Pub Date : 2025-08-25DOI: 10.1016/j.ymben.2025.08.011
Akinobu Katano , Ayana Mori , Daisuke Nonaka , Yutaro Mori , Shuhei Noda , Tsutomu Tanaka
Pyridine carboxylic acids, because of their structural similarity to aromatic carboxylic acids, have garnered increasing attention as alternative compounds in chemical synthesis. However, their broader utilization has been limited by challenges in biosynthetic production. In this study, we developed a metabolic pathway for biosynthesizing 2,5-pyridinedicarboxylate (2,5-PDCA) from glucose from p-aminobenzoate (PABA). The heterologous expression of 4-amino-3-hydroxybenzoate 2,3-dioxygenase (AhdA) in Escherichia coli enabled the conversion of 0.5 g/L of 4-amino-3-hydroxybenzoate (4A3HBA) into 0.47 g/L of 2,5-PDCA. Subsequent systematic evaluation of p-hydroxybenzoate hydroxylase (PobA) variants and optimization of pobA and ahdA co-expression facilitated the development of a 2,5-PDCA biosynthetic module for efficient production from PABA. Incorporating this module into a PABA biosynthesis pathway enabled direct 2,5-PDCA production from glucose. Further enhancements were achieved by increasing metabolic flux through the shikimate pathway and optimizing sodium pyruvate supplementation. Under optimized conditions, we achieved a titer of 1.84 g/L in test-tube cultures after 72 h and 10.6 g/L in bioreactor fermentation after 144 h. Overall, this study introduces a valuable strategy for the microbial production of pyridine carboxylates and establishes a promising platform for broader applications in aromatic compound biosynthesis.
{"title":"Biosynthesis of 2,5-pyridinedicarboxylate from glucose via p-aminobenzoic acid in Escherichia coli","authors":"Akinobu Katano , Ayana Mori , Daisuke Nonaka , Yutaro Mori , Shuhei Noda , Tsutomu Tanaka","doi":"10.1016/j.ymben.2025.08.011","DOIUrl":"10.1016/j.ymben.2025.08.011","url":null,"abstract":"<div><div>Pyridine carboxylic acids, because of their structural similarity to aromatic carboxylic acids, have garnered increasing attention as alternative compounds in chemical synthesis. However, their broader utilization has been limited by challenges in biosynthetic production. In this study, we developed a metabolic pathway for biosynthesizing 2,5-pyridinedicarboxylate (2,5-PDCA) from glucose from <em>p</em>-aminobenzoate (PABA). The heterologous expression of 4-amino-3-hydroxybenzoate 2,3-dioxygenase (AhdA) in <em>Escherichia coli</em> enabled the conversion of 0.5 g/L of 4-amino-3-hydroxybenzoate (4A3HBA) into 0.47 g/L of 2,5-PDCA. Subsequent systematic evaluation of <em>p</em>-hydroxybenzoate hydroxylase (PobA) variants and optimization of <em>pobA</em> and <em>ahdA</em> co-expression facilitated the development of a 2,5-PDCA biosynthetic module for efficient production from PABA. Incorporating this module into a PABA biosynthesis pathway enabled direct 2,5-PDCA production from glucose. Further enhancements were achieved by increasing metabolic flux through the shikimate pathway and optimizing sodium pyruvate supplementation. Under optimized conditions, we achieved a titer of 1.84 g/L in test-tube cultures after 72 h and 10.6 g/L in bioreactor fermentation after 144 h. Overall, this study introduces a valuable strategy for the microbial production of pyridine carboxylates and establishes a promising platform for broader applications in aromatic compound biosynthesis.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 252-261"},"PeriodicalIF":6.8,"publicationDate":"2025-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144908973","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 : 2025-08-22DOI: 10.1016/j.ymben.2025.08.009
Shivangi Mishra, Ke Xu, Madeline K. Kuckuk, William T. Cordell, Néstor J. Hernández-Lozada, Brian F. Pfleger
Poly(3-hydroxyoctanoate) (PHO) is a medium-chain-length PHA with low crystallinity and high elongation to break ratio, unlike the brittle short-chain-PHAs like PHB. These properties make PHO a promising candidate for industrial and biomedical applications. In this study, we demonstrated the production of PHO in Escherichia coli from a renewable and inexpensive glycerol feedstock by engineering fatty acid synthesis and β-oxidation to create a pool of 2,3-octenoyl-CoAs. In this base strain, E. coli ΔfadRABIJ, an (R)-specific enoyl-CoA hydratase (phaJ) and a PHA synthase (phaC) were expressed to produce PHO. Bioprospecting phaJ and phaC homologs from Pseudomonas aeruginosa and fadD homolog from Pseudomonas putida implicated a combination of phaJ2, phaC2, and PpfadD genes yielded the highest PHO content from exogenously fed octanoate. Finally, when a single copy of a previously described C8-specific thioesterase mutant CpFatB1.2-M4-287 was integrated into the chromosome of E. coli ΔfadRABIJ, the resulting E. coli strain NHL18 was capable of producing 3.69 ± 0.146 g/L of octanoic acid. Subsequently, the integration of PHA synthesis genes in NHL18 resulting in strain SM23 allowed the cell to accumulate 15 % cell dry weight of PHO with a final titer of 1.54 ± 0.234 g/L from glycerol in fed-batch fermentation.
{"title":"A metabolic engineering strategy for producing poly-(3-hydroxyoctanoic acid) in Escherichia coli from glycerol","authors":"Shivangi Mishra, Ke Xu, Madeline K. Kuckuk, William T. Cordell, Néstor J. Hernández-Lozada, Brian F. Pfleger","doi":"10.1016/j.ymben.2025.08.009","DOIUrl":"10.1016/j.ymben.2025.08.009","url":null,"abstract":"<div><div>Poly(3-hydroxyoctanoate) (PHO) is a medium-chain-length PHA with low crystallinity and high elongation to break ratio, unlike the brittle short-chain-PHAs like PHB. These properties make PHO a promising candidate for industrial and biomedical applications. In this study, we demonstrated the production of PHO in <em>Escherichia coli</em> from a renewable and inexpensive glycerol feedstock by engineering fatty acid synthesis and β-oxidation to create a pool of 2,3-octenoyl-CoAs. In this base strain, <em>E. coli</em> Δ<em>fadRABIJ,</em> an (R)-specific enoyl-CoA hydratase (<em>phaJ</em>) and a PHA synthase (<em>phaC</em>) were expressed to produce PHO. Bioprospecting <em>phaJ</em> and <em>phaC</em> homologs from <em>Pseudomonas aeruginosa</em> and <em>fadD</em> homolog from <em>Pseudomonas putida</em> implicated a combination of <em>phaJ2</em>, <em>phaC2</em>, and <sup>Pp</sup><em>fadD</em> genes yielded the highest PHO content from exogenously fed octanoate. Finally, when a single copy of a previously described C<sub>8</sub>-specific thioesterase mutant <em>CpFatB1.2-M4-287</em> was integrated into the chromosome of <em>E. coli</em> Δ<em>fadRABIJ,</em> the resulting <em>E. coli</em> strain NHL18 was capable of producing 3.69 ± 0.146 g/L of octanoic acid. Subsequently, the integration of PHA synthesis genes in NHL18 resulting in strain SM23 allowed the cell to accumulate 15 % cell dry weight of PHO with a final titer of 1.54 ± 0.234 g/L from glycerol in fed-batch fermentation.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 232-240"},"PeriodicalIF":6.8,"publicationDate":"2025-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144895573","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 : 2025-08-22DOI: 10.1016/j.ymben.2025.08.010
Xi Li , Daniel R. Weilandt , Felix C. Keber , Arjuna M. Subramanian , Shayne R. Loynes , Christopher V. Rao , Yihui Shen , Martin Wühr , Joshua D. Rabinowitz
Oleaginous yeasts are used commercially to produce oleochemicals and hold potential also for biodiesel production. In response to nitrogen or phosphorous limitation, oleaginous yeasts accumulate lipids in the form of triacylglycerols. Previous work has investigated potential mechanisms by which nutrient limitation induces lipid biosynthesis without verifying whether lipid biosynthesis flux is actually enhanced. Here we show, using 13C-glucose tracing, that in nitrogen or phosphorous limitation, lipid accumulation occurs without consistent increases in biosynthetic flux. Instead, the main driver of increased lipid pools is decreased growth-related dilution. This conclusion holds across two divergent oleaginous yeasts: Rhodotorula toruloides and Yarrowia lipolytica. Quantitative proteomics shows a substantial proteome reallocation in response to nitrogen and phosphorous limitation, with ribosomal proteins strongly downregulated, while lipid enzymes are preserved but not consistently upregulated in absolute quantity. Thus, nutrient limitation, rather than triggering greatly enhanced lipid synthesis, results in roughly sustained lipid enzyme levels and biosynthetic flux. Due to slower lipid dilution by cell division, this suffices to drive marked lipid accumulation.
{"title":"Lipid accumulation in nitrogen and phosphorus-limited yeast is caused by less growth-related dilution","authors":"Xi Li , Daniel R. Weilandt , Felix C. Keber , Arjuna M. Subramanian , Shayne R. Loynes , Christopher V. Rao , Yihui Shen , Martin Wühr , Joshua D. Rabinowitz","doi":"10.1016/j.ymben.2025.08.010","DOIUrl":"10.1016/j.ymben.2025.08.010","url":null,"abstract":"<div><div>Oleaginous yeasts are used commercially to produce oleochemicals and hold potential also for biodiesel production. In response to nitrogen or phosphorous limitation, oleaginous yeasts accumulate lipids in the form of triacylglycerols. Previous work has investigated potential mechanisms by which nutrient limitation induces lipid biosynthesis without verifying whether lipid biosynthesis flux is actually enhanced. Here we show, using <sup>13</sup>C-glucose tracing, that in nitrogen or phosphorous limitation, lipid accumulation occurs without consistent increases in biosynthetic flux. Instead, the main driver of increased lipid pools is decreased growth-related dilution. This conclusion holds across two divergent oleaginous yeasts: <em>Rhodotorula toruloides</em> and <em>Yarrowia lipolytica</em>. Quantitative proteomics shows a substantial proteome reallocation in response to nitrogen and phosphorous limitation, with ribosomal proteins strongly downregulated, while lipid enzymes are preserved but not consistently upregulated in absolute quantity. Thus, nutrient limitation, rather than triggering greatly enhanced lipid synthesis, results in roughly sustained lipid enzyme levels and biosynthetic flux. Due to slower lipid dilution by cell division, this suffices to drive marked lipid accumulation.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"93 ","pages":"Pages 60-72"},"PeriodicalIF":6.8,"publicationDate":"2025-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144900577","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 : 2025-08-20DOI: 10.1016/j.ymben.2025.08.005
Byung Tae Lee , Omkar S. Mohite , Mun Su Kwon , Hahk-Soo Kang , Tilmann Weber , Sang Yup Lee , Hyun Uk Kim
Secondary metabolites have crucial medicinal and industrial applications, but their alignment with primary metabolism remains unclear. As secondary metabolism depends on primary metabolism for precursor supply, we present a pan-reactome analysis of 242 Streptomyces strains to investigate their association and disconnection. This analysis includes phylogenetic grouping of the strains using genome data, and uniform manifold approximation and projection (UMAP) analysis of their genome-scale metabolic models (GEMs) and biosynthetic gene cluster (BGC) data, which represent biochemical reactions in primary and secondary metabolism. Subsequent correlation analysis of the preprocessed GEM and BGC data showed a Pearson correlation coefficient of 0.54, revealing both metabolic association and disconnection. In particular, among 47 precursors of polyketides, nonribosomal peptides, and hybrids, nine precursors required by these BGCs were predicted to be non-producible due to missing genes in primary metabolism or BGCs. The pan-reactome analysis facilitates the identification of precursor availability and metabolic gaps, providing insights into secondary metabolite biosynthesis.
{"title":"Pan-reactome analysis of Streptomyces strains reveals association and disconnection between primary and secondary metabolism","authors":"Byung Tae Lee , Omkar S. Mohite , Mun Su Kwon , Hahk-Soo Kang , Tilmann Weber , Sang Yup Lee , Hyun Uk Kim","doi":"10.1016/j.ymben.2025.08.005","DOIUrl":"10.1016/j.ymben.2025.08.005","url":null,"abstract":"<div><div>Secondary metabolites have crucial medicinal and industrial applications, but their alignment with primary metabolism remains unclear. As secondary metabolism depends on primary metabolism for precursor supply, we present a pan-reactome analysis of 242 <em>Streptomyces</em> strains to investigate their association and disconnection. This analysis includes phylogenetic grouping of the strains using genome data, and uniform manifold approximation and projection (UMAP) analysis of their genome-scale metabolic models (GEMs) and biosynthetic gene cluster (BGC) data, which represent biochemical reactions in primary and secondary metabolism. Subsequent correlation analysis of the preprocessed GEM and BGC data showed a Pearson correlation coefficient of 0.54, revealing both metabolic association and disconnection. In particular, among 47 precursors of polyketides, nonribosomal peptides, and hybrids, nine precursors required by these BGCs were predicted to be non-producible due to missing genes in primary metabolism or BGCs. The pan-reactome analysis facilitates the identification of precursor availability and metabolic gaps, providing insights into secondary metabolite biosynthesis.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 241-251"},"PeriodicalIF":6.8,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144895574","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 : 2025-08-18DOI: 10.1016/j.ymben.2025.08.008
Maurice Hädrich , Josef Hoff , Bastian Blombach
The exploitation of Vibrio natriegens as an unconventional host for biotechnology has progressed rapidly. This development is not only a result of the remarkable high growth rate of this marine bacterium on different substrates but is also possible due to good handling properties, a versatile metabolism and its inherent natural competence – features that have facilitated the development of a sophisticated genetic engineering and synthetic biology toolbox. The availability of robust metabolic and regulatory data enables a model-based quantitative description of metabolic routes and accelerates rational metabolic engineering of the facultative anaerobic bacterium. As reviewed here, numerous examples, ranging from small-molecule production over cell-free protein synthesis to bioremediation render V. natriegens a promising next-generation host for biotechnological applications.
{"title":"Advances in metabolic engineering of Vibrio natriegens as an unconventional host for biotechnology","authors":"Maurice Hädrich , Josef Hoff , Bastian Blombach","doi":"10.1016/j.ymben.2025.08.008","DOIUrl":"10.1016/j.ymben.2025.08.008","url":null,"abstract":"<div><div>The exploitation of <em>Vibrio natriegens</em> as an unconventional host for biotechnology has progressed rapidly. This development is not only a result of the remarkable high growth rate of this marine bacterium on different substrates but is also possible due to good handling properties, a versatile metabolism and its inherent natural competence – features that have facilitated the development of a sophisticated genetic engineering and synthetic biology toolbox. The availability of robust metabolic and regulatory data enables a model-based quantitative description of metabolic routes and accelerates rational metabolic engineering of the facultative anaerobic bacterium. As reviewed here, numerous examples, ranging from small-molecule production over cell-free protein synthesis to bioremediation render <em>V. natriegens</em> a promising next-generation host for biotechnological applications.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 217-231"},"PeriodicalIF":6.8,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144892258","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 : 2025-08-18DOI: 10.1016/j.ymben.2025.08.007
Federico De Marco , Ivy Rose Sebastian , Antonino Napoleone , Alexander Molin , Markus Riedl , Nina Bydlinski , Krishna Motheramgari , Mohamed K. Hussein , Lovro Kramer , Thomas Kelly , Thomas Jostock , Nicole Borth
The biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90 % of the genome and its impact on cellular phenotypes. This unannotated “dark matter” includes regulatory elements and other poorly characterized genomic features that may be potentially critical for cell behaviour. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO cell survival, many of which currently lack gene annotation or known functions. For these regions, we present their annotation status, transcriptional activity and annotated chromatin states. Selected regions, particularly those lacking all of the above, were individually deleted to confirm their essentiality. This work sheds a novel light on a substantial portion of the mammalian genome that has been traditionally difficult to investigate and therefore neglected. Notably, the fact that the deletion of some of these regions is lethal to cells suggests they encode critical regulatory functions. A better genome-wide understanding of these functions could open new avenues for engineering cells with improved bioprocess relevant properties.
{"title":"A genome-scale CRISPR deletion screen in Chinese hamster ovary cells reveals essential regions of the coding and non-coding genome","authors":"Federico De Marco , Ivy Rose Sebastian , Antonino Napoleone , Alexander Molin , Markus Riedl , Nina Bydlinski , Krishna Motheramgari , Mohamed K. Hussein , Lovro Kramer , Thomas Kelly , Thomas Jostock , Nicole Borth","doi":"10.1016/j.ymben.2025.08.007","DOIUrl":"10.1016/j.ymben.2025.08.007","url":null,"abstract":"<div><div>The biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90 % of the genome and its impact on cellular phenotypes. This unannotated “dark matter” includes regulatory elements and other poorly characterized genomic features that may be potentially critical for cell behaviour. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO cell survival, many of which currently lack gene annotation or known functions. For these regions, we present their annotation status, transcriptional activity and annotated chromatin states. Selected regions, particularly those lacking all of the above, were individually deleted to confirm their essentiality. This work sheds a novel light on a substantial portion of the mammalian genome that has been traditionally difficult to investigate and therefore neglected. Notably, the fact that the deletion of some of these regions is lethal to cells suggests they encode critical regulatory functions. A better genome-wide understanding of these functions could open new avenues for engineering cells with improved bioprocess relevant properties.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 194-207"},"PeriodicalIF":6.8,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144885422","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 : 2025-08-16DOI: 10.1016/j.ymben.2025.08.006
Jayce E. Taylor , Trevor Gannalo , Bryant Luu , Dileep Sai Kumar Palur , Augustine Arredondo , Ian C. Anderson , Twisha Dasgupta , John Didzbalis , Justin B. Siegel , Shota Atsumi
Monosaccharides exist in either “D” or “L” conformations, with L-sugars being much less abundant in nature and therefore classified as “rare sugars.” Rare sugars hold significant potential due to their unique interactions with biological systems, offering health, food, and crop benefits. One such sugar, L-sorbose, serves as a critical precursor to Vitamin C and offers a low-calorie, moderately sweet alternative to table sugar, being 60–70 % as sweet but with only 25 % of the caloric value. However, the broader study and application of rare sugars, including L-sorbose, are constrained by their high cost and limited availability. To address this challenge, we developed a biosynthetic strategy to convert the abundant and inexpensive D-sugar D-glucose into the rare L-sugar L-sorbose using microbial production. By utilizing phosphorylation and dephosphorylation steps to thermodynamically drive carbon flux, efficient production of 14.5 g L−1 L-sorbose was achieved under test tube conditions. Additionally, this pathway results in the co-production of D-sedoheptulose, a non-sweet, rare sugar shown to inhibit C6 sugar consumption in humans by modulating energy metabolism. The dual production of L-sorbose and D-sedoheptulose presents unique opportunities for applications in food and health sciences. This study demonstrates microbial production as a promising platform for rare L-sugar biosynthesis and provides a generalizable strategy for converting abundant D-sugars into underexplored L-sugars. Expanding access to L-sugars enables deeper investigations into their biological functions, metabolic pathways, and industrial applications. By advancing both fundamental sugar metabolism research and microbial production strategies, this study broadens the scope of rare sugar utilization.
单糖以“D”或“L”构象存在，L糖在自然界中的含量要少得多，因此被归类为“稀有糖”。稀有糖具有巨大的潜力，因为它们与生物系统的独特相互作用，提供健康，食品和作物效益。其中一种糖，l -山梨糖，是维生素C的关键前体，提供了一种低热量、中等甜味的替代食糖，甜味为60-70%，但热量只有食糖的25%。然而，包括l -山梨糖在内的稀有糖的广泛研究和应用受到其高成本和有限可用性的限制。为了解决这一挑战，我们开发了一种生物合成策略，利用微生物生产将丰富而廉价的d -糖d -葡萄糖转化为稀有的l -糖L-sorbose。利用磷酸化和去磷酸化步骤热力学驱动碳通量，在试管条件下实现了14.5 g L-1 l -海马糖的高效生产。此外，这一途径导致D-sedoheptulose的共同产生，D-sedoheptulose是一种非甜的稀有糖，通过调节能量代谢来抑制人体对C6糖的消耗。l -山梨糖和d -糖庚糖的双重生产为食品和健康科学的应用提供了独特的机会。本研究证明微生物生产是稀有l糖生物合成的一个有前途的平台，并为将丰富的d糖转化为未开发的l糖提供了一种通用策略。扩大对l糖的获取，可以更深入地研究它们的生物学功能、代谢途径和工业应用。本研究通过推进糖代谢基础研究和微生物生产策略，拓宽了稀有糖利用的范围。
{"title":"Retrofitting Escherichia coli for de novo production of rare L-sorbose from abundant D-glucose","authors":"Jayce E. Taylor , Trevor Gannalo , Bryant Luu , Dileep Sai Kumar Palur , Augustine Arredondo , Ian C. Anderson , Twisha Dasgupta , John Didzbalis , Justin B. Siegel , Shota Atsumi","doi":"10.1016/j.ymben.2025.08.006","DOIUrl":"10.1016/j.ymben.2025.08.006","url":null,"abstract":"<div><div>Monosaccharides exist in either “D” or “L” conformations, with L-sugars being much less abundant in nature and therefore classified as “rare sugars.” Rare sugars hold significant potential due to their unique interactions with biological systems, offering health, food, and crop benefits. One such sugar, L-sorbose, serves as a critical precursor to Vitamin C and offers a low-calorie, moderately sweet alternative to table sugar, being 60–70 % as sweet but with only 25 % of the caloric value. However, the broader study and application of rare sugars, including L-sorbose, are constrained by their high cost and limited availability. To address this challenge, we developed a biosynthetic strategy to convert the abundant and inexpensive D-sugar D-glucose into the rare L-sugar L-sorbose using microbial production. By utilizing phosphorylation and dephosphorylation steps to thermodynamically drive carbon flux, efficient production of 14.5 g L<sup>−1</sup> L-sorbose was achieved under test tube conditions. Additionally, this pathway results in the co-production of D-sedoheptulose, a non-sweet, rare sugar shown to inhibit C6 sugar consumption in humans by modulating energy metabolism. The dual production of L-sorbose and D-sedoheptulose presents unique opportunities for applications in food and health sciences. This study demonstrates microbial production as a promising platform for rare L-sugar biosynthesis and provides a generalizable strategy for converting abundant D-sugars into underexplored L-sugars. Expanding access to L-sugars enables deeper investigations into their biological functions, metabolic pathways, and industrial applications. By advancing both fundamental sugar metabolism research and microbial production strategies, this study broadens the scope of rare sugar utilization.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 208-216"},"PeriodicalIF":6.8,"publicationDate":"2025-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144873966","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 : 2025-08-12DOI: 10.1016/j.ymben.2025.08.004
Fabia Weiland, Kyoyoung Seo, Franka Janz, Marius Grad, Lea Geldmacher, Michael Kohlstedt, Judith Becker, Christoph Wittmann
Lignocellulosic biomass represents a promising renewable feedstock for sustainable biochemical production, with p-hydroxycinnamates emerging as key aromatic building blocks derived from agricultural residues and grassy plants. C. glutamicum has recently been engineered to produce cis, cis-muconate (MA), a high-value platform chemical used in biobased plastics, resins, and specialty chemicals. However, unlike other aromatics, the metabolism of the p-hydroxycinnamates p-coumarate, ferulate, and caffeate in MA-producing C. glutamicum is inefficient, limiting MA production performance. Here, we discovered that p-hydroxycinnamate metabolism, encoded by the phd operon, is repressed by the local repressor PhdR under glucose-rich conditions, while the global regulator GlxR activates the pathway in the absence of glucose. The deregulated C. glutamicum MA-10 lacking phdR exhibited an up to 98-fold increase in the conversion of p-coumarate, ferulate, and aromatic mixtures derived from plant waste into MA. Transcriptomic and metabolomic analyses revealed strong induction of the phd operon in strain MA-10 and a marked increase in intracellular aromatic CoA-esters and acetyl-CoA, indicating enhanced flux through the p-hydroxycinnamate degradation pathway. 13C-tracer studies demonstrated a substantial contribution of aromatic side-chain carbon to central metabolic pathways, supporting biomass formation and enabling MA production even in the absence of sugars. Additionally, MA-10 showed broadened substrate flexibility, degrading cinnamate into MA and methoxylated cinnamates into valuable benzoate derivatives. The strain also successfully converted aromatics from real straw lignin hydrolysates into MA. Our findings reveal the potential of targeted regulatory engineering to optimize C. glutamicum for lignin valorization. The newly developed strain MA-10 provides a robust platform for the biobased production of MA from lignocellulosic feedstocks, paving the way for sustainable and economically viable biorefinery processes.
{"title":"Metabolic engineering of Corynebacterium glutamicum for increased cis, cis-muconate production from plant-derived p-hydroxycinnamates via deregulated pathway flux and increased CoA intermediate availability","authors":"Fabia Weiland, Kyoyoung Seo, Franka Janz, Marius Grad, Lea Geldmacher, Michael Kohlstedt, Judith Becker, Christoph Wittmann","doi":"10.1016/j.ymben.2025.08.004","DOIUrl":"10.1016/j.ymben.2025.08.004","url":null,"abstract":"<div><div>Lignocellulosic biomass represents a promising renewable feedstock for sustainable biochemical production, with <em>p</em>-hydroxycinnamates emerging as key aromatic building blocks derived from agricultural residues and grassy plants. <em>C. glutamicum</em> has recently been engineered to produce <em>cis, cis</em>-muconate (MA), a high-value platform chemical used in biobased plastics, resins, and specialty chemicals. However, unlike other aromatics, the metabolism of the <em>p</em>-hydroxycinnamates <em>p</em>-coumarate, ferulate, and caffeate in MA-producing <em>C. glutamicum</em> is inefficient, limiting MA production performance. Here, we discovered that <em>p</em>-hydroxycinnamate metabolism, encoded by the <em>phd</em> operon, is repressed by the local repressor PhdR under glucose-rich conditions, while the global regulator GlxR activates the pathway in the absence of glucose. The deregulated <em>C. glutamicum</em> MA-10 lacking <em>phdR</em> exhibited an up to 98-fold increase in the conversion of <em>p</em>-coumarate, ferulate, and aromatic mixtures derived from plant waste into MA. Transcriptomic and metabolomic analyses revealed strong induction of the <em>phd</em> operon in strain MA-10 and a marked increase in intracellular aromatic CoA-esters and acetyl-CoA, indicating enhanced flux through the <em>p</em>-hydroxycinnamate degradation pathway. <sup>13</sup>C-tracer studies demonstrated a substantial contribution of aromatic side-chain carbon to central metabolic pathways, supporting biomass formation and enabling MA production even in the absence of sugars. Additionally, MA-10 showed broadened substrate flexibility, degrading cinnamate into MA and methoxylated cinnamates into valuable benzoate derivatives. The strain also successfully converted aromatics from real straw lignin hydrolysates into MA. Our findings reveal the potential of targeted regulatory engineering to optimize <em>C. glutamicum</em> for lignin valorization. The newly developed strain MA-10 provides a robust platform for the biobased production of MA from lignocellulosic feedstocks, paving the way for sustainable and economically viable biorefinery processes.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 262-283"},"PeriodicalIF":6.8,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144850834","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 : 2025-08-11DOI: 10.1016/j.ymben.2025.08.003
Kuo Zhao , Hailin Gao , Mengnan Han , Bo Zhang , Zhiqiang Liu , Shuping Zou , Yuguo Zheng
D-pantothenic acid (D-PA) is a vital water-soluble vitamin with diverse industrial applications, driving the demand for efficient microbial production. Here, we rationally engineered an Escherichia coli strain to enhance D-PA production through metabolic engineering. First, to enhance carbon utilization efficiency, competing byproduct pathways were deleted and the pentose phosphate pathway was downregulated. Next, the glucose and β-alanine transport systems were strategically enhanced, and cofactor availability was improved through engineering NADPH regeneration and ATP recycling pathways. Subsequently, pathway engineering was applied to fine-tune the expression of heterologous enzymes, thereby enhancing the metabolic pull toward D-PA biosynthesis. To enhance the supply of one-carbon donor required by the rate-limiting enzyme ketopantoate hydroxymethyltransferase (KPHMT), a heterologous 5,10-methylenetetrahydrofolate biosynthesis module was introduced. Finally, dynamic regulation of isocitrate synthase and pantothenate kinase was implemented to balance cell growth and D-PA production. As a result of the integrated metabolic engineering strategies, the final strain DPZ28/P31 achieved a D-PA titer of 98.6 g/L and a yield of 0.44 g/g glucose in a two-stage fed-batch fermentation. These findings provide valuable insights for industrial-scale production of D-PA and related compounds.
d -泛酸（D-PA）是一种重要的水溶性维生素，具有多种工业应用，推动了对高效微生物生产的需求。本研究通过代谢工程对大肠杆菌菌株进行合理改造，提高D-PA的产量。首先，为了提高碳利用效率，删除竞争性副产物途径，下调戊糖磷酸途径。接下来，葡萄糖和β-丙氨酸运输系统被战略性地增强，通过工程NADPH再生和ATP循环途径提高辅助因子的可用性。随后，途径工程应用于微调异种酶的表达，从而增强对D-PA生物合成的代谢拉动。为了提高酮托酸羟甲基转移酶（KPHMT）所需的单碳供体的供应，引入了异源的5,10-亚甲基四氢叶酸生物合成模块。最后，通过动态调节异柠檬酸合成酶和泛酸激酶来平衡细胞生长和D-PA的产生。通过综合代谢工程策略，最终菌株DPZ28/P31在两段补料分批发酵中获得了D-PA滴度为98.6 g/L，葡萄糖产量为0.44 g/g。这些发现为D-PA及其相关化合物的工业规模生产提供了有价值的见解。
{"title":"Heterologous integration-assisted metabolic engineering in Escherichia coli for elevated D-pantothenic acid production","authors":"Kuo Zhao , Hailin Gao , Mengnan Han , Bo Zhang , Zhiqiang Liu , Shuping Zou , Yuguo Zheng","doi":"10.1016/j.ymben.2025.08.003","DOIUrl":"10.1016/j.ymben.2025.08.003","url":null,"abstract":"<div><div>D-pantothenic acid (D-PA) is a vital water-soluble vitamin with diverse industrial applications, driving the demand for efficient microbial production. Here, we rationally engineered an <em>Escherichia coli</em> strain to enhance D-PA production through metabolic engineering. First, to enhance carbon utilization efficiency, competing byproduct pathways were deleted and the pentose phosphate pathway was downregulated. Next, the glucose and <em>β</em>-alanine transport systems were strategically enhanced, and cofactor availability was improved through engineering NADPH regeneration and ATP recycling pathways. Subsequently, pathway engineering was applied to fine-tune the expression of heterologous enzymes, thereby enhancing the metabolic pull toward D-PA biosynthesis. To enhance the supply of one-carbon donor required by the rate-limiting enzyme ketopantoate hydroxymethyltransferase (KPHMT), a heterologous 5,10-methylenetetrahydrofolate biosynthesis module was introduced. Finally, dynamic regulation of isocitrate synthase and pantothenate kinase was implemented to balance cell growth and D-PA production. As a result of the integrated metabolic engineering strategies, the final strain DPZ28/P31 achieved a D-PA titer of 98.6 g/L and a yield of 0.44 g/g glucose in a two-stage fed-batch fermentation. These findings provide valuable insights for industrial-scale production of D-PA and related compounds.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 161-173"},"PeriodicalIF":6.8,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144847622","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}
Adenosine triphosphate (ATP) regeneration by substrate-level phosphorylation is a general feature of cancer metabolism, even under normoxic conditions (aerobic glycolysis). However, it is unclear why cancer cells prefer inefficient aerobic glycolysis over the highly efficient process of oxidative phosphorylation for ATP regeneration. To investigate the metabolic principles underlying aerobic glycolysis, we performed 13C-metabolic flux analysis of 12 cultured cancer cell lines and explored the metabolic constraints required to reproduce the results using in silico metabolic simulations. We found that the measured flux distribution can be reproduced by maximizing the ATP consumption in the flux balance analysis considering a limitation of metabolic heat dissipation (enthalpy change). Consistent with the simulation, OXPHOS inhibition induced metabolic redirection to aerobic glycolysis while maintaining the intracellular temperature. Furthermore, the dependency on aerobic glycolysis was partly alleviated upon culturing at low temperatures. Our data suggest that metabolic thermogenesis is an important factor in understanding aerobic glycolysis in cancer cells and that an advantage of aerobic glycolysis is the reduction in metabolic heat generation during ATP regeneration.
{"title":"Metabolic flux and flux balance analyses indicate the relevance of metabolic thermogenesis and aerobic glycolysis in cancer cells","authors":"Nobuyuki Okahashi , Tomoki Shima , Yuya Kondo , Chie Araki , Shuma Tsuji , Akane Sawai , Hikaru Uehara , Susumu Kohno , Hiroshi Shimizu , Chiaki Takahashi , Fumio Matsuda","doi":"10.1016/j.ymben.2025.08.002","DOIUrl":"10.1016/j.ymben.2025.08.002","url":null,"abstract":"<div><div>Adenosine triphosphate (ATP) regeneration by substrate-level phosphorylation is a general feature of cancer metabolism, even under normoxic conditions (aerobic glycolysis). However, it is unclear why cancer cells prefer inefficient aerobic glycolysis over the highly efficient process of oxidative phosphorylation for ATP regeneration. To investigate the metabolic principles underlying aerobic glycolysis, we performed <sup>13</sup>C-metabolic flux analysis of 12 cultured cancer cell lines and explored the metabolic constraints required to reproduce the results using <em>in silico</em> metabolic simulations. We found that the measured flux distribution can be reproduced by maximizing the ATP consumption in the flux balance analysis considering a limitation of metabolic heat dissipation (enthalpy change). Consistent with the simulation, OXPHOS inhibition induced metabolic redirection to aerobic glycolysis while maintaining the intracellular temperature. Furthermore, the dependency on aerobic glycolysis was partly alleviated upon culturing at low temperatures. Our data suggest that metabolic thermogenesis is an important factor in understanding aerobic glycolysis in cancer cells and that an advantage of aerobic glycolysis is the reduction in metabolic heat generation during ATP regeneration.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 185-193"},"PeriodicalIF":6.8,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144812187","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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