Optimization of flux distribution in central carbon metabolism is important to improve the microbial productivity. As the number of precursors required for synthesis differs for each target compound, optimal flux distribution also varies. A library of mutant strains with diverse flux distributions can aid in optimal strain screening. Therefore, in this study, we aimed to construct a library of Escherichia coli strains with stepwise changes in flux distribution by introducing mutations into the ribosome-binding sites of key enzyme genes on its chromosome. We focused on the flux ratios at the glucose-6-phosphate and acetyl-CoA branch points to enhance mevalonate production. Mutations were introduced into the ribosome-binding sites of pgi and gltA to vary the flux ratios of the two pathway branches. Furthermore, a combinatorial repression library comprising 16 strains was constructed by varying pgi and gltA expression at four levels, and a plasmid containing mevalonate synthesis genes was introduced into each strain. Batch cultures were performed to obtain strains with mevalonate titers and yields 2.4- and 3.4-fold higher than those of the parent strain. Overall, our combinatorial suppression library of pgi and gltA facilitated the effective identification of mutants with optimal metabolism for mevalonate production.
{"title":"Fine modulation of carbon flow in central carbon metabolism via ribosome-binding site modification in Escherichia coli","authors":"Shogo Sawada, Tatsumi Imada, Hikaru Nagai, Philip Mundt, Fumio Matsuda, Hiroshi Shimizu, Yoshihiro Toya","doi":"10.1016/j.mec.2026.e00270","DOIUrl":"10.1016/j.mec.2026.e00270","url":null,"abstract":"<div><div>Optimization of flux distribution in central carbon metabolism is important to improve the microbial productivity. As the number of precursors required for synthesis differs for each target compound, optimal flux distribution also varies. A library of mutant strains with diverse flux distributions can aid in optimal strain screening. Therefore, in this study, we aimed to construct a library of <em>Escherichia coli</em> strains with stepwise changes in flux distribution by introducing mutations into the ribosome-binding sites of key enzyme genes on its chromosome. We focused on the flux ratios at the glucose-6-phosphate and acetyl-CoA branch points to enhance mevalonate production. Mutations were introduced into the ribosome-binding sites of <em>pgi</em> and <em>gltA</em> to vary the flux ratios of the two pathway branches. Furthermore, a combinatorial repression library comprising 16 strains was constructed by varying <em>pgi</em> and <em>gltA</em> expression at four levels, and a plasmid containing mevalonate synthesis genes was introduced into each strain. Batch cultures were performed to obtain strains with mevalonate titers and yields 2.4- and 3.4-fold higher than those of the parent strain. Overall, our combinatorial suppression library of <em>pgi</em> and <em>gltA</em> facilitated the effective identification of mutants with optimal metabolism for mevalonate production.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"22 ","pages":"Article e00270"},"PeriodicalIF":4.1,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146034884","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-06-01Epub Date: 2025-12-24DOI: 10.1016/j.mec.2025.e00268
Lingyun Li , Xin Chen , Yijie Zhang , Ning Qin , Yu Chen , Xu Ji , Jens Nielsen , Zihe Liu
Phosphofructokinase (Pfk), a key regulatory enzyme in glycolysis, is composed of Pfk1 and Pfk2 subunits in Saccharomyces cerevisiae. However, the distinct roles of these subunits in central carbon metabolism remain unclear. Here, we examined the metabolic consequences of deleting PFK1 or PFK2. The pfk2Δ strain exhibited more severe defects than pfk1Δ. Its maximum specific growth rate was reduced by approximately 54 % in pfk2Δ and by about 15 % in pfk1Δ, both relative to the reference strain. Ethanol production decreased by 36 % and 82 % in pfk1Δ strain and pfk2Δ strain, respectively, relative to the reference strain. Both deletion strains accumulated higher acetate levels compared to the reference strain, increasing by 25.4 % in the pfk1Δ strain and 82 % in the pfk2Δ strain. Flux balance analysis (FBA) revealed a markedly increased carbon flux to the tricarboxylic acid cycle (TCA) in the pfk2Δ strain, with respiration-associated carbon flux elevated 1.5-fold compared to the pfk1Δ strain. Consistently, transcriptomic profiling showed significant upregulation of respiration-related genes in the pfk2Δ strain compared to the reference strain. Notably, deletion of PFK2 enhanced acetyl-CoA-derived product formation, with free fatty acid (FFA) titers increasing from 412 mg L−1 to 517 mg L−1 (a 33.3 % increase). These findings establish PFK2 as a key regulatory node redirecting carbon flux from fermentation toward respiration and biosynthesis, offering new opportunities for metabolic engineering of acetyl-CoA-derived products.
{"title":"Mechanistic and applied study of phosphofructokinases, the “gatekeeper” of the glycolytic pathway on the central carbon metabolism","authors":"Lingyun Li , Xin Chen , Yijie Zhang , Ning Qin , Yu Chen , Xu Ji , Jens Nielsen , Zihe Liu","doi":"10.1016/j.mec.2025.e00268","DOIUrl":"10.1016/j.mec.2025.e00268","url":null,"abstract":"<div><div>Phosphofructokinase (Pfk), a key regulatory enzyme in glycolysis, is composed of Pfk1 and Pfk2 subunits in <em>Saccharomyces cerevisiae</em>. However, the distinct roles of these subunits in central carbon metabolism remain unclear. Here, we examined the metabolic consequences of deleting <em>PFK1</em> or <em>PFK2</em>. The <em>pfk2Δ</em> strain exhibited more severe defects than <em>pfk1Δ</em>. Its maximum specific growth rate was reduced by approximately 54 % in <em>pfk2Δ</em> and by about 15 % in <em>pfk1Δ</em>, both relative to the reference strain. Ethanol production decreased by 36 % and 82 % in <em>pfk1Δ</em> strain and <em>pfk2Δ</em> strain, respectively, relative to the reference strain. Both deletion strains accumulated higher acetate levels compared to the reference strain, increasing by 25.4 % in the <em>pfk1Δ</em> strain and 82 % in the <em>pfk2Δ</em> strain. Flux balance analysis (FBA) revealed a markedly increased carbon flux to the tricarboxylic acid cycle (TCA) in the <em>pfk2Δ</em> strain, with respiration-associated carbon flux elevated 1.5-fold compared to the <em>pfk1Δ</em> strain. Consistently, transcriptomic profiling showed significant upregulation of respiration-related genes in the <em>pfk2Δ</em> strain compared to the reference strain. Notably, deletion of <em>PFK2</em> enhanced acetyl-CoA-derived product formation, with free fatty acid (FFA) titers increasing from 412 mg L<sup>−1</sup> to 517 mg L<sup>−1</sup> (a 33.3 % increase). These findings establish <em>PFK2</em> as a key regulatory node redirecting carbon flux from fermentation toward respiration and biosynthesis, offering new opportunities for metabolic engineering of acetyl-CoA-derived products.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"22 ","pages":"Article e00268"},"PeriodicalIF":4.1,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145897977","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-06-01Epub Date: 2025-12-24DOI: 10.1016/j.mec.2025.e00269
S. Bilal Jilani , Nandhini Ashok , Yannick J. Bomble , Adam M. Guss , Daniel G. Olson
Clostridium thermocellum is a promising host for consolidated bioprocessing due to its ability to directly ferment cellulose into fuels and chemicals. However, natural product formation in this organism is limited. Here, we report engineering C. thermocellum for the production of 2,3-butanediol (23BD), a valuable industrial chemical. We functionally expressed a thermophilic 23BD pathway in this organism resulting in a 23BD titer of 19.7 mM from cellulose, representing a metabolic yield of 24%. We used a cell-free systems biology approach to identify limiting steps in the 23BD pathway, revealing that exogenous 23BD dehydrogenase (BDH) activity was essential for production, while native acetolactate synthase (ALS) and acetolactate decarboxylase (ALDC) activities were present but limiting in the parent strain. This approach also revealed redox balance limitations. We demonstrated that this improved understanding of redox balance limitations could be used to increase 23BD titer in vivo, showing that adding acetate could be used to increase 23BD yield. This work establishes a foundation for developing C. thermocellum into a robust platform for 23BD production directly from cellulose and highlights the utility of cell-free systems for guiding metabolic engineering in non-model organisms.
{"title":"Engineering Clostridium thermocellum for production of 2,3-butanediol from cellulose","authors":"S. Bilal Jilani , Nandhini Ashok , Yannick J. Bomble , Adam M. Guss , Daniel G. Olson","doi":"10.1016/j.mec.2025.e00269","DOIUrl":"10.1016/j.mec.2025.e00269","url":null,"abstract":"<div><div><em>Clostridium thermocellum</em> is a promising host for consolidated bioprocessing due to its ability to directly ferment cellulose into fuels and chemicals. However, natural product formation in this organism is limited. Here, we report engineering <em>C. thermocellum</em> for the production of 2,3-butanediol (23BD), a valuable industrial chemical. We functionally expressed a thermophilic 23BD pathway in this organism resulting in a 23BD titer of 19.7 mM from cellulose, representing a metabolic yield of 24%. We used a cell-free systems biology approach to identify limiting steps in the 23BD pathway, revealing that exogenous 23BD dehydrogenase (BDH) activity was essential for production, while native acetolactate synthase (ALS) and acetolactate decarboxylase (ALDC) activities were present but limiting in the parent strain. This approach also revealed redox balance limitations. We demonstrated that this improved understanding of redox balance limitations could be used to increase 23BD titer in vivo, showing that adding acetate could be used to increase 23BD yield. This work establishes a foundation for developing <em>C. thermocellum</em> into a robust platform for 23BD production directly from cellulose and highlights the utility of cell-free systems for guiding metabolic engineering in non-model organisms.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"22 ","pages":"Article e00269"},"PeriodicalIF":4.1,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145979336","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-06-01Epub Date: 2026-01-17DOI: 10.1016/j.mec.2026.e00271
Yifeng Hu , Tae Seok Moon
Dimethyl terephthalate (DMT) serves as the precursor in the production of polyethylene terephthalate, polytrimethylene terephthalate, and polybutylene terephthalate. The widespread use of DMT in the polymer industry and its ubiquitous existence in end products raise alarms about its potential harm to humans and animals. DMT can enter the environment through the degradation of polymers and their end products, and cause endocrine disruption, oxidative stress, and an elevated risk of cancer. In recent years, DMT has also gained renewed interest in its potential for plastic recycling and upcycling. In this study, we identified two strains of Rhodococcus that possess DMT-degrading capabilities and utilized transcriptomic analysis and gene knockout to elucidate the mechanisms of DMT degradation. R. opacus PD630 and R. jostii RPET were found to convert up to 1 g/L DMT into mono-methyl terephthalate (MMT). A putative DMTase (RS34275) was identified for this conversion. R. jostii RPET also demonstrates the ability to convert DMT into MMT and to utilize MMT for its cellular growth via the terephthalate pathway. A putative MMTase (RS21885) as the sole enzyme was identified for the conversion of MMT into terephthalate in the RPET strain. In addition, we successfully produced lycopene and lipids from an engineered RPET strain using DMT as a substrate. Our findings will facilitate future DMT bioremediation and bio-upcycling of DMT-associated plastics, enabling the production of value-added products.
{"title":"Elucidating biodegradation of dimethyl terephthalate by two Rhodococcus strains for its valorization applications","authors":"Yifeng Hu , Tae Seok Moon","doi":"10.1016/j.mec.2026.e00271","DOIUrl":"10.1016/j.mec.2026.e00271","url":null,"abstract":"<div><div>Dimethyl terephthalate (DMT) serves as the precursor in the production of polyethylene terephthalate, polytrimethylene terephthalate, and polybutylene terephthalate. The widespread use of DMT in the polymer industry and its ubiquitous existence in end products raise alarms about its potential harm to humans and animals. DMT can enter the environment through the degradation of polymers and their end products, and cause endocrine disruption, oxidative stress, and an elevated risk of cancer. In recent years, DMT has also gained renewed interest in its potential for plastic recycling and upcycling. In this study, we identified two strains of <em>Rhodococcus</em> that possess DMT-degrading capabilities and utilized transcriptomic analysis and gene knockout to elucidate the mechanisms of DMT degradation. <em>R. opacus</em> PD630 and <em>R. jostii</em> RPET were found to convert up to 1 g/L DMT into mono-methyl terephthalate (MMT). A putative DMTase (RS34275) was identified for this conversion. <em>R. jostii</em> RPET also demonstrates the ability to convert DMT into MMT and to utilize MMT for its cellular growth via the terephthalate pathway. A putative MMTase (RS21885) as the sole enzyme was identified for the conversion of MMT into terephthalate in the RPET strain. In addition, we successfully produced lycopene and lipids from an engineered RPET strain using DMT as a substrate. Our findings will facilitate future DMT bioremediation and bio-upcycling of DMT-associated plastics, enabling the production of value-added products.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"22 ","pages":"Article e00271"},"PeriodicalIF":4.1,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146034885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01Epub Date: 2025-07-08DOI: 10.1016/j.mec.2025.e00266
Leena Patel , Bryan P. Marzullo , Jonathan Barlow , Himani Rana , Amar J. Azad , Patricia Thomas , Daniel A. Tennant , Katja Gehmlich
Many cardiac pathologies are characterised by increased stiffness of the myocardium, due to excess deposition of extracellular matrix (ECM) proteins and structural remodelling, impacting the behaviour of cardiomyocytes (CMs). Metabolism of CMs shifts in cardiac pathologies, with the healthy heart primarily utilising fatty acids as its source of energy production, whilst the diseased heart switches to utilise glucose. The shift in metabolic source with stiffness of the myocardium has not been investigated.
To investigate the effect of ECM stiffnesses on iPSC-CM metabolism, iPSC-CMs were cultured on polydimethylsiloxane (PDMS) substrates of healthy and fibrotic stiffness (20 kPa and 130 kPa respectively) and plastic. Cellular metabolism of iPSC-CMs was assessed through isotope-labelled mass spectrometry with central carbon tracing as well as real-time cellular bioenergetics using extracellular flux analysis. Key metabolic genes were investigated at transcript and protein level, with proteomics analysis conducted to identify protein profiles on substrate stiffnesses.
Mass spectrometry data revealed greater utilisation of glucose in iPSC-CMs cultured on plastic compared to softer PDMS substrates, indicating greater glycolytic activity. Extracellular flux analysis demonstrated greater lactic acid efflux from iPSC-CMs cultured on plastic substrates, reflective of increased glycolytic flux and a shift towards aerobic glycolysis as the primary source of ATP synthesis. This study revealed culture of iPSC-CMs on traditional cell culture plastics or glass coverslips displaying pathological metabolism, highlighting the use of physiological substrates for metabolic investigation.
{"title":"Substrate stiffness-dependent metabolic reprogramming of iPSC-derived cardiomyocytes on physiological PDMS polymers","authors":"Leena Patel , Bryan P. Marzullo , Jonathan Barlow , Himani Rana , Amar J. Azad , Patricia Thomas , Daniel A. Tennant , Katja Gehmlich","doi":"10.1016/j.mec.2025.e00266","DOIUrl":"10.1016/j.mec.2025.e00266","url":null,"abstract":"<div><div>Many cardiac pathologies are characterised by increased stiffness of the myocardium, due to excess deposition of extracellular matrix (ECM) proteins and structural remodelling, impacting the behaviour of cardiomyocytes (CMs). Metabolism of CMs shifts in cardiac pathologies, with the healthy heart primarily utilising fatty acids as its source of energy production, whilst the diseased heart switches to utilise glucose. The shift in metabolic source with stiffness of the myocardium has not been investigated.</div><div>To investigate the effect of ECM stiffnesses on iPSC-CM metabolism, iPSC-CMs were cultured on polydimethylsiloxane (PDMS) substrates of healthy and fibrotic stiffness (20 kPa and 130 kPa respectively) and plastic. Cellular metabolism of iPSC-CMs was assessed through isotope-labelled mass spectrometry with central carbon tracing as well as real-time cellular bioenergetics using extracellular flux analysis. Key metabolic genes were investigated at transcript and protein level, with proteomics analysis conducted to identify protein profiles on substrate stiffnesses.</div><div>Mass spectrometry data revealed greater utilisation of glucose in iPSC-CMs cultured on plastic compared to softer PDMS substrates, indicating greater glycolytic activity. Extracellular flux analysis demonstrated greater lactic acid efflux from iPSC-CMs cultured on plastic substrates, reflective of increased glycolytic flux and a shift towards aerobic glycolysis as the primary source of ATP synthesis. This study revealed culture of iPSC-CMs on traditional cell culture plastics or glass coverslips displaying pathological metabolism, highlighting the use of physiological substrates for metabolic investigation.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"21 ","pages":"Article e00266"},"PeriodicalIF":3.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144631369","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Heterologous compound production is a complex trait since the native metabolic fluxes supplying the precursors, redox power, and energy are under multilevel cellular regulation. Improving complex traits using targeted engineering needs combinatorially charting the complex genetic underpinnings. While this is laborious, adaptive laboratory evolution (ALE) has been used to improve many traits of microbial strains that are of application relevance such as tolerance of harsh conditions and nutrient utilization. However, in contrast to such traits, heterologous production can seldom be intuitively coupled with cellular fitness.
Here, a novel method EvolveXGA was developed for genome-scale metabolic model guided design of strategies combining chemical environments and genetic engineering of the metabolic network to allow ALE of desired traits. Adaptive evolution of traits occurs when the co-variance between the traits and fitness involves a genetic dependency like a flux coupling would indicate. Thus, combinations of chemical environments and metabolic network structures were searched using a genetic algorithm to identify those that render desired traits (i.e., sets of metabolic fluxes) flux-coupled with fitness. The search was performed for the production of 29 heterologous compounds in yeast Saccharomyces cerevisiae. Strategies for coupling the production routes of 13 compounds with fitness were found with four metabolic reaction knock outs and three components in the chemical environment. In addition, strategies for fitness-coupling native fluxes involved in the production was found for the remaining compounds. In addition, a model-guided strategy was implemented for fitness-coupling of heterologous glycolic acid (GA) synthesis in S. cerevisiae via oxaloacetase, oxalyl-CoA synthetase, and oxalyl-CoA reductase (i.e., oxalate pathway). ALE was performed and evolved populations and isolated clones were characterized using whole-genome sequencing and quantitative metabolite analysis. Three out of six isolates had better GA yield from glucose than a non-optimized control strain expressing the oxalate pathway and glyoxylate reductase.
EvolveXGA generalizes metabolic model-guided design of strategies to couple production routes with cell fitness. The strategies bring optimizing heterologous production in engineered microbial cells in the realm of ALE. Slow and expensive strain optimization is a major hinder of novel processes using engineered microbial cells reaching industrial realization. Thus, EvolveXGA contributes to biotechnological solutions for the brighter future.
{"title":"Model-guided chemical environment and metabolic network design to couple pathways with cell fitness","authors":"Natalia Kakko von Koch , Tuula Tenkanen , Sandra Castillo , Virve Vidgren , Tino Koponen , Kristoffer Krogerus , Merja Penttilä , Paula Jouhten","doi":"10.1016/j.mec.2025.e00267","DOIUrl":"10.1016/j.mec.2025.e00267","url":null,"abstract":"<div><div>Heterologous compound production is a complex trait since the native metabolic fluxes supplying the precursors, redox power, and energy are under multilevel cellular regulation. Improving complex traits using targeted engineering needs combinatorially charting the complex genetic underpinnings. While this is laborious, adaptive laboratory evolution (ALE) has been used to improve many traits of microbial strains that are of application relevance such as tolerance of harsh conditions and nutrient utilization. However, in contrast to such traits, heterologous production can seldom be intuitively coupled with cellular fitness.</div><div>Here, a novel method EvolveXGA was developed for genome-scale metabolic model guided design of strategies combining chemical environments and genetic engineering of the metabolic network to allow ALE of desired traits. Adaptive evolution of traits occurs when the co-variance between the traits and fitness involves a genetic dependency like a flux coupling would indicate. Thus, combinations of chemical environments and metabolic network structures were searched using a genetic algorithm to identify those that render desired traits (i.e., sets of metabolic fluxes) flux-coupled with fitness. The search was performed for the production of 29 heterologous compounds in yeast <em>Saccharomyces cerevisiae</em>. Strategies for coupling the production routes of 13 compounds with fitness were found with four metabolic reaction knock outs and three components in the chemical environment. In addition, strategies for fitness-coupling native fluxes involved in the production was found for the remaining compounds. In addition, a model-guided strategy was implemented for fitness-coupling of heterologous glycolic acid (GA) synthesis in <em>S</em>. <em>cerevisiae</em> via oxaloacetase, oxalyl-CoA synthetase, and oxalyl-CoA reductase (i.e., oxalate pathway). ALE was performed and evolved populations and isolated clones were characterized using whole-genome sequencing and quantitative metabolite analysis. Three out of six isolates had better GA yield from glucose than a non-optimized control strain expressing the oxalate pathway and glyoxylate reductase.</div><div>EvolveXGA generalizes metabolic model-guided design of strategies to couple production routes with cell fitness. The strategies bring optimizing heterologous production in engineered microbial cells in the realm of ALE. Slow and expensive strain optimization is a major hinder of novel processes using engineered microbial cells reaching industrial realization. Thus, EvolveXGA contributes to biotechnological solutions for the brighter future.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"21 ","pages":"Article e00267"},"PeriodicalIF":4.1,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145620495","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methanol has attracted attention as an alternative carbon source to petroleum. Komagataella phaffii, a methanol-assimilating yeast, is a useful host for the chemical production from methanol. A previous study successfully constructed a metabolically engineered K. phaffii GS115/S8/Z3 strain capable of producing D-lactic acid from methanol. In this study, we aimed to develop a strain with improved D-lactic acid production by applying ultra-violet mutagenesis to the D-lactic acid-producing strain, GS115/S8/Z3. The resulting mutant strain DLac_Mut2_221 produced 5.38 g/L of D-lactic acid from methanol, a 1.52-fold increase compared to the parent strain GS115/S8/Z3. The transcriptome analysis of the mutant DLac_Mut2_221 identified 158 differentially expressed genes, providing insights into key mechanisms contributing to enhanced D-lactic acid production. Metabolic engineering strategies for K. phaffii based on the knowledge gained from this study will contribute to improving the productivity of various useful chemicals from methanol.
{"title":"Improvement of D-lactic acid production from methanol by metabolically engineered Komagataella phaffii via ultra-violet mutagenesis","authors":"Yoshifumi Inoue, Kaito Nakamura, Ryosuke Yamada, Takuya Matsumoto, Hiroyasu Ogino","doi":"10.1016/j.mec.2025.e00262","DOIUrl":"10.1016/j.mec.2025.e00262","url":null,"abstract":"<div><div>Methanol has attracted attention as an alternative carbon source to petroleum. <em>Komagataella phaffii</em>, a methanol-assimilating yeast, is a useful host for the chemical production from methanol. A previous study successfully constructed a metabolically engineered <em>K. phaffii</em> GS115/S8/Z3 strain capable of producing D-lactic acid from methanol. In this study, we aimed to develop a strain with improved D-lactic acid production by applying ultra-violet mutagenesis to the D-lactic acid-producing strain, GS115/S8/Z3. The resulting mutant strain DLac_Mut2_221 produced 5.38 g/L of D-lactic acid from methanol, a 1.52-fold increase compared to the parent strain GS115/S8/Z3. The transcriptome analysis of the mutant DLac_Mut2_221 identified 158 differentially expressed genes, providing insights into key mechanisms contributing to enhanced D-lactic acid production. Metabolic engineering strategies for <em>K. phaffii</em> based on the knowledge gained from this study will contribute to improving the productivity of various useful chemicals from methanol.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"20 ","pages":"Article e00262"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144089620","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-01Epub Date: 2025-02-12DOI: 10.1016/j.mec.2025.e00257
Tobias B. Alter , Pascal A. Pieters , Colton J. Lloyd , Adam M. Feist , Emre Özdemir , Bernhard O. Palsson , Daniel C. Zielinski
Whole-cell biocatalysis facilitates the production of a wide range of industrially and pharmaceutically relevant molecules from sustainable feedstocks such as plastic wastes, carbon dioxide, lignocellulose, or plant-based sugar sources. The identification and use of efficient enzymes in the applied biocatalyst is key to establishing economically feasible production processes. The generation and selection of favorable enzyme variants in adaptive laboratory evolution experiments using growth as a selection criterion is facilitated by tightly coupling enzyme catalytic activity to microbial metabolic activity. Here, we present a computational workflow to design strains that have a severe, growth-limiting metabolic chokepoint through a shared class of enzymes. The resulting chassis cell, termed enzyme selection system (ESS), is a platform for growth-coupling any enzyme from the respective enzyme class, thus offering cross-pathway application for enzyme engineering purposes. By applying the constraint-based modeling workflow, a publicly accessible database of 25,505 potential and experimentally tractable ESS designs was built for Escherichia coli and a broad range of production pathways with biotechnological relevance. A model-based analysis of the generated design database reveals a general design principle that the target enzyme activity is linked to overall microbial metabolic activity, not just the synthesis of one biomass precursor. It can be observed that the stronger the predicted coupling between target enzyme and metabolic activity, the lower the maximum growth rate and therefore the viability of an ESS. Consequently, growth-coupling strategies with only suboptimal coupling strengths, as are included in the ESS design database, may be of interest for practical applications of ESSs in order to circumvent overly restrictive growth defects. In summary, the computed design database, which is accessible via https://biosustain.github.io/ESS-Designs/, and its analysis provide a foundation for the generation of valuable in vivo ESSs for enzyme optimization purposes and a range of biotechnological applications in general.
{"title":"Metabolic growth-coupling strategies for in vivo enzyme selection systems","authors":"Tobias B. Alter , Pascal A. Pieters , Colton J. Lloyd , Adam M. Feist , Emre Özdemir , Bernhard O. Palsson , Daniel C. Zielinski","doi":"10.1016/j.mec.2025.e00257","DOIUrl":"10.1016/j.mec.2025.e00257","url":null,"abstract":"<div><div>Whole-cell biocatalysis facilitates the production of a wide range of industrially and pharmaceutically relevant molecules from sustainable feedstocks such as plastic wastes, carbon dioxide, lignocellulose, or plant-based sugar sources. The identification and use of efficient enzymes in the applied biocatalyst is key to establishing economically feasible production processes. The generation and selection of favorable enzyme variants in adaptive laboratory evolution experiments using growth as a selection criterion is facilitated by tightly coupling enzyme catalytic activity to microbial metabolic activity. Here, we present a computational workflow to design strains that have a severe, growth-limiting metabolic chokepoint through a shared class of enzymes. The resulting chassis cell, termed enzyme selection system (ESS), is a platform for growth-coupling any enzyme from the respective enzyme class, thus offering cross-pathway application for enzyme engineering purposes. By applying the constraint-based modeling workflow, a publicly accessible database of 25,505 potential and experimentally tractable ESS designs was built for <em>Escherichia coli</em> and a broad range of production pathways with biotechnological relevance. A model-based analysis of the generated design database reveals a general design principle that the target enzyme activity is linked to overall microbial metabolic activity, not just the synthesis of one biomass precursor. It can be observed that the stronger the predicted coupling between target enzyme and metabolic activity, the lower the maximum growth rate and therefore the viability of an ESS. Consequently, growth-coupling strategies with only suboptimal coupling strengths, as are included in the ESS design database, may be of interest for practical applications of ESSs in order to circumvent overly restrictive growth defects. In summary, the computed design database, which is accessible via <span><span>https://biosustain.github.io/ESS-Designs/</span><svg><path></path></svg></span>, and its analysis provide a foundation for the generation of valuable <em>in vivo</em> ESSs for enzyme optimization purposes and a range of biotechnological applications in general.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"20 ","pages":"Article e00257"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143464329","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-01Epub Date: 2024-12-12DOI: 10.1016/j.mec.2024.e00253
Michael Weldon, Sanniv Ganguly, Christian Euler
The growing plastics end-of-life crisis threatens ecosystems and human health globally. Microbial plastic degradation and upcycling have emerged as potential solutions to this complex challenge, but their industrial feasibility and limitations thereon have not been fully characterized. In this perspective paper, we review literature describing both plastic degradation and transformation of plastic monomers into value-added products by microbes. We aim to understand the current feasibility of combining these into a single, closed-loop process. Our analysis shows that microbial plastic degradation is currently the rate-limiting step to “closing the loop”, with reported rates that are orders of magnitude lower than those of pathways to upcycle plastic degradation products. We further find that neither degradation nor upcycling have been demonstrated at rates sufficiently high to justify industrialization at present. As a potential way to address these limitations, we suggest more investigation into mixotrophic approaches, showing that those which leverage the unique properties of plastic degradation products such as ethylene glycol might improve rates sufficiently to motivate industrial process development.
{"title":"Co-consumption for plastics upcycling: A perspective","authors":"Michael Weldon, Sanniv Ganguly, Christian Euler","doi":"10.1016/j.mec.2024.e00253","DOIUrl":"10.1016/j.mec.2024.e00253","url":null,"abstract":"<div><div>The growing plastics end-of-life crisis threatens ecosystems and human health globally. Microbial plastic degradation and upcycling have emerged as potential solutions to this complex challenge, but their industrial feasibility and limitations thereon have not been fully characterized. In this perspective paper, we review literature describing both plastic degradation and transformation of plastic monomers into value-added products by microbes. We aim to understand the current feasibility of combining these into a single, closed-loop process. Our analysis shows that microbial plastic degradation is currently the rate-limiting step to “closing the loop”, with reported rates that are orders of magnitude lower than those of pathways to upcycle plastic degradation products. We further find that neither degradation nor upcycling have been demonstrated at rates sufficiently high to justify industrialization at present. As a potential way to address these limitations, we suggest more investigation into mixotrophic approaches, showing that those which leverage the unique properties of plastic degradation products such as ethylene glycol might improve rates sufficiently to motivate industrial process development.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"20 ","pages":"Article e00253"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717657/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142971620","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-01Epub Date: 2025-06-04DOI: 10.1016/j.mec.2025.e00264
Jaqueline Aline Gerhardt , Marcelo Ventura Rubio , Cesar Rafael Fanchini Terrasan , Natalia Sayuri Wassano , Aryadne Rodrigues , Fernanda Lopes de Figueiredo , Everton Paschoal Antoniel , Fabiano Jares Contesini , Artur Hermano Sampaio Dias , Uffe Hasbro Mortensen , Munir Salomão Skaf , André Damasio
Filamentous fungi are cell factories traditionally used for enzyme production in various industrial sectors, including food and beverages, biopolymers, biofuels, and animal feed. Despite significant progress in optimizing enzyme production, challenges related to cost-effectiveness persist. Genes involved in the fungal secretory pathway have been modified to address productivity barriers, including post-translational modifications such as N-glycosylation of proteins. N-glycosylation can significantly affect protein stability, production yield, and functionality. This study investigated the isolated and combined deletion of genes involved in N-glycan assembly on protein production in Aspergillus nidulans. To test this hypothesis, we utilized CRISPR/Cas9 technology to knock out 14 genes related to N-glycan assembly (AN5888, AN11802, AN5346, AN6874, AN5725, AN7425, algC, algI, algL, algF and AN5748) and protein quality control (clxA, gtbA, and AN4623), resulting in eight viable mutants. Next, we integrated a GH3 beta-xylosidase encoding gene (bxlb; AN8401) into these mutants and the reference strain for constitutive expression and secretion. Single deletion of most target genes did not affect protein secretion and fungal growth. Interestingly, the specific activity of BxlB in the secretome of single mutants was influenced by culture time, while BxlB secretion remained unaffected. Conversely, the combined deletion of algC and algI increased BxlB secretion, whereas the kinetic parameters remained unaffected relative to the enzyme produced by the reference strain. Multiple deletions of algC, algF, and algI did not affect BxlB secretion but reduced catalytic efficiency. After analyzing the secretomes of double and triple mutant strains produced on plant biomass using mass spectrometry, we observed that these knockouts reduced the overall secretion of a specific set of carbohydrate-active enzymes (CAZymes). Other clusters were upregulated in the mutant strains, indicating severe secretome alterations. Overall, the combined deletion of algC and algI may be a promising strategy for increasing the secretion of recombinant proteins in A. nidulans while also enhancing downstream processes, such as protein purification, by reducing the protein background in the secretome of the mutant strain.
{"title":"Improving recombinant protein secretion in Aspergillus nidulans by targeting the N-glycosylation machinery","authors":"Jaqueline Aline Gerhardt , Marcelo Ventura Rubio , Cesar Rafael Fanchini Terrasan , Natalia Sayuri Wassano , Aryadne Rodrigues , Fernanda Lopes de Figueiredo , Everton Paschoal Antoniel , Fabiano Jares Contesini , Artur Hermano Sampaio Dias , Uffe Hasbro Mortensen , Munir Salomão Skaf , André Damasio","doi":"10.1016/j.mec.2025.e00264","DOIUrl":"10.1016/j.mec.2025.e00264","url":null,"abstract":"<div><div>Filamentous fungi are cell factories traditionally used for enzyme production in various industrial sectors, including food and beverages, biopolymers, biofuels, and animal feed. Despite significant progress in optimizing enzyme production, challenges related to cost-effectiveness persist. Genes involved in the fungal secretory pathway have been modified to address productivity barriers, including post-translational modifications such as N-glycosylation of proteins. N-glycosylation can significantly affect protein stability, production yield, and functionality. This study investigated the isolated and combined deletion of genes involved in N-glycan assembly on protein production in <em>Aspergillus nidulans</em>. To test this hypothesis, we utilized CRISPR/Cas9 technology to knock out 14 genes related to N-glycan assembly (AN5888, AN11802, AN5346, AN6874, AN5725, AN7425, <em>algC</em>, <em>algI</em>, <em>algL</em>, <em>algF</em> and AN5748) and protein quality control (<em>clxA</em>, <em>gtbA</em>, and AN4623), resulting in eight viable mutants. Next, we integrated a GH3 beta-xylosidase encoding gene (<em>bxlb;</em> AN8401) into these mutants and the reference strain for constitutive expression and secretion. Single deletion of most target genes did not affect protein secretion and fungal growth. Interestingly, the specific activity of BxlB in the secretome of single mutants was influenced by culture time, while BxlB secretion remained unaffected. Conversely, the combined deletion of <em>algC</em> and <em>algI</em> increased BxlB secretion, whereas the kinetic parameters remained unaffected relative to the enzyme produced by the reference strain. Multiple deletions of <em>algC</em>, <em>algF</em>, and <em>algI</em> did not affect BxlB secretion but reduced catalytic efficiency. After analyzing the secretomes of double and triple mutant strains produced on plant biomass using mass spectrometry, we observed that these knockouts reduced the overall secretion of a specific set of carbohydrate-active enzymes (CAZymes). Other clusters were upregulated in the mutant strains, indicating severe secretome alterations. Overall, the combined deletion of <em>algC</em> and <em>algI</em> may be a promising strategy for increasing the secretion of recombinant proteins in <em>A. nidulans</em> while also enhancing downstream processes, such as protein purification, by reducing the protein background in the secretome of the mutant strain.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"20 ","pages":"Article e00264"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144254586","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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