Metabolic Engineering Communications最新文献

{"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":"2025-04-02","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":"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":"2025-04-01","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 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":"2025-03-31","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":"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":"2025-03-30","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":"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":"2025-02-12","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":"2024-12-12","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":"From plastic waste to bioprocesses: Using ethylene glycol from polyethylene terephthalate biodegradation to fuel Escherichia coli metabolism and produce value-added compounds","authors":"Alexandra Balola,&nbsp;Sofia Ferreira,&nbsp;Isabel Rocha","doi":"10.1016/j.mec.2024.e00254","DOIUrl":"10.1016/j.mec.2024.e00254","url":null,"abstract":"<div><div>Polyethylene Terephthalate (PET) is a petroleum-based plastic polymer that, by design, can last decades, if not hundreds of years, when released into the environment through plastic waste leakage. In the pursuit of sustainable solutions to plastic waste recycling and repurposing, the enzymatic depolymerization of PET has emerged as a promising green alternative. However, the metabolic potential of the resulting PET breakdown molecules, such as the two-carbon (C2) molecule ethylene glycol (EG), remains largely untapped. Here, we review and discuss the current state of research regarding existing natural and synthetic microbial pathways that enable the assimilation of EG as a carbon and energy source for <em>Escherichia coli</em>. Leveraging the metabolic versatility of <em>E. coli</em>, we explore the viability of this widely used industrial strain in harnessing EG as feedstock for the synthesis of target value-added compounds <em>via</em> metabolic and protein engineering strategies. Consequently, we assess the potential of EG as a versatile alternative to conventional carbon sources like glucose, facilitating the closure of the loop between the highly available PET waste and the production of valuable biochemicals. This review explores the interplay between PET biodegradation and EG metabolism, as well as the key challenges and opportunities, while offering perspectives and suggestions for propelling advancements in microbial EG assimilation for circular economy applications.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"19 ","pages":"Article e00254"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11667706/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142886049","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":"Cutting-edge developments in plastic biodegradation and upcycling via engineering approaches","authors":"Zeinab Rezaei,&nbsp;Amir Soleimani Dinani,&nbsp;Hamid Moghimi","doi":"10.1016/j.mec.2024.e00256","DOIUrl":"10.1016/j.mec.2024.e00256","url":null,"abstract":"<div><div>The increasing use of plastics has resulted in the production of high quantities of plastic waste that pose a serious risk to the environment. The upcycling of plastics into value-added products offers a potential solution for resolving the plastics environmental crisis. Recently, various microorganisms and their enzymes have been identified for their ability to degrade plastics effectively. Furthermore, many investigations have revealed the application of plastic monomers as carbon sources for bio-upcycling to generate valuable materials such as biosurfactants, bioplastics, and biochemicals. With the advancement in the fields of synthetic biology and metabolic engineering, the construction of high-performance microbes and enzymes for plastic removal and bio-upcycling can be achieved. Plastic valorization can be optimized by improving uptake and conversion efficiency, engineering transporters and enzymes, metabolic pathway reconstruction, and also using a chemo-biological hybrid approach. This review focuses on engineering approaches for enhancing plastic removal and the methods of depolymerization and upcycling processes of various microplastics. Additionally, the major challenges and future perspectives for facilitating the development of a sustainable circular plastic economy are highlighted.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"19 ","pages":"Article e00256"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142759625","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":"Genetically encoded biosensors for the circular plastics bioeconomy","authors":"Micaela Chacón,&nbsp;Neil Dixon","doi":"10.1016/j.mec.2024.e00255","DOIUrl":"10.1016/j.mec.2024.e00255","url":null,"abstract":"<div><div>Current plastic production and consumption routes are unsustainable due to impact upon climate change and pollution, and therefore reform across the entire value chain is required. Biotechnology offers solutions for production from renewable feedstocks, and to aid end of life recycling/upcycling of plastics. Biology sequence/design space is complex requiring high-throughput analytical methods to facilitate the iterative optimisation, design-build, test-learn (DBTL), cycle of Synthetic Biology. Furthermore, genetic regulatory tools can enable harmonisation between biotechnological demands and the physiological constraints of the selected production host. Genetically encoded biosensors offer a solution for both requirements to facilitate the circular plastic bioeconomy. In this review we present a summary of biosensors developed to date reported to be responsive to plastic precursors/monomers. In addition, we provide a summary of the demonstrated and prospective applications of these biosensors for the construction and deconstruction of plastics. Collectively, this review provides a valuable resource of biosensor tools and enabled applications to support the development of the circular plastics bioeconomy.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"19 ","pages":"Article e00255"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11683335/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142907271","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":"Reconstruction and analyses of genome-scale halomonas metabolic network yield a highly efficient PHA production","authors":"Luhui Zhang ,&nbsp;Xinpei Sun ,&nbsp;Jianwen Ye ,&nbsp;QianQian Yuan ,&nbsp;Xin Zhang ,&nbsp;Fei Sun ,&nbsp;Yongpan An ,&nbsp;Yutong Chen ,&nbsp;Yuehui Qian ,&nbsp;Daqian Yang ,&nbsp;Qian Wang ,&nbsp;Miaomiao Gao ,&nbsp;Tao Chen ,&nbsp;Hongwu Ma ,&nbsp;Guoqiang Chen ,&nbsp;Zhengwei Xie","doi":"10.1016/j.mec.2024.e00251","DOIUrl":"10.1016/j.mec.2024.e00251","url":null,"abstract":"<div><div>In pursuit of reliable and efficient industrial microbes, this study integrates cutting-edge systems biology tools with <em>Halomonas bluephagenesis</em> TD01, a robust halophilic bacterium. We generated the complete and annotated circular genome sequence for this model organism, constructed and meticulously curated a genome-scale metabolic network, achieving striking 86.32% agreement with Biolog Phenotype Microarray data and visualize the network via an interactive Electron/Thrift server architecture. We then analyzed the genome-scale network using vertex sampling analysis (VSA) and found that productions of biomass, polyhydroxyalkanoates (PHA), citrate, acetate, and pyruvate are mutually competing. Recognizing the dynamic nature of <em>H. bluephagenesis</em> TD01, we further developed and implemented the hyper-cube-shrink-analysis (HCSA) framework to predict effects of nutrient availabilities and metabolic reactions in the model on biomass and PHA accumulation. We then, based on the analysis results, proposed and validate multi-step feeding strategies tailored to different fermentation stages. This integrated approach yielded remarkable results, with fermentation culminating in a cell dry weight of 100.4 g/L and 70% PHA content, surpassing previous benchmarks. Our findings exemplify the powerful potential of system-level tools in the design and optimization of industrial microorganisms, paving the way for more efficient and sustainable bio-based processes.</div></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"19 ","pages":"Article e00251"},"PeriodicalIF":3.7,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142707018","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}