Metabolic engineering最新文献

{"title":"Efficient utilization of xylose requires CO2 fixation in Synechococcus elongatus PCC 7942","authors":"","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":null,"pages":null},"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":"Engineering ethanologenicity into the extremely thermophilic bacterium Anaerocellum (f. Caldicellulosiriuptor) bescii","authors":"","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":null,"pages":null},"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":"","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":null,"pages":null},"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":"","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":null,"pages":null},"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":"","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":null,"pages":null},"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":"","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":null,"pages":null},"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}

{"title":"A precise and sustainable doxycycline-inducible cell line development platform for reliable mammalian cell engineering with gain-of-function mutations","authors":"","doi":"10.1016/j.ymben.2024.09.004","DOIUrl":"10.1016/j.ymben.2024.09.004","url":null,"abstract":"<div><p>For mammalian synthetic biology research, multiple orthogonal and tunable gene expression systems have been developed, among which the tetracycline (Tet)-inducible system is a key tool for gain-of-function mutations. Precise and long-lasting regulation of genetic circuits is necessary for the effective use of these systems in genetically engineered stable cell lines. However, current cell line development strategies, which depend on either random or site-specific integration along with antibiotic selection, are unpredictable and unsustainable, limiting their widespread use. To overcome these issues, we aimed to establish a <u>R</u>obust <u>O</u>verexpression via <u>S</u>ite-specific integration of <u>E</u>ffector (ROSE) system, a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated streamlined Tet-On3G-inducible master cell line (MCL) development platform. ROSE MCLs equipped with a landing pad facilitated the transcriptional regulation of various effector genes via recombinase-mediated cassette exchange. Long-term investigation revealed that the modular design of genetic payloads and integration sites significantly affected the induction capacity and stability, with ROSE MCLs exhibiting exceptional induction performance. To demonstrate the versatility of our platform, we explored its efficiency for the precise regulation of selection stringency, manufacturing of therapeutic antibodies with tunable expression levels and timing, and transcription factor engineering. Overall, this study demonstrated the effectiveness and reliability of the ROSE platform, highlighting its potential for various biological and biotechnological applications.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":6.8,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717624001137/pdfft?md5=f92842f3df45fa5c66a2dcae4976ed5d&pid=1-s2.0-S1096717624001137-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142145975","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":"A machine learning framework for extracting information from biological pathway images in the literature","authors":"","doi":"10.1016/j.ymben.2024.09.001","DOIUrl":"10.1016/j.ymben.2024.09.001","url":null,"abstract":"<div><p>There have been significant advances in literature mining, allowing for the extraction of target information from the literature. However, biological literature often includes biological pathway images that are difficult to extract in an easily editable format. To address this challenge, this study aims to develop a machine learning framework called the “Extraction of Biological Pathway Information” (EBPI). The framework automates the search for relevant publications, extracts biological pathway information from images within the literature, including genes, enzymes, and metabolites, and generates the output in a tabular format. For this, this framework determines the direction of biochemical reactions, and detects and classifies texts within biological pathway images. Performance of EBPI was evaluated by comparing the extracted pathway information with manually curated pathway maps. EBPI will be useful for extracting biological pathway information from the literature in a high-throughput manner, and can be used for pathway studies, including metabolic engineering.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":6.8,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142133113","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":"A novel step towards the heterologous biosynthesis of paclitaxel: Characterization of T1βOH taxane hydroxylase","authors":"","doi":"10.1016/j.ymben.2024.08.005","DOIUrl":"10.1016/j.ymben.2024.08.005","url":null,"abstract":"<div><p>In the quest for innovative cancer therapeutics, paclitaxel remains a cornerstone in clinical oncology. However, its complex biosynthetic pathway, particularly the intricate oxygenation steps, has remained a puzzle in the decades following the characterization of the last taxane hydroxylase. The high divergence and promiscuity of enzymes involved have posed significant challenges. In this study, we adopted an innovative approach, combining <em>in silico</em> methods and functional gene analysis, to shed light on this elusive pathway. Our molecular docking investigations using a library of potential ligands uncovered TB574 as a potential missing enzyme in the paclitaxel biosynthetic pathway, demonstrating auspicious interactions. Complementary in vivo assays utilizing engineered <em>S. cerevisiae</em> strains as novel microbial cell factory consortia not only validated TB574's critical role in forging the elusive paclitaxel intermediate, T5αAc-1β,10β-diol, but also achieved the biosynthesis of paclitaxel precursors at an unprecedented yield including T5αAc-1β,10β-diol with approximately 40 mg/L. This achievement is highly promising, offering a new direction for further exploration of a novel metabolic engineering approaches using microbial consortia. In conclusion, our study not only furthers study the roles of previously uncharacterized enzymes in paclitaxel biosynthesis but also forges a path for pioneering advancements in the complete understanding of paclitaxel biosynthesis and its heterologous production. The characterization of <em>T1βOH</em> underscores a significant leap forward for future advancements in paclitaxel production using heterologous systems to improve cancer treatment and pharmaceutical production, thereby holding immense promise for enhancing the efficacy of cancer therapies and the efficiency of pharmaceutical manufacturing.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":6.8,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717624001083/pdfft?md5=a00b74a70ccfa7e8b2b2e1d09c950a8f&pid=1-s2.0-S1096717624001083-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142093533","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":"Engineering yeast for high-level production of β-farnesene from sole methanol","authors":"","doi":"10.1016/j.ymben.2024.08.006","DOIUrl":"10.1016/j.ymben.2024.08.006","url":null,"abstract":"<div><p>Methanol, a rich one-carbon feedstock, can be massively produced from CO₂ by the liquid sunshine route, which is helpful to realize carbon neutrality. β-Farnesene is widely used in the production of polymers, surfactants, lubricants, and also serves as a suitable substitute for jet fuel. Constructing an efficient cell factory is a feasible approach for β-farnesene production through methanol biotransformation. Here, we extensively engineered the methylotrophic yeast <em>Ogataea polymorpha</em> for the efficient bio-production of β-farnesene using methanol as the sole carbon source. Our study demonstrated that sufficient supply of precursor acetyl-CoA and cofactor NADPH in an excellent yeast chassis had a 1.3-fold higher β-farnesene production than that of wild-type background strain. Further optimization of the mevalonate pathway and enhancement of acetyl-CoA supply led to a 7-fold increase in β-farnesene accumulation, achieving the highest reported sesquiterpenoids production (14.7 g/L with a yield of 46 mg/g methanol) from one-carbon feedstock under fed-batch fermentation in bioreactor. This study demonstrates the great potential of engineering <em>O. polymorpha</em> for high-level terpenoid production from methanol.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":6.8,"publicationDate":"2024-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142056048","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}