Metabolic Engineering Communications最新文献

{"title":"Metabolic growth-coupling strategies for in vivo enzyme selection systems","authors":"Tobias B. Alter ,&nbsp;Pascal A. Pieters ,&nbsp;Colton J. Lloyd ,&nbsp;Adam M. Feist ,&nbsp;Emre Özdemir ,&nbsp;Bernhard O. Palsson ,&nbsp;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}

{"title":"Co-consumption for plastics upcycling: A perspective","authors":"Michael Weldon,&nbsp;Sanniv Ganguly,&nbsp;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}

{"title":"Improving recombinant protein secretion in Aspergillus nidulans by targeting the N-glycosylation machinery","authors":"Jaqueline Aline Gerhardt ,&nbsp;Marcelo Ventura Rubio ,&nbsp;Cesar Rafael Fanchini Terrasan ,&nbsp;Natalia Sayuri Wassano ,&nbsp;Aryadne Rodrigues ,&nbsp;Fernanda Lopes de Figueiredo ,&nbsp;Everton Paschoal Antoniel ,&nbsp;Fabiano Jares Contesini ,&nbsp;Artur Hermano Sampaio Dias ,&nbsp;Uffe Hasbro Mortensen ,&nbsp;Munir Salomão Skaf ,&nbsp;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}

{"title":"Synthetic pathways for microbial biosynthesis of valuable pyrazine derivatives using genetically modified Pseudomonas putida KT2440","authors":"Vytautas Petkevičius,&nbsp;Justė Juknevičiūtė,&nbsp;Domas Mašonis,&nbsp;Rolandas Meškys","doi":"10.1016/j.mec.2025.e00258","DOIUrl":"10.1016/j.mec.2025.e00258","url":null,"abstract":"<div><div>Using engineered microbes for synthesizing high-valued chemicals from renewable sources is a foundation in synthetic biology, however, it is still in its early stages. Here, we present peculiarities and troubleshooting of the construction of novel synthetic metabolic pathways in genetically modified work-horse <em>Pseudomonas putida</em> KT2440. The combination of this microbial host and heterologous expressed non-heme diiron monooxygenases enabled <em>de novo</em> biosynthesis of 2,5-dimethylpyrazine (2,5-DMP) carboxylic acid and <em>N</em>-oxides as target products. A key intermediate, 2,5-DMP, was obtained by using <em>Pseudomonas putida</em> KT2440Δ6 strain containing six gene deletions in the L-threonine pathway, along with the overexpression of <em>thrA</em>^{<em>S345F</em>} and <em>tdh</em> from <em>E. coli</em>. Thus, the carbon surplus was redirected from glucose through L-threonine metabolism toward the formation of 2,5-DMP, resulting in a product titre of 106 ± 30 mg <span>L</span>⁻¹. By introducing two native genes (<em>thrB</em> and <em>thrC</em> from <em>P. putida</em> KT2440) from the L-threonine biosynthesis pathway, the production of 2,5-DMP was increased to 168 ± 20 mg L⁻¹. The resulting 2,5-DMP was further derivatized through two separate pathways. Recombinant <em>P. putida</em> KT2440 strain harboring xylene monooxygenase (XMO) produced 5-methyl-2-pyrazinecarboxylic acid from glucose as a targeted compound in a product titre of 204 ± 24 mg L⁻¹. The microbial host containing genes of PmlABCDEF monooxygenase (Pml) biosynthesized <em>N</em>-oxides – 2,5-dimethylpyrazine 1-oxide as a main product, and 2,5-dimethylpyrazine 1,4-dioxide as a minor product, reaching product titres of 82 ± 8 mg L⁻¹ and 11 ± 2 mg L⁻¹ respectively.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"20 ","pages":"Article e00258"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143748119","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}

{"title":"Production of borneol, camphor, and bornyl acetate using engineered Saccharomyces cerevisiae","authors":"Masahiro Tominaga ,&nbsp;Kazuma Kawakami ,&nbsp;Hiro Ogawa ,&nbsp;Tomomi Nakamura ,&nbsp;Akihiko Kondo ,&nbsp;Jun Ishii","doi":"10.1016/j.mec.2025.e00259","DOIUrl":"10.1016/j.mec.2025.e00259","url":null,"abstract":"<div><div>Microbial production of bicyclic monoterpenes is of great interest because their production primarily utilizes non-sustainable resources. Here, we report an engineered <em>Saccharomyces cerevisiae</em> yeast that produces bicyclic monoterpenes, including borneol, camphor, and bornyl acetate. The engineered yeast expresses a bornyl pyrophosphatase synthase from <em>Salvia officinalis</em> fused with mutated farnesyl pyrophosphate synthase from <em>S</em>. <em>cerevisiae</em> and two mevalonate pathway enzymes (an acetoacetyl-CoA thiolase/hydroxymethylglutaryl-CoA [HMG-CoA] reductase and an HMG-CoA synthase) from <em>Enterococcus faecalis</em>. The yeast produced up to 23.0 mg/L of borneol in shake-flask fermentation. By additionally expressing borneol dehydrogenase from <em>Pseudomonas</em> sp. TCU-HL1 or bornyl acetyltransferase from <em>Wurfbainia villosa</em>, the engineered yeast produced 23.5 mg/L of camphor and 21.1 mg/L of bornyl acetate, respectively. This is the first report of heterologous production of camphor and bornyl acetate.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"20 ","pages":"Article e00259"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143767910","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}

{"title":"Engineering Pseudomonas putida for production of 3-hydroxyacids using hybrid type I polyketide synthases","authors":"Matthias Schmidt ,&nbsp;Aaron A. Vilchez ,&nbsp;Namil Lee ,&nbsp;Leah S. Keiser ,&nbsp;Allison N. Pearson ,&nbsp;Mitchell G. Thompson ,&nbsp;Yolanda Zhu ,&nbsp;Robert W. Haushalter ,&nbsp;Adam M. Deutschbauer ,&nbsp;Satoshi Yuzawa ,&nbsp;Lars M. Blank ,&nbsp;Jay D. Keasling","doi":"10.1016/j.mec.2025.e00261","DOIUrl":"10.1016/j.mec.2025.e00261","url":null,"abstract":"<div><div>Engineered type I polyketide synthases (T1PKSs) are a potentially transformative platform for the biosynthesis of small molecules. Due to their modular nature, T1PKSs can be rationally designed to produce a wide range of bulk or specialty chemicals. While heterologous PKS expression is best studied in microbes of the genus <em>Streptomyces</em>, recent studies have focused on the exploration of non-native PKS hosts. The biotechnological production of chemicals in fast growing and industrial relevant hosts has numerous economic and logistic advantages. With its native ability to utilize alternative feedstocks, <em>Pseudomonas putida</em> has emerged as a promising workhorse for the sustainable production of small molecules. Here, we outline the assessment of <em>P. putida</em> as a host for the expression of engineered T1PKSs and production of 3-hydroxyacids. After establishing the functional expression of an engineered T1PKS, we successfully expanded and increased the pool of available acyl-CoAs needed for the synthesis of polyketides using transposon sequencing and protein degradation tagging. This work demonstrates the potential of T1PKSs in <em>P. putida</em> as a production platform for the sustainable biosynthesis of unnatural polyketides.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"20 ","pages":"Article e00261"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143785397","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}

{"title":"Multi-step pathway engineering in probiotic Saccharomyces boulardii for abscisic acid production in the gut","authors":"Femke Van Gaever ,&nbsp;Paul Vandecruys ,&nbsp;Yasmine Driege ,&nbsp;Seo Woo Kim ,&nbsp;Johan M. Thevelein ,&nbsp;Rudi Beyaert ,&nbsp;Jens Staal","doi":"10.1016/j.mec.2025.e00263","DOIUrl":"10.1016/j.mec.2025.e00263","url":null,"abstract":"<div><div>The plant hormone abscisic acid (ABA) has gained attention for its role in animals and humans, particularly due to its protective effects in various immune and inflammatory disorders. Given its high concentrations in fruits like figs, bilberries and apricots, ABA shows promise as a nutraceutical. However scalability, short half-life and cost limit the use of ABA-enriched fruit extracts and synthetic supplements. In this study, we propose an alternative ABA administration method to overcome these challenges. We genetically engineered a strain of the probiotic <em>Saccharomyces boulardii to produce and deliver ABA directly to the gut of mice. Using t</em>he biosynthesis pathway from <em>Botrytis cinerea</em>, four genes (<em>bcaba1-4</em>) were integrated into <em>S. boulardii</em>, enabling ABA production at 30 °C, as previously described in <em>Saccharomyces cerevisiae</em>. Introducing an additional cytochrome P450 reductase gene resulted in a 7-fold increase in ABA titers, surpassing previous ABA-producing <em>S. cerevisiae</em> strains. Supplementation of the ABA-producing <em>S. boulardii</em> in the diet of mice (at a concentration of 5 × 10⁸ CFU/g) led to effective gut colonization but resulted in low serum ABA levels (approximately 1.8 ng/mL). The absence of detectable serum ABA after administration of the ABA-producing probiotic through oral gavage, prompted further investigation to determine the underlying cause. The physiological body temperature (37 °C) was identified as a major bottleneck for ABA production. Modifications to enhance the mevalonate pathway flux improved ABA levels at 37 °C. However, additional modifications are needed to optimize ABA production before testing this probiotic in disease contexts in mice.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"20 ","pages":"Article e00263"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144231628","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}

{"title":"13C-metabolic flux analysis of Saccharomyces cerevisiae in complex media","authors":"Hayato Fujiwara ,&nbsp;Nobuyuki Okahashi ,&nbsp;Taisuke Seike ,&nbsp;Fumio Matsuda","doi":"10.1016/j.mec.2025.e00260","DOIUrl":"10.1016/j.mec.2025.e00260","url":null,"abstract":"<div><div><em>Saccharomyces cerevisiae</em> is often cultivated in complex media for applications in food and other biochemical production. However, ¹³C-metabolic flux analysis (¹³C-MFA) has been conducted for <em>S. cerevisiae</em> cultivated in synthetic media, resulting in a limited understanding of the metabolic flux distributions under the complex media. In this study, ¹³C-MFA was applied to <em>S. cerevisiae</em> cultivated in complex media to quantify the metabolic fluxes in the central metabolic network. <em>S. cerevisiae</em> was cultivated in a synthetic dextrose (SD) medium supplemented with 20 amino acids (SD + AA) and yeast extract peptone dextrose (YPD) medium. The results revealed that glutamic acid, glutamine, aspartic acid, and asparagine are incorporated into the TCA cycle as carbon sources in parallel with glucose consumption. Based on these findings, we successfully conducted ¹³C-MFA of <em>S. cerevisiae</em> cultivated in SD + AA and YPD media using parallel labeling and measured amino acid uptake rates. Furthermore, we applied the developed approach to ¹³C-MFA of yeast cultivated in malt extract medium. The analysis revealed that the metabolic flux through the anaplerotic and oxidative pentose phosphate pathways was lower in complex media than in synthetic media. Owing to the reduced carbon loss by the branching pathways, carbon flow toward ethanol production via glycolysis could be elevated. ¹³C-MFA of <em>S. cerevisiae</em> cultured in complex media provides valuable insights for metabolic engineering and process optimization in industrial yeast fermentation.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"20 ","pages":"Article e00260"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143791672","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}

{"title":"Production of (R)-citramalate by engineered Saccharomyces cerevisiae","authors":"Ryosuke Mitsui ,&nbsp;Akihiko Kondo ,&nbsp;Tomokazu Shirai","doi":"10.1016/j.mec.2024.e00247","DOIUrl":"10.1016/j.mec.2024.e00247","url":null,"abstract":"<div><p>The budding yeast, <em>Saccharomyces cerevisiae</em>, has a high tolerance to organic acids and alcohols, and thus grows well under toxic concentrations of various compounds in the culture medium, potentially allowing for highly efficient compound production. (<em>R</em>)-citramalate is a raw material for methyl methacrylate and can be used as a metabolic intermediate in the biosynthesis of higher alcohols. (<em>R</em>)-citramalate is synthesized from pyruvate and acetyl-CoA. Unlike <em>Escherichia coli</em>, <em>S. cerevisiae</em> has organelles, and its intracellular metabolites are compartmentalized, preventing full use of intracellular acetyl-CoA. Therefore, in this study, to increase the amount of cytosolic acetyl-CoA for highly efficient production of (<em>R</em>)-citramalate, we inhibited the transport of cytosolic acetyl-CoA and pyruvate to the mitochondria. We also constructed a heterologous pathway to supply cytosolic acetyl-CoA. Additionally, we attempted to export (<em>R</em>)-citramalate from cells by expressing a heterologous dicarboxylate transporter gene. We evaluated the effects of these approaches on (<em>R</em>)-citramalate production and constructed a final strain by combining these positive approaches. The resulting strain produced 16.5 mM (<em>R</em>)-citramalate in batch culture flasks. This is the first report of (<em>R</em>)-citramalate production by recombinant <em>S. cerevisiae</em>, and the (<em>R</em>)-citramalate production by recombinant yeast achieved in this study was the highest reported to date.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"19 ","pages":"Article e00247"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000166/pdfft?md5=8e77960467f6df90982ae565f50fc7ce&pid=1-s2.0-S2214030124000166-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141985411","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}

{"title":"Expression and characterization of monofunctional alcohol dehydrogenase enzymes in Clostridium thermocellum","authors":"Daniela Prates Chiarelli ,&nbsp;Bishal Dev Sharma ,&nbsp;Shuen Hon ,&nbsp;Luana Walravens Bergamo ,&nbsp;Lee R. Lynd ,&nbsp;Daniel G. Olson","doi":"10.1016/j.mec.2024.e00243","DOIUrl":"https://doi.org/10.1016/j.mec.2024.e00243","url":null,"abstract":"<div><p><em>Clostridium thermocellum</em> is a thermophilic anaerobic bacterium that could be used for cellulosic biofuel production due to its strong native ability to consume cellulose, however its ethanol production ability needs to be improved to enable commercial application. In our previous strain engineering work, we observed a spontaneous mutation in the native <em>adhE</em> gene that reduced ethanol production. Here we attempted to complement this mutation by heterologous expression of 18 different alcohol dehydrogenase (<em>adh)</em> genes. We were able to express all of them successfully in <em>C. thermocellum</em>. Surprisingly, however, none of them increased ethanol production, and several actually <em>decreased</em> it. Our findings contribute to understanding the correlation between <em>C. thermocellum</em> ethanol production and Adh enzyme cofactor preferences. The identification of a set of <em>adh</em> genes that can be successfully expressed in this organism provides a foundation for future investigations into how the properties of Adh enzymes affect ethanol production.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"19 ","pages":"Article e00243"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000129/pdfft?md5=5d222b62409146f886808888e57c6440&pid=1-s2.0-S2214030124000129-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141480759","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}