Metabolic engineering最新文献

{"title":"Improved biosynthesis of C4 derivatives by engineered thiolase","authors":"Zeyao Chen ,&nbsp;Changxi Zhang ,&nbsp;Bing Xu ,&nbsp;Zhiping Ma ,&nbsp;Jing Zhao ,&nbsp;Mengzhen Nie ,&nbsp;Yaping Mao ,&nbsp;Kechun Zhang","doi":"10.1016/j.ymben.2025.05.001","DOIUrl":"10.1016/j.ymben.2025.05.001","url":null,"abstract":"<div><div>Ethylene glycol (EG), a major product of the enzymatic degradation of polyethylene terephthalate (PET), provides a promising feedstock for sustainable biomanufacturing. Herein, we developed a novel metabolic pathway using <em>Escherichia coli</em>(<em>E. coli</em>) as a host for the biosynthesis of four-carbon compounds such as 1,4-butanediol (1,4-BDO), 1,2,4-butanetriol (1,2,4-BTO), and succinate, from two-carbon substrates such as glycolate and EG. This represents an efficient strategy of using C2 precursors for these high-value chemicals. Through directed evolution of the β-ketoacyl thiolase B of <em>Cupriavidus necator</em> (CnBktB), one of the rate-limiting enzymes in the pathway, via an established growth-coupled screening platform, we identified the L89S mutant, which exhibits significantly enhanced catalytic efficiency in assimilating glycolyl-CoA and acetyl-CoA. Using glycolate and glucose as substrates, the route achieves production titers of &gt;200 mg/L for 1,4-BDO, 266 mg/L for 1,2,4-BTO, and 9.22 g/L for succinate. Furthermore, integrating an upstream module for EG conversion to glycolate allows direct utilization of PET-derived EG, yielding 11.4 g/L succinate with 93 % conversion efficiency from EG. This work bridges the fields of synthetic biology and plastic waste recycling, demonstrating a sustainable and scalable route for converting PET-derived EG into valuable four-carbon compounds. The novel biosynthetic pathways developed in this study offer a foundation for advancing circular bioeconomy strategies and reducing the environmental impact of plastic waste.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 192-203"},"PeriodicalIF":6.8,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143911541","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":"Harnessing CO2 fixation and reducing power recycling for enhanced polyhydroxyalkanoates industrial bioproduction","authors":"Jing Feng ,&nbsp;Xueshan Li ,&nbsp;Xin Teng ,&nbsp;Dingding Fan ,&nbsp;Jin Yin ,&nbsp;Yanci Qiu ,&nbsp;Ziling Yi ,&nbsp;Li Chen ,&nbsp;Haoqian M. Zhang ,&nbsp;Chitong Rao","doi":"10.1016/j.ymben.2025.04.009","DOIUrl":"10.1016/j.ymben.2025.04.009","url":null,"abstract":"<div><div>Palm oil is an attractive feedstock for bioproduction due to its high carbon content and low cost. However, its metabolism generates excess reducing power, leading to redox imbalances and reduced metabolic efficiency in industrial fermentations. Through a model-driven approach integrating flux balance analysis, we activated the Calvin-Benson-Bassham (CBB) cycle in <em>Cupriavidus necator</em> to recycle surplus reducing power and restore metabolic balance in polyhydroxyalkanoate (PHA) bioproduction. Computational simulations predicted that constitutive activation of the CBB cycle enhanced CO₂ fixation and accelerated biomass generation when utilizing palm oil as the carbon source. Model-guided optimization revealed that precise tuning of CBB activation strength was critical, as both insufficient and excessive activation led to metabolic inefficiencies. At the 2-liter bench-scale, CBB activation tuning resulted in biomass changes ranging from −18 % to 21 % and PHA yield changes ranging from −36 % to 25 %. Mechanistic studies demonstrated that CBB activation improves metabolic efficiency through reducing power recycling and carbon redistribution. In the 15 m³ industrial-scale fermentations, the engineered strain achieved a 20 % higher PHA yield. These results demonstrate that recycling surplus reducing power is a scalable and robust strategy for enhanced bioproduction efficiency.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 204-216"},"PeriodicalIF":6.8,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143917466","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":"Ribo-seq guided design of enhanced protein secretion in Komagataella phaffii","authors":"Aida Tafrishi ,&nbsp;Troy Alva ,&nbsp;Justin Chartron ,&nbsp;Ian Wheeldon","doi":"10.1016/j.ymben.2025.04.007","DOIUrl":"10.1016/j.ymben.2025.04.007","url":null,"abstract":"<div><div>The production of recombinant proteins requires the precise coordination of various biological processes, including protein synthesis, folding, trafficking, and secretion. The overproduction of a heterologous protein can impose various bottlenecks on these networks. Identifying and alleviating these bottlenecks can guide strain engineering efforts to enhance protein production. The methylotrophic yeast <em>Komagataella phaffii</em> is used for its high capacity to produce recombinant proteins. Here, we use ribosome profiling to identify bottlenecks in protein secretion during heterologous expression of human serum albumin (HSA). Validation of this analysis showed that the knockout of non-essential genes whose gene products target the ER, through co- and post-translational mechanisms, and have high ribosome utilization can increase production of a heterologous protein, HSA. A triple knockout in co-translationally translocated carbohydrate and acetate transporter Gal2p, cell wall maintenance protein Ydr134cp, and the post-translationally translocated cell wall protein <em>Aoa65896.1</em> increased HSA production by 35 %. This data-driven strain engineering approach uses cell-level information to identify gene targets for phenotype improvement. This specific case identifies hits and creates strains with improved HSA production, with Ribo-seq and bioinformatic analysis to identify non-essential ER targeted proteins that are high ribosome utilizers.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 228-241"},"PeriodicalIF":6.8,"publicationDate":"2025-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143923300","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 design of a platform Pseudomonas strain producing various phenazine derivatives","authors":"Sheng-Jie Yue ,&nbsp;Ying Liu ,&nbsp;Wei Wang ,&nbsp;Hong-Bo Hu ,&nbsp;Xue-Hong Zhang","doi":"10.1016/j.ymben.2025.04.010","DOIUrl":"10.1016/j.ymben.2025.04.010","url":null,"abstract":"<div><div>Phenazine derivatives, a class of nitrogen-containing heterocyclic compounds, exhibit broad-spectrum antifungal, anticancer, and antimalarial activities. <em>Pseudomonas</em> and <em>Streptomyces</em> are the primary microbial strains responsible for the synthesis of phenazine derivatives. In general, <em>Pseudomonas</em> strains use phenazine-1-carboxylic acid (PCA) as a precursor for enzymatic modification, while <em>Streptomyces</em> strains employ phenazine-1,6-dicarboxylic acid (PDC) as the precursor. <em>Pseudomonas</em> is considered an ideal platform for the efficient biosynthesis of various phenazine derivatives due to its rapid growth rate, ease of genetic manipulation, and well-established fermentation systems. However, the synthesis of phenazine derivatives in <em>Pseudomonas</em> largely relies on previously reported natural biosynthetic pathways from other microbial strains. The biosynthesis of phenazine derivatives through unknown pathways often presents significant challenges for researchers. The concept of combinatorial biosynthesis offers a promising solution to overcome these difficulties. In this study, we designed and constructed a platform <em>Pseudomonas</em> strain producing 15 phenazine derivatives by exchanging and combining the modifying enzymes of PCA and PDC, besides 16 constructed modification pathways. Among these, three derivatives feature novel chemical structures, while 13 represent previously unreported biosynthetic pathways. With the discovery of new phenazine modifying enzymes, they can be quickly incorporated into our platform, enabling the rapid synthesis of a wide variety of phenazine derivatives. This work demonstrates the potential of designing non-natural metabolic pathways to enable the production of diverse phenazine derivatives, thereby enhancing bacterial capacity for the synthesis of high-value phenazine compounds. This combinatorial biosynthetic approach provides a potential alternative for exploring unknown biosynthetic routes and for the development of unexplored natural biosynthetic pathways for phenazine derivatives.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 217-227"},"PeriodicalIF":6.8,"publicationDate":"2025-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143903092","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 Escherichia coli for 4-nitrophenylalanine production via the 4-aminophenylalanine synthetic pathway","authors":"Ayana Mori ,&nbsp;Yuuki Hirata ,&nbsp;Mayumi Kishida ,&nbsp;Daisuke Nonaka ,&nbsp;Akihiko Kondo ,&nbsp;Yutaro Mori ,&nbsp;Shuhei Noda ,&nbsp;Tsutomu Tanaka","doi":"10.1016/j.ymben.2025.04.006","DOIUrl":"10.1016/j.ymben.2025.04.006","url":null,"abstract":"<div><div>The non-natural amino acid 4-nitrophenylalanine is a crucial pharmaceutical ingredient and has extensive utility in protein engineering. Here, we demonstrated the production of 4-nitrophenylalanine by <em>Escherichia coli</em> with AurF, 4-aminobenzoate <em>N</em>-oxygenase from <em>Streptomyces thioluteus</em>. Firstly, eight distinct gene combinations, encompassing four variants of <em>papA</em> and two of <em>papBC</em>, were evaluated to optimize the production of 4-aminophenylalanine, a precursor of 4-nitrophenylalanine. The strain co-expressing both <em>pabAB</em> from <em>E. coli</em> and <em>papBC</em> from <em>Streptomyces venezuelae</em> attained the highest 4-aminophenylalanine production. In a fed-batch fermenter cultivation, 4-aminophenylalanine production of 22.5 g/L was achieved. To produce 4-nitrophenylalanine from glucose, we constructed strains co-expressing AurF alongside the genes responsible for 4-aminophenylalanine synthesis. The subsequent optimization of the plasmid copy numbers carrying each gene set resulted in an increase in the 4-nitrophenylalanine production titer. Transcription analysis revealed that the expression level of the 4-aminophenylalanine biosynthetic genes markedly contributed to 4-nitrophenylalanine production. After optimizing batch fermentation conditions, the titer of 4-nitrophenylalanine increased to 2.22 g/L. Overall, these results provide the basis for industrial microbial production of 4-nitrophenylalanine, contributing to the advancement of biotechnological methodologies for generating non-natural amino acids with specific functionalities.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 171-180"},"PeriodicalIF":6.8,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143893294","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":"Modulating fatty acid metabolism and composition of CHO cells by feeding high levels of fatty acids complexed using methyl-β-cyclodextrin","authors":"Bradley Priem ,&nbsp;Xiangchen Cai ,&nbsp;Yu-Jun Hong ,&nbsp;Karl Gilmore ,&nbsp;Zijun Deng ,&nbsp;Sabrina Chen ,&nbsp;Harnish Mukesh Naik ,&nbsp;Michael J. Betenbaugh ,&nbsp;Maciek R. Antoniewicz","doi":"10.1016/j.ymben.2025.04.005","DOIUrl":"10.1016/j.ymben.2025.04.005","url":null,"abstract":"<div><div>Chinese Hamster Ovary (CHO) cells are widely used in the pharmaceutical industry to produce therapeutic proteins. Increasing the productivity of CHO cells through media development and genetic engineering is a significant industry objective. Past research demonstrated the benefits of modulating fatty acid composition of CHO cells through genetic engineering. In this study, we describe an alternative approach to modulate fatty acid composition by directly feeding high levels of fatty acids in CHO cell culture. To accomplish this, we developed and optimized a pharmaceutically relevant feeding strategy using methyl-β-cyclodextrin (MBCD) to solubilize fatty acids. To quantify fatty acid composition of CHO cells, a new GC-MS protocol was developed and validated. In fed batch cultures, we found that the degree of saturation of fatty acids in CHO cell mass, i.e. the relative abundances of saturated, monounsaturated and polyunsaturated fatty acids, can be controlled by the choice of fatty acid supplement and feeding strategy. Feeding unsaturated fatty acids such as palmitoleic acid, oleic acid, and linoleic acid had the greatest impact the fatty acid composition of CHO cells, increasing their respective abundances in cell mass by upwards of 25x, 1.5x, and 50x, respectively. ¹³C-Tracing further revealed that the supplemented fatty acids were involved in a range of elongation, desaturation, and β-oxidation reactions to yield both common and uncommon fatty acids such as vaccenic acid and hypogeic acid. Finally, we show that CHO-K1 and CHO-GS cells take up fatty acids solubilized with MBCD at rates comparable to delivery using bovine serum albumin. Taken together, this work paves the way for new feed media formulations containing fatty acids to optimize CHO cell physiology in industrial cell cultures.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 158-169"},"PeriodicalIF":6.8,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143881601","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":"Psilocybin biosynthesis enhancement through gene source optimization","authors":"Madeleine R. Keller ,&nbsp;Madeline G. McKinney ,&nbsp;Abhishek K. Sen ,&nbsp;Felicia G. Guagliardo ,&nbsp;Elle B. Hellwarth ,&nbsp;Khondokar Nowshin Islam ,&nbsp;Nicholas A. Kaplan ,&nbsp;William J. Gibbons Jr. ,&nbsp;Grace E. Kemmerly ,&nbsp;Chance Meers ,&nbsp;Xin Wang ,&nbsp;J. Andrew Jones","doi":"10.1016/j.ymben.2025.04.003","DOIUrl":"10.1016/j.ymben.2025.04.003","url":null,"abstract":"<div><div>Psilocybin, the prodrug to the psychoactive compound in ‘magic’ mushrooms, is currently being studied in clinical trials as a treatment for severe mental health conditions, such as depression and anxiety. Previous reports of psilocybin biosynthesis as reconstituted in <em>E. coli</em> reported maximum titers of 1.16 g/L, exclusively using genes from the most common recreationally used mushroom, <em>Psilocybe cubensis</em>. This study explores the effect of gene species variation on psilocybin and baeocystin production using various exogenous genes sourced from psilocybin-producing mushrooms <em>Psilocybe cubensis</em>, <em>Psilocybe cyanescens</em>, <em>Panaeolus cyanescens</em>, and <em>Gymnopilus dilepis</em>. The <em>psiD</em> and <em>psiK</em> genes sourced from <em>P. cubensis</em> demonstrated unequivocally superior performance, while <em>psiM</em> showed varied production levels of psilocybin and the pathway intermediate baeocystin with changes in gene source. Strains containing a <em>psiM</em> gene sourced from <em>Psilocybe cyanescens</em> demonstrated a higher degree of baeocystin selectivity as compared to other <em>psiM</em> genes, demonstrating a key difference between species. Most notably, the strain <em>Gymdi30</em>, containing <em>psiM</em> sourced from <em>G. dilepis</em>, achieved a psilocybin titer of 1.46 ± 0.13 g/L, the highest reported to date. Comparative proteomic analysis of Gymdi30 during periods of high and low productivity was also performed to investigate bottlenecks in cellular metabolism, which could be limiting strain performance. This work represents a significant improvement in psilocybin biosynthesis, a key step towards the development of a biosynthetic manufacturing route for psilocybin.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 119-129"},"PeriodicalIF":6.8,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143850804","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":"Awakening of the RuMP cycle for partial methylotrophy in the thermophile Parageobacillus thermoglucosidasius","authors":"Miguel Paredes-Barrada ,&nbsp;Annemieke Mathissen ,&nbsp;Roland A. van der Molen ,&nbsp;Pablo J. Jiménez-Huesa ,&nbsp;Machiel Eduardo Polano ,&nbsp;Stefano Donati ,&nbsp;Miriam Abele ,&nbsp;Christina Ludwig ,&nbsp;Richard van Kranenburg ,&nbsp;Nico J. Claassens","doi":"10.1016/j.ymben.2025.04.002","DOIUrl":"10.1016/j.ymben.2025.04.002","url":null,"abstract":"<div><div>Given sustainability and scalability concerns of using sugar feedstocks for microbial bioproduction of bulk chemicals, widening the feedstock range for microbial cell factories is of high interest. Methanol is a one-carbon alcohol that stands out as an alternative feedstock for the bioproduction of chemicals, as it is electron-rich, water-miscible and can be produced from several renewable resources. Bioconversion of methanol into products under thermophilic conditions (&gt;50 °C) could be highly advantageous for industrial biotechnology. Although progress is being made with natural, thermophilic methylotrophic microorganisms, they are not yet optimal for bioproduction and establishing alternative thermophilic methylotrophic bioproduction platforms can widen possibilities. Hence, we set out to implement methanol assimilation in the emerging thermophilic model organism <em>Parageobacillus thermoglucosidasius.</em> We engineered <em>P. thermoglucosidasius</em> to be strictly dependent for its growth on methanol assimilation via the core of the highly efficient ribulose monophosphate (RuMP) cycle, while co-assimilating ribose. Surprisingly, this did not require heterologous expression of RuMP enzymes. Instead, by laboratory evolution we awakened latent, native enzyme activities to form the core of the RuMP cycle. We obtained fast methylotrophic growth in which ∼17 % of biomass was strictly obtained from methanol. This work lays the foundation for developing a versatile thermophilic bioproduction platform based on renewable methanol.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 145-157"},"PeriodicalIF":6.8,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143881600","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-omics driven genome-scale metabolic modeling improves viral vector yield in HEK293","authors":"L. Zehetner ,&nbsp;D. Széliová ,&nbsp;B. Kraus ,&nbsp;J.A. Hernandez Bort ,&nbsp;J. Zanghellini","doi":"10.1016/j.ymben.2025.03.011","DOIUrl":"10.1016/j.ymben.2025.03.011","url":null,"abstract":"<div><div>HEK293 cells are a versatile cell line extensively used in the production of recombinant proteins and viral vectors, notably Adeno-associated virus (AAV) (Bulcha et al., 2021). Despite their high transfection efficiency and adaptability to various culture conditions, challenges remain in achieving sufficient yields of active viral particles. This study presents a comprehensive multi-omics analysis of two HEK293 strains under good manufacturing practice conditions, focusing on the metabolic and cellular responses during AAV production. The investigation included lipidomic, exometabolomic, and transcriptomic profiling across different conditions and time points. Genome-scale metabolic models (GSMMs) were reconstructed for these strains to elucidate metabolic shifts and identify potential bottlenecks in AAV production. Notably, the study revealed significant differences between a High-producing (HP) and a Low-producing (LP) HEK293 strains, highlighting pseudohypoxia in the LP strain. Key findings include the identification of hypoxia-inducible factor 1-alpha (HIF-1<span><math><mi>α</mi></math></span>) as a critical regulator in the LP strain, linking pseudohypoxia to poor AAV productivity. Inhibition of HIF-1<span><math><mi>α</mi></math></span> resulted in immediate cessation of cell growth and a 2.5-fold increase in viral capsid production, albeit with a decreased number of viral genomes, impacting the full-to-empty particle ratio. This trade-off is significant because it highlights a key challenge in AAV production: achieving a balance between capsid assembly and genome packaging to optimize the yield of functional viral vectors. Overall this suggests that while HIF-1<span><math><mi>α</mi></math></span> inhibition enhances capsid assembly, it simultaneously hampers nucleotide synthesis via the pentose phosphate pathway (PPP), necessary for nucleotide synthesis, and therefore for AAV genome replication.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 103-118"},"PeriodicalIF":6.8,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143838491","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":"Carbon-conserving bioproduction of malate in an E. coli-based cell-free system","authors":"Ryan A.L. Cardiff ,&nbsp;Shaafique Chowdhury ,&nbsp;Widianti Sugianto ,&nbsp;Benjamin I. Tickman ,&nbsp;Diego Alba Burbano ,&nbsp;Pimphan A. Meyer ,&nbsp;Margaret Cook ,&nbsp;Brianne King ,&nbsp;David Garenne ,&nbsp;Alexander S. Beliaev ,&nbsp;Vincent Noireaux ,&nbsp;Peralta-Yahya Pamela ,&nbsp;James M. Carothers","doi":"10.1016/j.ymben.2025.03.020","DOIUrl":"10.1016/j.ymben.2025.03.020","url":null,"abstract":"<div><div>Formate, a biologically accessible form of CO₂, has attracted interest as a renewable feedstock for bioproduction. However, approaches are needed to investigate efficient routes for biological formate assimilation due to its toxicity and limited utilization by microorganisms. Cell-free systems hold promise due to their potential for efficient use of carbon and energy sources and compatibility with diverse feedstocks. However, bioproduction using purified cell-free systems is limited by costly enzyme purification, whereas lysate-based systems must overcome loss of flux to background reactions in the cell extract. Here, we engineer an <em>E. coli</em>-based system for an eight-enzyme pathway from DNA and incorporate strategies to regenerate cofactors and minimize loss of flux through background reactions. We produce the industrial di-acid malate from glycine, bicarbonate, and formate by engineering the carbon-conserving reductive TCA and formate assimilation pathways. We show that <em>in situ</em> regeneration of NADH drives metabolic flux towards malate, improving titer by 15-fold. Background reactions can also be reduced 6-fold by diluting the lysate following expression and introducing chemical inhibitors of competing reactions. Together, these results establish a carbon-conserving, lysate-based cell-free platform for malate production, producing 64 μM malate after 8 h. This system conserves 43 % of carbon otherwise lost as CO₂ through the TCA cycle and incorporates 0.13 mol CO₂ equivalents/mol glycine fed. Finally, techno-economic analysis of cell-free malate production from formate revealed that the high cost of lysate is a key challenge to the economic feasibility of the process, even assuming efficient cofactor recycling. This work demonstrates the capabilities of cell-free expression systems for both the prototyping of carbon-conserving pathways and the sustainable bioproduction of platform chemicals.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 59-76"},"PeriodicalIF":6.8,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143825983","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}