
<div><div>Alkanes are considered among the most promising candidates for next-generation biofuels. Amongst various pathways discovered for alkane production, the cyanobacterial AAR (acyl ACP reductase) - ADO (aldehyde deformylating oxygenase) pathway has been the most studied pathway. Considering that cyanobacteria have the innate ability to produce alkanes, they can serve as an excellent chassis for sustainable biofuel production. In the first report of the AAR-ADO pathway, it was recorded that there are 17 unique genes present in the genome of only alkane-producing cyanobacteria. However, except for the role of AAR and ADO, none of the other genes have been implicated in alkane production so far. In this study, we performed overexpression and/or deletion of all 17 unique genes in <em>Synechococcus elongatus</em> PCC7942 and evaluated their role in growth, photosynthetic efficiency and alkane production. Based on the essentiality feature of genes in the cell survival of PCC7942, 9 essential genes were overexpressed, and 8 non-essential genes were knocked out in PCC7942. Among the essential genes that made a significant impact on alkane production, the overexpression of <em>Synpcc7942_1772</em> (encoding small subunit ribosomal protein) and <em>Synpcc7942_2212</em> (encoding large subunit ribosomal protein) led to ∼3.3-fold and ∼4.1-fold increased alkane production, respectively, suggesting a previously unrecognized link between translational machinery and metabolic pathway, while co-expression of <em>aar</em> and <em>ado</em> together increased alkane production by ∼5-fold. For the non-essential genes, the deletion of <em>Synpcc7942_0452</em> (encoding hypothetical protein), <em>Synpcc7942_1223</em> (encoding DevC), and <em>Synpcc7942_1918</em> (encoding UDP‐glucose: tetrahydro biopterin glucosyltransferase) led to the complete abolition of alkane production, indicating their critical roles. On the other hand, the deletion of <em>Synpcc7942_0544</em> and <em>Synpcc7942_0619,</em> both encoding hypothetical proteins, led to a ∼5.6-fold and ∼4.4-fold increase in intracellular alkane production, respectively. Measurement of photosynthetic efficiency via Dual PAM (Pulse-Amplitude Modulated) fluorometry revealed a correlation between higher alkane production and increased photosynthetic efficiency, which the genome-scale metabolic model of PCC7942 also validated. Upon further detailed investigation of the genes making a large impact on alkane production, we identified <em>Synpcc7942_0619</em> and <em>Synpcc7942_1223</em> gene products as potential transporters based on the AlphaFold structure model and TMHMM (Transmembrane Hidden Markov Model) plot. The <em>Synpcc7942_0619</em> encodes a DedA family transporter whose overexpression led to ∼1.5-fold higher extracellular alkane production, while deletion had a reverse effect. On the other hand, <em>Synpcc7942_1223</em> encodes the DevC component of the tripartite efflux system, whose overexpression inc
{"title":"Unlocking the potential of unique genes in cyanobacterial alkane synthesis","authors":"Humaira Parveen , Vineesha Garg , Piyush Pachauri , Mohd Azeem Khan , Syed Shams Yazdani","doi":"10.1016/j.ymben.2025.11.016","DOIUrl":"10.1016/j.ymben.2025.11.016","url":null,"abstract":"<div><div>Alkanes are considered among the most promising candidates for next-generation biofuels. Amongst various pathways discovered for alkane production, the cyanobacterial AAR (acyl ACP reductase) - ADO (aldehyde deformylating oxygenase) pathway has been the most studied pathway. Considering that cyanobacteria have the innate ability to produce alkanes, they can serve as an excellent chassis for sustainable biofuel production. In the first report of the AAR-ADO pathway, it was recorded that there are 17 unique genes present in the genome of only alkane-producing cyanobacteria. However, except for the role of AAR and ADO, none of the other genes have been implicated in alkane production so far. In this study, we performed overexpression and/or deletion of all 17 unique genes in <em>Synechococcus elongatus</em> PCC7942 and evaluated their role in growth, photosynthetic efficiency and alkane production. Based on the essentiality feature of genes in the cell survival of PCC7942, 9 essential genes were overexpressed, and 8 non-essential genes were knocked out in PCC7942. Among the essential genes that made a significant impact on alkane production, the overexpression of <em>Synpcc7942_1772</em> (encoding small subunit ribosomal protein) and <em>Synpcc7942_2212</em> (encoding large subunit ribosomal protein) led to ∼3.3-fold and ∼4.1-fold increased alkane production, respectively, suggesting a previously unrecognized link between translational machinery and metabolic pathway, while co-expression of <em>aar</em> and <em>ado</em> together increased alkane production by ∼5-fold. For the non-essential genes, the deletion of <em>Synpcc7942_0452</em> (encoding hypothetical protein), <em>Synpcc7942_1223</em> (encoding DevC), and <em>Synpcc7942_1918</em> (encoding UDP‐glucose: tetrahydro biopterin glucosyltransferase) led to the complete abolition of alkane production, indicating their critical roles. On the other hand, the deletion of <em>Synpcc7942_0544</em> and <em>Synpcc7942_0619,</em> both encoding hypothetical proteins, led to a ∼5.6-fold and ∼4.4-fold increase in intracellular alkane production, respectively. Measurement of photosynthetic efficiency via Dual PAM (Pulse-Amplitude Modulated) fluorometry revealed a correlation between higher alkane production and increased photosynthetic efficiency, which the genome-scale metabolic model of PCC7942 also validated. Upon further detailed investigation of the genes making a large impact on alkane production, we identified <em>Synpcc7942_0619</em> and <em>Synpcc7942_1223</em> gene products as potential transporters based on the AlphaFold structure model and TMHMM (Transmembrane Hidden Markov Model) plot. The <em>Synpcc7942_0619</em> encodes a DedA family transporter whose overexpression led to ∼1.5-fold higher extracellular alkane production, while deletion had a reverse effect. On the other hand, <em>Synpcc7942_1223</em> encodes the DevC component of the tripartite efflux system, whose overexpression inc","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 136-152"},"PeriodicalIF":6.8,"publicationDate":"2025-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145611806","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 : 2025-11-21DOI: 10.1016/j.ymben.2025.11.011
Joseph H. Lynch , Shaunak Ray , Clint Chapple , Natalia Dudareva , John A. Morgan
2-Phenylethanol (2-PE) is a natural aromatic compound with properties that make it a potential biological oxygenate for petroleum-derived gasoline. In plants, 2-PE biosynthesis competes with the phenylpropanoid pathway for the common precursor, phenylalanine (Phe). The phenylpropanoid pathway directs significant carbon flux towards the formation of lignin, a major biopolymer in plant cell walls that impedes the process of biofuel production. Prior genetic engineering in Arabidopsis used acetaldehyde synthase (AAS) in tandem with phenylacetaldehyde reductase (PAR) to redirect part of the carbon flux from lignin towards 2-PE production as a value-added product. To identify the bottleneck(s) in 2-PE biosynthesis, we established a baseline by generating transgenic Arabidopsis overexpressing AAS and PAR. Next, fluxes to 2-PE and lignin were calculated based on the time course of isotopic enrichment of downstream metabolites after feeding with 13C6-ring labeled Phe, which revealed a limitation in Phe precursor availability. To increase substrate availability in plants, we tested two independent strategies by crossing lines with the highest AAS/PAR expression to: (1) Arabidopsis overexpressing a feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase known to increase Phe production, and (2) the pal1/pal2 double mutant known to reduce the activity of the competing enzyme, phenylalanine ammonia lyase (PAL). Although both strategies increased 2-PE production in both Arabidopsis stem and leaves, the second strategy had a higher impact. To identify additional metabolic targets, we performed a metabolic control analysis, which revealed that the plastidial Phe transporter limits flux towards 2-PE formation. To avoid this transport step, the PAR/AAS tandem construct was fused to a sequence encoding a chloroplast signal peptide to target 2-PE biosynthesis to plastids. The direct availability of Phe to AAS in plastids combined with the lack of competition with cytosolic PAL resulted in significantly elevated 2-PE levels. Thus, integrating metabolic control analysis with experimental validation of model predictions establishes a foundation for the rational engineering of 2-PE in plants.
{"title":"Model-guided metabolic engineering of 2-phenylethanol in Arabidopsis","authors":"Joseph H. Lynch , Shaunak Ray , Clint Chapple , Natalia Dudareva , John A. Morgan","doi":"10.1016/j.ymben.2025.11.011","DOIUrl":"10.1016/j.ymben.2025.11.011","url":null,"abstract":"<div><div>2-Phenylethanol (2-PE) is a natural aromatic compound with properties that make it a potential biological oxygenate for petroleum-derived gasoline. In plants, 2-PE biosynthesis competes with the phenylpropanoid pathway for the common precursor, phenylalanine (Phe). The phenylpropanoid pathway directs significant carbon flux towards the formation of lignin, a major biopolymer in plant cell walls that impedes the process of biofuel production. Prior genetic engineering in Arabidopsis used acetaldehyde synthase (AAS) in tandem with phenylacetaldehyde reductase (PAR) to redirect part of the carbon flux from lignin towards 2-PE production as a value-added product. To identify the bottleneck(s) in 2-PE biosynthesis, we established a baseline by generating transgenic Arabidopsis overexpressing AAS and PAR. Next, fluxes to 2-PE and lignin were calculated based on the time course of isotopic enrichment of downstream metabolites after feeding with <sup>13</sup>C<sub>6</sub>-ring labeled Phe, which revealed a limitation in Phe precursor availability. To increase substrate availability in plants, we tested two independent strategies by crossing lines with the highest <em>AAS/PAR</em> expression to: (1) Arabidopsis overexpressing a feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase known to increase Phe production, and (2) the <em>pal1/pal2</em> double mutant known to reduce the activity of the competing enzyme, phenylalanine ammonia lyase (PAL). Although both strategies increased 2-PE production in both Arabidopsis stem and leaves, the second strategy had a higher impact. To identify additional metabolic targets, we performed a metabolic control analysis, which revealed that the plastidial Phe transporter limits flux towards 2-PE formation. To avoid this transport step, the PAR/AAS tandem construct was fused to a sequence encoding a chloroplast signal peptide to target 2-PE biosynthesis to plastids. The direct availability of Phe to AAS in plastids combined with the lack of competition with cytosolic PAL resulted in significantly elevated 2-PE levels. Thus, integrating metabolic control analysis with experimental validation of model predictions establishes a foundation for the rational engineering of 2-PE in plants.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 124-135"},"PeriodicalIF":6.8,"publicationDate":"2025-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145567417","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 : 2025-11-19DOI: 10.1016/j.ymben.2025.11.015
Jinpeng Wang , Yuxiang Hong , Zizhao Wu , Ayelet Fishman , Peng Xu
Malonyl-CoA is a central precursor involved in the synthesis of various bio-based chemicals, including polyketides, fatty acids, and flavonoids. However, the production of these chemicals is often limited by the availability of malonyl-CoA. Based on retrosynthesis principles, we designed two thermodynamically favorable malonyl-CoA pathways using L-glutamate and L-aspartate as substrates. The novel pathways leverage oxidative deamination and decarboxylation reactions and efficiently channel metabolic flux toward malonyl-CoA, resulting in increased production of total polyketides beyond the capacity of the native acetyl-CoA carboxylase route using glucose as substrate. We also discovered a new-to-nature polyketide (4-hydroxy-6-hydroxyethyl-2-pyrone) derived from the side activity of the TAL pathway, reaching 6.4 g/L in Y. lipolytica. This work highlights the utility of the novel malonyl-CoA pathways in enhancing polyketide production, and the possibility of upcycling abundant amino acids or protein waste in the animal farming or meat industry to produce high-value nonnatural polyketides.
{"title":"Engineering amino acid-derived malonyl-CoA pathways to boost polyketide production in Yarrowia lipolytica","authors":"Jinpeng Wang , Yuxiang Hong , Zizhao Wu , Ayelet Fishman , Peng Xu","doi":"10.1016/j.ymben.2025.11.015","DOIUrl":"10.1016/j.ymben.2025.11.015","url":null,"abstract":"<div><div>Malonyl-CoA is a central precursor involved in the synthesis of various bio-based chemicals, including polyketides, fatty acids, and flavonoids. However, the production of these chemicals is often limited by the availability of malonyl-CoA. Based on retrosynthesis principles, we designed two thermodynamically favorable malonyl-CoA pathways using L-glutamate and L-aspartate as substrates. The novel pathways leverage oxidative deamination and decarboxylation reactions and efficiently channel metabolic flux toward malonyl-CoA, resulting in increased production of total polyketides beyond the capacity of the native acetyl-CoA carboxylase route using glucose as substrate. We also discovered a new-to-nature polyketide (4-hydroxy-6-hydroxyethyl-2-pyrone) derived from the side activity of the TAL pathway, reaching 6.4 g/L in <em>Y. lipolytica</em>. This work highlights the utility of the novel malonyl-CoA pathways in enhancing polyketide production, and the possibility of upcycling abundant amino acids or protein waste in the animal farming or meat industry to produce high-value nonnatural polyketides.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 99-109"},"PeriodicalIF":6.8,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145553880","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 : 2025-11-19DOI: 10.1016/j.ymben.2025.11.010
Roy Eerlings , Tobias Karmainski , Andreas Müsgens , Philipp Demling , Samira van den Bogaard , Makarius Baier , Alexander Deitert , Amila Vejzovic , Vanessa Veccari , Lillith Yöndem , Tobias Alter , Lars M. Blank
Itaconic acid, a versatile platform chemical, has garnered significant attention due to its broad use in polymers, resins, and bio-based materials. Although fungi, especially Aspergillus and Ustilago, are the main producers of itaconic acid, reprogramming yeast species like Saccharomyces cerevisiae and Yarrowia lipolytica as alternative production platforms offers advantages for industrial bioproduction, including rapid growth, accessible genetic tools, and well-established fermentation methods. In this study, we systematically investigated the fungal itaconic acid pathway dynamics in S. cerevisiae, identifying the Ustilago metabolic route and mitochondrial transport mechanism as the most efficient. Notably, the heterologous produced itaconic acid was predominantly secreted by the yeast transformants. With in silico methods, we confirmed the role of Aqr1p, Dtr1p, and Qdr3p in itaconic acid transport in S. cerevisiae. Significant increases in itaconic acid biosynthesis were obtained when the main promiscuous itaconic acid transporter Dtr1p was swapped with the specialized Ustilago itaconic acid transporter Itp1, reaching titers of up to 1.3 g/L in shake flask cultivations. Transferring to 3.8 L bioreactor fermentations achieved a final titer of 4.7 g/L, the highest itaconic acid titer reported in S. cerevisiae to date. Although current yeast production levels are still below those of natural fungal producers, the molecular and mechanistic insights gained here are useful for improving itaconic acid biosynthesis, both in yeast and in the existing fungal production systems.
{"title":"Elucidating the itaconic acid pathway dynamics in Saccharomyces cerevisiae","authors":"Roy Eerlings , Tobias Karmainski , Andreas Müsgens , Philipp Demling , Samira van den Bogaard , Makarius Baier , Alexander Deitert , Amila Vejzovic , Vanessa Veccari , Lillith Yöndem , Tobias Alter , Lars M. Blank","doi":"10.1016/j.ymben.2025.11.010","DOIUrl":"10.1016/j.ymben.2025.11.010","url":null,"abstract":"<div><div>Itaconic acid, a versatile platform chemical, has garnered significant attention due to its broad use in polymers, resins, and bio-based materials. Although fungi, especially <em>Aspergillus</em> and <em>Ustilago</em>, are the main producers of itaconic acid, reprogramming yeast species like <em>Saccharomyces cerevisiae</em> and <em>Yarrowia lipolytica</em> as alternative production platforms offers advantages for industrial bioproduction, including rapid growth, accessible genetic tools, and well-established fermentation methods. In this study, we systematically investigated the fungal itaconic acid pathway dynamics in <em>S. cerevisiae</em>, identifying the <em>Ustilago</em> metabolic route and mitochondrial transport mechanism as the most efficient. Notably, the heterologous produced itaconic acid was predominantly secreted by the yeast transformants. With <em>in silico</em> methods, we confirmed the role of Aqr1p, Dtr1p, and Qdr3p in itaconic acid transport in <em>S. cerevisiae</em>. Significant increases in itaconic acid biosynthesis were obtained when the main promiscuous itaconic acid transporter Dtr1p was swapped with the specialized <em>Ustilago</em> itaconic acid transporter Itp1, reaching titers of up to 1.3 g/L in shake flask cultivations. Transferring to 3.8 L bioreactor fermentations achieved a final titer of 4.7 g/L, the highest itaconic acid titer reported in <em>S. cerevisiae</em> to date. Although current yeast production levels are still below those of natural fungal producers, the molecular and mechanistic insights gained here are useful for improving itaconic acid biosynthesis, both in yeast and in the existing fungal production systems.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 110-123"},"PeriodicalIF":6.8,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145559771","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 : 2025-11-17DOI: 10.1016/j.ymben.2025.11.013
Cheon Woo Moon , Mohammad Rifqi Ghiffary , Cindy Pricilia Surya Prabowo , Hyun Uk Kim , Sang Yup Lee
3-Hydroxypropionic acid (3-HP) is a versatile platform chemical with broad applications, serving as a precursor for the synthesis of value-added chemicals as well as the biodegradable polymers. However, current industrial production of 3-HP relies on chemical synthesis, which requires harmful raw materials and harsh reaction conditions. As a sustainable alternative, microbial biosynthesis of 3-HP has gained increasing attention. Yet, most reported pathways remain constrained by their dependence on vitamin B12, a costly cofactor that limits scalability in industrial applications. Here, we report the development of a Corynebacterium glutamicum strain capable of high-level fermentative production of 3-HP from glucose via the introduction of a vitamin B12-independent, β-alanine-derived pathway. Candidate genes for the conversion of β-alanine to 3-HP were first screened, and the optimized pathway was subsequently introduced into a previously developed β-alanine-overproducing BAL10 strain. By eliminating competing pathways to increase precursor availability, redirecting carbon flux through the pentose phosphate pathway to improve cofactor balance, strengthening the β-alanine biosynthetic pathway, and identifying a previously uncharacterized 3-HP transporter followed by fine-tuning its expression, the final engineered strain produced 126.3 g/L of 3-HP in high-inoculum fed-batch fermentation, with a yield of 0.36 g/g glucose and an overall productivity of 1.75 g/L/h. These results demonstrate the feasibility of a vitamin B12-independent pathway for high-level 3-HP production, highlighting its potential for sustainable and scalable industrial application.
{"title":"Metabolic engineering of Corynebacterium glutamicum for vitamin B12-independent production of 3-hydroxypropionic acid","authors":"Cheon Woo Moon , Mohammad Rifqi Ghiffary , Cindy Pricilia Surya Prabowo , Hyun Uk Kim , Sang Yup Lee","doi":"10.1016/j.ymben.2025.11.013","DOIUrl":"10.1016/j.ymben.2025.11.013","url":null,"abstract":"<div><div>3-Hydroxypropionic acid (3-HP) is a versatile platform chemical with broad applications, serving as a precursor for the synthesis of value-added chemicals as well as the biodegradable polymers. However, current industrial production of 3-HP relies on chemical synthesis, which requires harmful raw materials and harsh reaction conditions. As a sustainable alternative, microbial biosynthesis of 3-HP has gained increasing attention. Yet, most reported pathways remain constrained by their dependence on vitamin B<sub>12</sub>, a costly cofactor that limits scalability in industrial applications. Here, we report the development of a <em>Corynebacterium glutamicum</em> strain capable of high-level fermentative production of 3-HP from glucose via the introduction of a vitamin B<sub>12</sub>-independent, β-alanine-derived pathway. Candidate genes for the conversion of β-alanine to 3-HP were first screened, and the optimized pathway was subsequently introduced into a previously developed β-alanine-overproducing BAL10 strain. By eliminating competing pathways to increase precursor availability, redirecting carbon flux through the pentose phosphate pathway to improve cofactor balance, strengthening the β-alanine biosynthetic pathway, and identifying a previously uncharacterized 3-HP transporter followed by fine-tuning its expression, the final engineered strain produced 126.3 g/L of 3-HP in high-inoculum fed-batch fermentation, with a yield of 0.36 g/g glucose and an overall productivity of 1.75 g/L/h. These results demonstrate the feasibility of a vitamin B<sub>12</sub>-independent pathway for high-level 3-HP production, highlighting its potential for sustainable and scalable industrial application.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 90-98"},"PeriodicalIF":6.8,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145536194","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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