Metabolic engineering最新文献

{"title":"Hyperproduction of 7-dehydrocholesterol by rewiring the post-squalene module in lipid droplets of Saccharomyces cerevisiae","authors":"Xiang Xiu ,&nbsp;Xianhao Xu ,&nbsp;Yaokang Wu ,&nbsp;Yanfeng Liu ,&nbsp;Jianghua Li ,&nbsp;Guocheng Du ,&nbsp;Jian Chen ,&nbsp;Xueqin Lv ,&nbsp;Long Liu","doi":"10.1016/j.ymben.2024.10.001","DOIUrl":"10.1016/j.ymben.2024.10.001","url":null,"abstract":"<div><div>Lipid droplets (LDs) are specialized organelles that store neutral lipids to reduce the negative effects of lipotoxicity on cells. However, many neutral lipids are precursors for the synthesis of sterols and complex terpenoids, and this sequestration often greatly limits the efficient biosynthesis of sterols and complex terpenoids. In this study, taking 7-dehydrocholesterol (7-DHC) synthesis in <em>Saccharomyces cerevisiae</em> as an example, we revealed the blocking mechanism of LD sequestration on the efficient synthesis of metabolic products and found that LDs can sequester a significant amount of squalene, the precursor of 7-DHC, effectively preventing it from being directed toward the post-squalene pathway. Based on this, a post-squalene pathway was reconstructed on LDs, which resulted in a 28.7% increase in the 7-DHC titer, reaching 684.1 mg/L, whereas the squalene titer was reduced by approximately 97%. Subsequently, the triacylglycerol degradation pathway was weakened to release the storage space in LDs, and the esterification pathway was concurrently strengthened to guide 7-DHC storage within LDs, which further increased 7-DHC production, reaching 792.9 mg/L. Finally, by reducing the NADH/NAD + ratio to alleviate the redox imbalance, the 7-DHC titer reached 867.6 mg/L in shake flask and 5.1 g/L in a 3-L bioreactor, which is the highest reported titer to date. In summary, this study provides new insights into the important role of LDs in sterol synthesis and offers a novel strategy for constructing cell factories for the efficient synthesis of sterol compounds.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 147-156"},"PeriodicalIF":6.8,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142385364","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 yeast for de novo production of kratom monoterpene indole alkaloids","authors":"Maxence Holtz ,&nbsp;Daniela Rago ,&nbsp;Ida Nedermark ,&nbsp;Frederik G. Hansson ,&nbsp;Beata J. Lehka ,&nbsp;Lea G. Hansen ,&nbsp;Nils E.J. Marcussen ,&nbsp;Wouter J. Veneman ,&nbsp;Linda Ahonen ,&nbsp;Juraithip Wungsintaweekul ,&nbsp;Carlos G. Acevedo-Rocha ,&nbsp;Ron P. Dirks ,&nbsp;Jie Zhang ,&nbsp;Jay D. Keasling ,&nbsp;Michael K. Jensen","doi":"10.1016/j.ymben.2024.09.011","DOIUrl":"10.1016/j.ymben.2024.09.011","url":null,"abstract":"<div><div>Monoterpene indole alkaloids (MIAs) from <em>Mitragyna speciosa</em> (“kratom”), such as mitragynine and speciogynine, are promising novel scaffolds for opioid receptor ligands for treatment of pain, addiction, and depression. While kratom leaves have been used for centuries in South-East Asia as stimulant and pain management substance, the biosynthetic pathway of these psychoactives have only recently been partially elucidated. Here, we demonstrate the <em>de novo</em> production of mitragynine and speciogynine in <em>Saccharomyces cerevisiae</em> through the reconstruction of a five-step synthetic pathway from common MIA precursor strictosidine comprising fungal tryptamine 4-monooxygenase to bypass an unknown kratom hydroxylase. Upon optimizing cultivation conditions, a titer of ∼290 μg/L kratom MIAs from glucose was achieved. Untargeted metabolomics analysis of lead production strains led to the identification of numerous shunt products derived from the activity of strictosidine synthase (STR) and dihydrocorynantheine synthase (DCS), highlighting them as candidates for enzyme engineering to further improve kratom MIAs production in yeast. Finally, by feeding fluorinated tryptamine and expressing a human tailoring enzyme, we further demonstrate production of fluorinated and hydroxylated mitragynine derivatives with potential applications in drug discovery campaigns. Altogether, this study introduces a yeast cell factory platform for the biomanufacturing of complex natural and new-to-nature kratom MIAs derivatives with therapeutic potential.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 135-146"},"PeriodicalIF":6.8,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142375608","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":"α-Substituted 3-hydroxy acid production from glucose in Escherichia coli","authors":"K'yal R. Bannister,&nbsp;Kristala L.J. Prather","doi":"10.1016/j.ymben.2024.09.006","DOIUrl":"10.1016/j.ymben.2024.09.006","url":null,"abstract":"<div><div>Polyhydroxyalkanoates (PHAs) are renewably-derived, microbial polyesters composed of hydroxy acids (HAs). Demand for sustainable plastics alternatives, combined with the unfavorable thermal properties exhibited by some PHAs, motivates the discovery of novel PHA-based materials. Incorporation of α-substituted HAs yields thermostable PHAs; however, the reverse β-oxidation (rBOX) pathway, the canonical pathway for HA production, is unable to produce these monomers because it utilizes thiolases with narrow substrate specificity. Here, we present a thiolase-independent pathway to two α-substituted HAs, 3-hydroxyisobutyric acid (3HIB) and 3-hydroxy-2-methylbutyric acid (3H2MB). This pathway involves the conversion of glucose to various branched acyl-CoAs and ultimately to 3HIB or 3H2MB. As proof of concept, we engineered <em>Escherichia coli</em> for the specific production of 3HIB and 3H2MB from glucose at titers as high as 66 ± 5 mg/L and 290 ± 40 mg/L, respectively. Optimizing this pathway for 3H2MB production via a novel byproduct recycle increased titer by 60%. This work illustrates the utility of novel pathway design HA production leading to PHAs with industrially relevant properties.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 124-134"},"PeriodicalIF":6.8,"publicationDate":"2024-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142308103","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":"Efficient utilization of xylose requires CO2 fixation in Synechococcus elongatus PCC 7942","authors":"Shannon R. Pressley,&nbsp;Jake N. Gonzales,&nbsp;Shota Atsumi","doi":"10.1016/j.ymben.2024.09.010","DOIUrl":"10.1016/j.ymben.2024.09.010","url":null,"abstract":"<div><div>Cyanobacteria show great promise as autotrophic hosts for the renewable biosynthesis of useful chemicals from CO₂ and light. While they can efficiently fix CO₂, cyanobacteria are generally outperformed by heterotrophic production hosts in terms of productivity and titer. Photomixotrophy, or co-utilization of sugars and CO₂ as carbon feedstocks, has been implemented in cyanobacteria to greatly improve productivity and titers of several chemical products. We introduced xylose photomixotrophy to a 2,3-butanediol producing strain of <em>Synechococcus elongatus</em> PCC 7942 and characterized the effect of gene knockouts, changing pathway expression levels, and changing growth conditions on chemical production. Interestingly, 2,3-butanediol production was almost completely inhibited in the absence of added CO₂. Untargeted metabolomics implied that RuBisCO was a significant bottleneck, especially at ambient CO₂ levels, restricting the supply of lower glycolysis metabolites needed for 2,3-butanediol production. The dependence of the strain on elevated CO₂ levels suggests some practical limitations on how xylose photomixotrophy can be efficiently carried out in <em>S. elongatus</em>.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 115-123"},"PeriodicalIF":6.8,"publicationDate":"2024-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717624001277/pdfft?md5=d948f70faeea495436d780992a7c5a88&pid=1-s2.0-S1096717624001277-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142308101","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishing a coumarin production platform by protein and metabolic engineering","authors":"Chong Xie ,&nbsp;Ning An ,&nbsp;Lei Zhou ,&nbsp;Xiaolin Shen ,&nbsp;Jia Wang ,&nbsp;Yajun Yan ,&nbsp;Xinxiao Sun ,&nbsp;Qipeng Yuan","doi":"10.1016/j.ymben.2024.09.009","DOIUrl":"10.1016/j.ymben.2024.09.009","url":null,"abstract":"<div><div>Coumarins are a vast family of natural products with diverse biological activities. Cinnamyl-CoA <em>ortho</em>-hydroxylases (CCHs) catalyze the gateway and rate-limiting step in coumarin biosynthesis. However, engineering CCHs is challenging due to the large size of the substrates and the vague structure-activity relationship. Herein, directed evolution and structure-guided engineering were performed to engineer a CCH (<em>At</em>F6′H from <em>Arabidopsis thaliana</em>) using a fluorescence-based screening method, yielding the transplantable surface mutations and the substrate-specific pocket mutations with improved activity. Structural analysis and molecular dynamics simulations elucidated the conformational changes that led to increased catalytic efficiency. Applying appropriate variants with the optimized upstream biosynthetic pathways improved the titers of three simple coumarins by 5 to 22-fold. Further introducing glycosylation modules resulted in the production of four coumarin glucosides, among which the titer of aesculin was increased by 15.7-fold and reached 3 g/L in scale-up fermentation. This work unleashed the potential of CCHs and established an <em>Escherichia coli</em> platform for coumarins production.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 89-98"},"PeriodicalIF":6.8,"publicationDate":"2024-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142308102","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":"Engineering ethanologenicity into the extremely thermophilic bacterium Anaerocellum (f. Caldicellulosiriuptor) bescii","authors":"Ryan G. Bing ,&nbsp;Kathryne C. Ford ,&nbsp;Daniel J. Willard ,&nbsp;Mohamad J.H. Manesh ,&nbsp;Christopher T. Straub ,&nbsp;Tunyaboon Laemthong ,&nbsp;Benjamin H. Alexander ,&nbsp;Tania Tanwee ,&nbsp;Hailey C. O'Quinn ,&nbsp;Farris L. Poole ,&nbsp;Jason Vailionis ,&nbsp;Ying Zhang ,&nbsp;Dmitry Rodionov ,&nbsp;Michael W.W. Adams ,&nbsp;Robert M. Kelly","doi":"10.1016/j.ymben.2024.09.007","DOIUrl":"10.1016/j.ymben.2024.09.007","url":null,"abstract":"<div><div>The anaerobic bacterium <em>Anaerocellum</em> (f. <em>Caldicellulosiruptor</em>) <em>bescii</em> natively ferments the carbohydrate content of plant biomass (including microcrystalline cellulose) into predominantly acetate, H₂, and CO₂, and smaller amounts of lactate, alanine and valine. While this extreme thermophile (growth T_opt 78 °C) is not natively ethanologenic, it has been previously metabolically engineered with this property, albeit initially yielding low solvent titers (∼15 mM). Herein we report significant progress on improving ethanologenicity in <em>A. bescii</em>, such that titers above 130 mM have now been achieved, while concomitantly improving selectivity by minimizing acetate formation. Metabolic engineering progress has benefited from improved molecular genetic tools and better understanding of <em>A. bescii</em> growth physiology. Heterologous expression of a mutated thermophilic alcohol dehydrogenase (AdhE) modified for co-factor requirement, coupled with bioreactor operation strategies related to pH control, have been key to enhanced ethanol generation and fermentation product specificity. Insights gained from metabolic modeling of <em>A. bescii</em> set the stage for its further improvement as a metabolic engineering platform.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 99-114"},"PeriodicalIF":6.8,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142276837","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":"Architecting a transcriptional repressor-based genetic inverter for tryptophan derived pathway regulation in Escherichia coli","authors":"Xinyu Gong ,&nbsp;Yuxi Teng ,&nbsp;Jianli Zhang ,&nbsp;Qi Gan ,&nbsp;Ming Song ,&nbsp;Ameen Alaraj ,&nbsp;Peter Kner ,&nbsp;Yajun Yan","doi":"10.1016/j.ymben.2024.09.008","DOIUrl":"10.1016/j.ymben.2024.09.008","url":null,"abstract":"<div><div>Efficient microbial cell factories require intricate and precise metabolic regulations for optimized production, which can be significantly aided by implementing regulatory genetic circuits with versatile functions. However, constructing functionally diverse genetic circuits in host strains is challenging. Especially, functional diversification based on transcriptional repressors has been rarely explored due to the difficulty in inverting their repression properties. To address this, we proposed a design logic to create transcriptional repressor-based genetic inverters for functional enrichment. As proof of concept, a tryptophan-inducible genetic inverter was constructed by integrating two sets of transcriptional repressors, <em>PtrpO1</em>-TrpR1 and <em>PtetO1</em>-TetR. In this genetic inverter, the repression of TetR towards <em>PtetO1</em> could be alleviated by the tryptophan-TrpR1 complex in the presence of tryptophan, leading to the activated output. Subsequently, we optimized the dynamic performance of the inverter and constructed tryptophan-triggered dynamic activation systems. Further coupling of the original repression function of <em>PtrpO1</em>-TrpR1 with inverter variants realized the tryptophan-triggered bifunctional regulation system. Finally, the dynamic regulation systems enabled tryptophan production monitoring. These systems also remarkably increased the titers of the tryptophan derivatives tryptamine and violacein by 2.0-fold and 7.4-fold, respectively. The successful design and application of the genetic inverter enhanced the applicability of transcriptional repressors.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 66-77"},"PeriodicalIF":6.8,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142246789","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":"Optimized genome-wide CRISPR screening enables rapid engineering of growth-based phenotypes in Yarrowia lipolytica","authors":"Nicholas R. Robertson ,&nbsp;Varun Trivedi ,&nbsp;Brian Lupish ,&nbsp;Adithya Ramesh ,&nbsp;Yuna Aguilar ,&nbsp;Stephanie Carrera ,&nbsp;Sangcheon Lee ,&nbsp;Anthony Arteaga ,&nbsp;Alexander Nguyen ,&nbsp;Chase Lenert-Mondou ,&nbsp;Marcus Harland-Dunaway ,&nbsp;Robert Jinkerson ,&nbsp;Ian Wheeldon","doi":"10.1016/j.ymben.2024.09.005","DOIUrl":"10.1016/j.ymben.2024.09.005","url":null,"abstract":"<div><div>CRISPR-Cas9 functional genomic screens uncover gene targets linked to various phenotypes for metabolic engineering with remarkable efficiency. However, these genome-wide screens face a number of design challenges, including variable guide RNA activity, ensuring sufficient genome coverage, and maintaining high transformation efficiencies to ensure full library representation. These challenges are prevalent in non-conventional yeast, many of which exhibit traits that are well suited to metabolic engineering and bioprocessing. To address these hurdles in the oleaginous yeast <em>Yarrowia lipolytica</em>, we designed a compact, high-activity genome-wide sgRNA library. The library was designed using DeepGuide, a sgRNA activity prediction algorithm and a large dataset of ∼50,000 sgRNAs with known activity. Three guides per gene enables redundant targeting of 98.8% of genes in the genome in a library of 23,900 sgRNAs. We deployed the optimized library to uncover genes essential to the tolerance of acetate, a promising alternative carbon source, and various hydrocarbons present in many waste streams. Our screens yielded several gene knockouts that improve acetate tolerance on their own and as double knockouts in media containing acetate as the sole carbon source. Analysis of the hydrocarbon screens revealed genes related to fatty acid and alkane metabolism in <em>Y. lipolytica</em>. The optimized CRISPR gRNA library and its successful use in <em>Y. lipolytica</em> led to the discovery of alternative carbon source-related genes and provides a workflow for creating high-activity, compact genome-wide libraries for strain engineering.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 55-65"},"PeriodicalIF":6.8,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717624001228/pdfft?md5=a98b4da5c76bbe1a998a4bf5f0062b56&pid=1-s2.0-S1096717624001228-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142290818","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Combinatorial iterative method for metabolic engineering of Yarrowia lipolytica: Application for betanin biosynthesis","authors":"Wei Jiang ,&nbsp;Shengbao Wang ,&nbsp;Paulo Avila ,&nbsp;Tue Sparholt Jørgensen ,&nbsp;Zhijie Yang ,&nbsp;Irina Borodina","doi":"10.1016/j.ymben.2024.09.003","DOIUrl":"10.1016/j.ymben.2024.09.003","url":null,"abstract":"<div><div>Combinatorial library-based metabolic engineering approaches allow lower cost and faster strain development. We developed a genetic toolbox EXPRESS^YALI for combinatorial engineering of the oleaginous yeast <em>Yarrowia lipolytica</em>. The toolbox enables consecutive rounds of engineering, where up to three combinatorially assembled gene expression cassettes can be integrated into each yeast clone per round. The cassettes are integrated into distinct intergenic sites or an open reading frame of a target gene if a simultaneous gene knockout is desired. We demonstrate the application of the toolbox by optimizing the <em>Y. lipolytica</em> to produce the red beet color betanin via six consecutive rounds of genome editing and screening. The library size varied between 24 and 360. Library screening was facilitated by automated color-based colony picking. In the first round, betanin pathway genes were integrated, resulting in betanin titer of around 20 mg/L. Through the following five consecutive rounds, additional biosynthetic genes were integrated, and the precursor supply was optimized, resulting in a titer of 70 mg/L. Three beta-glucosidases were deleted to prevent betanin deglycosylation, which led to a betanin titer of 130 mg/L in a small scale and a titer of 1.4 g/L in fed-batch bioreactors. The EXPRESS^YALI toolbox can facilitate metabolic engineering efforts in <em>Y. lipolytica</em> (available via AddGene Cat. Nr. 212682–212704, Addgene kit ID # 1000000245).</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 78-88"},"PeriodicalIF":6.8,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142174631","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":"The 6-phosphofructokinase reaction in Acetivibrio thermocellus is both ATP- and pyrophosphate-dependent","authors":"Jeroen G. Koendjbiharie,&nbsp;Teun Kuil,&nbsp;Carolus M.K. Nurminen,&nbsp;Antonius J.A. van Maris","doi":"10.1016/j.ymben.2024.09.002","DOIUrl":"10.1016/j.ymben.2024.09.002","url":null,"abstract":"<div><p><em>Acetivibrio thermocellus</em> (formerly <em>Clostridium thermocellum</em>) is a potential platform for lignocellulosic ethanol production. Its industrial application is hampered by low product titres, resulting from a low thermodynamic driving force of its central metabolism. It possesses both a functional ATP- and a functional PP_i-dependent 6-phosphofructokinase (PP_i-Pfk), of which only the latter is held responsible for the low driving force. Here we show that, following the replacement of PP_i-Pfk by cytosolic pyrophosphatase and transaldolase, the native ATP-Pfk is able to carry the full glycolytic flux. Interestingly, the barely-detectable <em>in vitro</em> ATP-Pfk activities are only a fraction of what would be required, indicating its contribution to glycolysis has consistently been underestimated. A kinetic model demonstrated that the strong inhibition of ATP-Pfk by PP_i can prevent futile cycling that would arise when both enzymes are active simultaneously. As such, there seems to be no need for a long-sought-after PP_i-generating mechanism to drive glycolysis, as PP_i-Pfk can simply use whatever PP_i is available, and ATP-Pfk complements the rest of the PFK-flux. Laboratory evolution of the ΔPP_i-Pfk strain, unable to valorize PP_i, resulted in a mutation in the GreA transcription elongation factor. This mutation likely results in reduced RNA-turnover, hinting at transcription as a significant (and underestimated) source of anabolic PP_i. Together with other mutations, this resulted in an <em>A</em>. <em>thermocellus</em> strain with the hitherto highest biomass-specific cellobiose uptake rate of 2.2 g/g_x/h. These findings are both relevant for fundamental insight into dual ATP/PP_i Pfk-nodes, which are not uncommon in other microorganisms, as well as for further engineering of <em>A</em>. <em>thermocellus</em> for consolidated bioprocessing.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"86 ","pages":"Pages 41-54"},"PeriodicalIF":6.8,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717624001113/pdfft?md5=3c30f0db5f89c40bfd4e53984ece9b90&pid=1-s2.0-S1096717624001113-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142154542","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}