Metabolic engineering最新文献

{"title":"Demonstration and technoeconomic analysis of dodecanol production from acetate using metabolically engineered Escherichia coli","authors":"Paul M. Perkovich,&nbsp;Yoel R. Cortés-Peña,&nbsp;Justin J. Baerwald,&nbsp;Thomas H. Graupmann,&nbsp;Theodore A. Chavkin,&nbsp;Shivangi Mishra,&nbsp;William T. Cordell,&nbsp;Victor M. Zavala,&nbsp;Brian F. Pfleger","doi":"10.1016/j.ymben.2025.10.007","DOIUrl":"10.1016/j.ymben.2025.10.007","url":null,"abstract":"<div><div>In a circular bioeconomy, the one-way conversion of petroleum to chemicals and CO₂ is replaced with processes that reduce CO₂ to energy carriers and useful materials that are returned to CO₂ upon combustion. A circular bioeconomy that relies on photosynthesis to generate sugars as the chief energy carrier and precursor to chemical building blocks has yet to overcome many recalcitrant aspects of plant-based photosynthesis, namely, high feedstock costs, arable land scarcity, food competition, and fertilizer overuse. Acetate is a potential sustainable energy carrier because it can be produced from CO₂ either electrocatalytically or by acetogens via the Wood-Ljungdahl pathway. In this work, we conducted a metabolic engineering study of <em>Escherichia coli's</em> ability to convert acetate into dodecanol as a model oleochemical product. We performed techno-economic and life cycle analyses to determine break-even points with alternative fossil fuel-based strategies and identified critical process performance parameters for supporting an industrial acetate-based bioprocess. These analyses showed that oleochemical yield is the primary driver of minimum oleochemical selling price and carbon intensity. Therefore, to increase yield on acetate, we deleted the <em>aceBAK</em> operon, which facilitates funneling of acetate into biomass instead of product. We performed additional strain engineering to increase flux towards dodecanol and increase acetate uptake. Finally, we demonstrated increased yield in controlled bioreactors, improving from 13 % of the maximum theoretical yield to 37 %. Rigorous uncertainty analyses assuming a range of market conditions and future technological performances resulted in 88 % and 37 % of simulated scenarios having lower carbon intensities than fossil fuel-based routes and lower minimum selling prices than the market price.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"93 ","pages":"Pages 243-259"},"PeriodicalIF":6.8,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145383251","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}

{"title":"Microbial production of propionic acid through a novel β-alanine route","authors":"Da-Hee Ahn ,&nbsp;Yoo-Sung Ko ,&nbsp;Cindy Pricilia Surya Prabowo ,&nbsp;Sang Yup Lee","doi":"10.1016/j.ymben.2025.10.011","DOIUrl":"10.1016/j.ymben.2025.10.011","url":null,"abstract":"<div><div>Propionic acid is a key three carbon platform chemical with broad applications in food preservation, pharmaceuticals, and polymer production. Traditional microbial production of propionic acid employing <em>Propionibacterium</em> species is constrained by slow growth, and limited genetic engineering tools, thereby restricting its industrial use. Here, we report the development of a novel biosynthetic pathway for propionic acid production via the β-alanine route. This pathway was engineered into two modules: an upstream β-alanine-forming module and a downstream propionic acid-forming module. The downstream pathway was first constructed and validated in <em>Escherichia coli</em> W3110. Subsequently, co-expression of the upstream module enabled <em>de novo</em> propionic acid production from glucose. Through enzyme screening, precursor flux enhancement, and optimization of phosphoenolpyruvate carboxylase (PPC) flux, the final engineered <em>E. coli</em> strain achieved 14.8 g/L of propionic acid in fed-batch fermentation. Furthermore, we explored <em>Corynebacterium glutamicum</em> ATCC 13032 as an alternative host due to its superior tolerance to propionic acid. The same downstream pathway was introduced into a previously developed β-alanine-overproducing <em>C. glutamicum</em> strain to enable propionic acid production from glucose. Additional engineering strategies, such as enzyme screening, disruption of competing pathways (<em>ack-pta</em>), and elimination of propionic acid catabolic pathways (<em>prpD2B2C2</em>), led to the production of 47.4 g/L of propionic acid in fed-batch fermentation, representing the highest reported titer of heterologous propionic acid production. This work establishes a novel and vitamin B₁₂-independent strategy for bio-based propionic acid production, offering a sustainable alternative to conventional processes.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"93 ","pages":"Pages 219-231"},"PeriodicalIF":6.8,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145396648","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}

{"title":"Corrigendum to “Metabolic and enzyme rewiring enables high-production of vanillin in unconventional yeast” [Metabol. Eng. 93 (2026) 158–167]","authors":"Yan Guo ,&nbsp;Liyang Zhou ,&nbsp;Wanshu Lai ,&nbsp;Zhilan Qian ,&nbsp;Haishuang Yu ,&nbsp;Menghao Cai","doi":"10.1016/j.ymben.2025.10.006","DOIUrl":"10.1016/j.ymben.2025.10.006","url":null,"abstract":"","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"93 ","pages":"Page 168"},"PeriodicalIF":6.8,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145314644","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}

{"title":"Metabolic and enzyme rewiring enables high-production of vanillin in unconventional yeast","authors":"Yan Guo ,&nbsp;Liyang Zhou ,&nbsp;Wanshu Lai ,&nbsp;Zhilan Qian ,&nbsp;Haishuang Yu ,&nbsp;Menghao Cai","doi":"10.1016/j.ymben.2025.10.002","DOIUrl":"10.1016/j.ymben.2025.10.002","url":null,"abstract":"<div><div>Vanillin is an aromatic flavor compound widely used in the food, pharmaceutical, and cosmetic industries. Microbial biosynthesis offers a sustainable alternative to traditional plant extraction and chemical synthesis; however, the susceptibility of vanillin to redox reactions and the weak enzyme activity in cells severely limit the vanillin production capacity by microbial biosynthesis. This study presents the first successful attempt at <em>de novo</em> synthesis of vanillin in the unconventional yeast <em>Komagataella phaffii</em>. The initial titer was quite low (0.5 mg/L), but removal of 14 endogenous oxidoreductases to block vanillin conversion resulted in an 11.1-fold improvement in vanillin production. The combination of pathway rewiring and cofactor (nicotinamide adenine dinucleotide phosphate [NADPH] and <em>S</em>-adenosylmethionine) regeneration redirected the metabolic flux toward vanillin synthesis and achieved a further 19.9-fold improvement in vanillin production. Rational rewiring of the rate-limiting enzyme, caffeic acid <em>O</em>-methyltransferase (NtCOMT), generated a dominant mutant NtCOMT^N312A/H315N from 70 variants, which promoted activity by 49.7 % and prevented intermediate accumulation. These strategies eventually enabled the co-coupling of <em>de novo</em> biosynthesis and caffeic acid conversion, achieving the highest reported production of vanillin (1055.9 mg/L) by <em>K. phaffii</em> fermentation in a bioreactor. These findings highlight the potential of unconventional yeast as a chassis host for aromatic aldehyde synthesis and the construction of a versatile microbial platform for the production of carbonyl compounds.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"93 ","pages":"Pages 158-167"},"PeriodicalIF":6.8,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145261529","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}

{"title":"Multi-layered transcriptional and post-translational fine-tuning of metabolic pathways for overproduction of L-valine","authors":"Xutao Lang ,&nbsp;Wenwen Yu ,&nbsp;Xuewen Zhu ,&nbsp;Xianhao Xu ,&nbsp;Yanfeng Liu ,&nbsp;Jianghua Li ,&nbsp;Guocheng Du ,&nbsp;Jian Chen ,&nbsp;Xueqin Lv ,&nbsp;Long Liu","doi":"10.1016/j.ymben.2025.09.005","DOIUrl":"10.1016/j.ymben.2025.09.005","url":null,"abstract":"<div><div>L-valine is an essential amino acid widely used in the food, pharmaceutical, and animal feed industries. Currently, engineering microbial cell factories to produce L-valine from low-cost feedstocks has emerged as a leading strategy. However, there is still a lack of an L-valine-producing strain that simultaneously exhibits high titer, high yield, and high productivity. The metabolic engineering strategies reported for L-valine biosynthesis primarily rely on conventional unidimensional, transcriptional-level modifications, which limit fine-tuning and do not provide comprehensive, multi-layered regulation. In this study, we constructed an <em>Escherichia coli</em> hyperproducer of L-valine by multiplexed transcriptional and post-translational fine-tuning of metabolic pathways. Initially, the transcriptional repression in L-valine synthetic pathway was eliminated by using promoter engineering strategy. Then, the “push-pull-inhibit” and transport engineering strategies were used to improve L-valine accumulation, achieving a flask fermentation titer of 21.6 g/L and a productivity of 0.45 g/L/h. Subsequently, we rationally designed membraneless organelles (MLOs) to enable spatial regulation of metabolic biosynthesis, which enhanced the targeted recruitment of dihydroxy-acid dehydratase and branched-chain amino acid aminotransferase. This spatial reorganization led to a 95.6 % increase in productivity, reaching 0.88 g/L/h. Finally, the best-performing strain produced 90.6 g/L L-valine in a 3-L bioreactor at 28 h, with a yield of 0.48 g/g glucose and a productivity of 3.24 g/L/h. To the best of our knowledge, this represents the highest L-valine productivity achieved to date. Our strategy provides a practical and effective approach for advancing microbial amino acid biosynthesis by multi-layered transcriptional and post-translational regulation of metabolic pathways.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"93 ","pages":"Pages 80-88"},"PeriodicalIF":6.8,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145093553","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}

{"title":"Development of a thermophilic l-arabinose-inducible system in Acetivibrio thermocellus (Clostridium thermocellum)","authors":"Fenghua Liu ,&nbsp;Chao Chen ,&nbsp;Ya-Jun Liu ,&nbsp;Edward A. Bayer ,&nbsp;Itzhak Mizrahi ,&nbsp;Yingang Feng","doi":"10.1016/j.ymben.2025.09.008","DOIUrl":"10.1016/j.ymben.2025.09.008","url":null,"abstract":"<div><div>Inducible genetic operation systems constitute essential tools in microbial synthetic biology and metabolic engineering. However, inducible systems in non-model microbes, particularly thermophiles, are rarely reported. <em>Acetivibrio thermocellus</em> (previously termed <em>Clostridium thermocellum</em>), a representative strain of thermophilic non-model microbes, currently serves as a promising chassis organism in biorefinery. Although various genetic tools are available for <em>A. thermocellus</em>, superior thermophilic inducible systems are in high demand. In this study, we developed a thermostable <span>l</span>-arabinose-inducible system (ThermoARAi) in <em>A. thermocellus</em> by utilizing the inducible promoter P_abnE and repressor AraR from <em>Geobacillus stearothermophilus</em> T-6. Through systematic promoter engineering and optimization of induction conditions using a thermostable β-glucuronidase as reporter, the system exhibited dynamic range improvement from a 5.4-fold induction to a 175-fold induction with negligible leakage. Furthermore, the ThermoARAi system was appropriate for use in metabolic engineering, as validated by its applications in whole-cell saccharification of cellulosic substrates and degradation of amorphous polyethylene terephthalate films. The ThermoARAi system significantly expands the genetic toolkit for precise gene expression modulation, metabolic engineering, and biotechnological applications in <em>A. thermocellus</em>. Importantly, this approach may also serve as a foundation for developing genetic tools in other Clostridia that play key roles in diverse ecosystems, including the gut.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"93 ","pages":"Pages 89-99"},"PeriodicalIF":6.8,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145103541","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}

{"title":"Metabolic engineering of Acinetobacter baylyi ADP1 for efficient utilization of pentose sugars and production of glutamic acid","authors":"Jin Luo,&nbsp;Elena Efimova,&nbsp;Ville Santala,&nbsp;Suvi Santala","doi":"10.1016/j.ymben.2025.10.001","DOIUrl":"10.1016/j.ymben.2025.10.001","url":null,"abstract":"<div><div>Efficient utilization of pentose sugars is critical for advancing sustainable biomanufacturing using lignocellulose. However, many host strains capable of consuming glucose and lignin-derived monomers are unable to utilize pentose sugars. Here, we engineered <em>Acinetobacter baylyi</em> ADP1 for the utilization of D-xylose and L-arabinose. We first modelled different pentose utilization pathways using flux balance analysis to choose the most optimal pathway. A marker-free approach combining transformation and selection facilitated the integration of the pentose catabolic gene clusters of the selected Weimberg pathway into the <em>A. baylyi</em> genome, generating strains capable of efficiently utilizing both D-xylose and L-arabinose as sole carbon sources without any additional engineering or adaptation. For D-xylose, the cells achieved the highest growth rate (μ = 0.73 h⁻¹) reported to date for non-native hosts engineered for pentose utilization. For L-arabinose, a growth rate of μ = 0.40 h⁻¹ was achieved, which also surpassed the growth rate on a native substrate of <em>A. baylyi</em>, glucose (μ = 0.37 h⁻¹). Importantly, pentose utilization occurred simultaneously with glucose utilization. We then applied metabolic flux analysis using ¹³C labeled xylose to reveal D-xylose metabolism in the engineered strain. To demonstrate the potential for bioproduction, L-glutamate was selected as a target compound. Deletion of <em>sucAB</em> and <em>gabT</em>, and the overexpression of <em>gdhA</em> enabled L-glutamate production. With the engineered strain, a carbon yield of 34 % during co-utilization with succinate and 70 % via whole-cell catalysis using resting cells was achieved. Notably, L-glutamate production directly from industrially relevant hemicellulose hydrolysate was demonstrated. This study establishes a robust platform for pentose utilization and bioproduction in <em>A. baylyi</em> ADP1 and highlights the potential for metabolic optimization.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"93 ","pages":"Pages 145-157"},"PeriodicalIF":6.8,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145258527","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}

{"title":"From soil to biomanufacturing: Systems-driven metabolic pathway rewiring in non-model bacteria for gram-scale antibiotic production","authors":"Tingfeng Cheng ,&nbsp;Suihao Yan ,&nbsp;Min Xu ,&nbsp;Lei Zhao","doi":"10.1016/j.ymben.2025.11.001","DOIUrl":"10.1016/j.ymben.2025.11.001","url":null,"abstract":"<div><div>Microbial natural products (NPs) are a pivotal reservoir for drugs used in human health and agriculture. Andrimid, a polyketide-non-ribosomal peptide hybrid antibiotic inhibiting bacterial acetyl-CoA carboxylase, shows enormous potential in antibiotic drug development to mitigate antimicrobial resistance. However, industrial-scale manufacturing and downstream development of andrimid are largely prohibited due to its milligram level production in microorganisms. Herein, using an integrative multi-omics approach, we improved the yield of andrimid remarkably from milligram to gram level in a non-model environmental soil bacterium, <em>Erwinia persicina</em> BST187, isolated from the rhizosphere of tomato. Systematic reprogramming of the pathways for carbon source uptake, competing metabolites biosynthesis, supply of essential building blocks including phenylalanine, glycine, valine and malonyl-CoA and cofactor biosynthesis using CRIPSR/Cas9 based gene editing tools, coupled with fine-tuning the transcription of the biosynthetic genes of andrimid, resulted in the generation of the optimal producer, G17. Combined with fermentation optimization, andrimid was produced to a highest level of 1099.42 mg/L with a productivity of 15.3 mg/L/h using a 5 L bioreactor, representing a 628-fold increase compared to the parental strain. This study showcases the genome wide engineering of non-model bacteria and generates a plasmid- and inducer-free <em>E. persicina</em> strain for high-level andrimid production, providing a blueprint for systems-driven metabolic engineering of complex bioactive NPs for biomanufacturing.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"93 ","pages":"Pages 271-285"},"PeriodicalIF":6.8,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145442020","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Systematic rewiring of Bacillus subtilis for efficient de novo biosynthesis of the neuroprotectant cytidine-5′-diphosphocholine","authors":"Shaomei Yang ,&nbsp;Xu Feng ,&nbsp;Hao Wei ,&nbsp;Yanshuang Wang ,&nbsp;Shouying Fu ,&nbsp;Xiuzhen Gao ,&nbsp;Qinyuan Ma","doi":"10.1016/j.ymben.2025.09.004","DOIUrl":"10.1016/j.ymben.2025.09.004","url":null,"abstract":"<div><div>Cytidine-5′-diphosphocholine (CDP-choline) is a crucial neuroprotective agent. Current industrial production relies on chemical and enzymatic methods that face inherent sustainability challenges and share a dependence on the costly precursor, cytidine monophosphate (CMP). Here, we report the systems metabolic engineering of <em>Bacillus subtilis</em> for the efficient, <em>de novo</em> biosynthesis of CDP-choline from glucose, completely obviating the need for CMP. A synthetic pathway was first established by introducing heterologous choline kinase and phosphocholine cytidylyltransferase. Subsequently, a multi-module engineering strategy was implemented, focusing on enhancing precursor supply and redirecting carbon metabolism. This involved systematically optimizing choline uptake by overexpressing the transporter OpuD and deleting the transcriptional repressor <em>opcR</em>, fortifying the cytidine triphosphate (CTP) pool by overexpressing feedback-resistant CTP synthase gene <em>pyrG</em>^E156K, and deleting the transcriptional repressor <em>pyrR</em> along with other pyrimidine nucleotide consumption genes, and channeling carbon flux towards the TCA cycle by reducing pyruvate and malate consumption. The final engineered strain achieved a titer of 4.79 ± 0.24 g/L CDP-choline in a 5 L fed-batch bioreactor, with a high specific yield of 149.0 ± 5.8 mg/g DCW. Notably, the process exhibited a highly advantageous intracellular accumulation of 92.7 %, which simplifies downstream purification. This study represents the first successful demonstration of CDP-choline production from simple sugars in a microbial host, establishing a robust and economically competitive platform for its industrial manufacture.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"93 ","pages":"Pages 35-45"},"PeriodicalIF":6.8,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145056657","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}

{"title":"Reconstruction of a resource balance analysis model of Clostridium thermocellum examines the metabolic cost of glycolytic and cellulosome enzymes","authors":"Thomas C. Willis ,&nbsp;Wheaton L. Schroeder ,&nbsp;Daven B. Khana ,&nbsp;Xuejun Qian ,&nbsp;Sanjeev Dahal ,&nbsp;Daniel Amador-Noguez ,&nbsp;Costas D. Maranas","doi":"10.1016/j.ymben.2025.09.001","DOIUrl":"10.1016/j.ymben.2025.09.001","url":null,"abstract":"<div><div><em>Clostridium thermocellum</em> is an increasingly well-studied organism with considerable advantages for consolidated bioprocessing towards ethanol production. Here, a genome-scale resource balance analysis (RBA) model of <em>C. thermocellum</em>, ctRBA, is reconstructed based on a recently published stoichiometric model (<em>i</em>CTH669), global proteomics, and ¹³C MFA datasets to analyze proteome allocation and the burden imposed on metabolism with regard to ethanol yield and titer. Glycolytic and fermentation enzyme concentrations were accurately quantified by the model, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and acetaldehyde-alcohol dehydrogenase (AdhE) having predicted and measured higher concentrations relative to other enzymes in glycolysis and fermentation. The metabolic burden associated with the formation of the cellulosome, the enzyme complex responsible for carbon source degradation and solubilization, was assessed and found to be consequential in constraining ethanol yield and titer, but not biomass formation. Putative enzyme substitution strains were modeled, with each strain replacing a single enzyme in <em>C. thermocellum</em> with a variant that uses more favorable cofactors. Strains substituting GAPDH and phosphofructokinase (PFK) predicted 30 % and 86 % increases in maximum theoretical ethanol yield and titer, respectively, a result unavailable to typical stoichiometric modeling. Model ctRBA acts as a predictive tool for assessing the effect of genetic perturbations on proteome allocation and ethanol yield and titer.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"93 ","pages":"Pages 14-23"},"PeriodicalIF":6.8,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145031857","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}