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Genome-Scale Metabolic Reconstruction and Metabolic Versatility of an Obligate Methanotroph Methylococcus Capsulatus Str. Bath

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Genome-Scale Metabolic Reconstruction and Metabolic Versatility of an Obligate Methanotroph Methylococcus Capsulatus Str. Bath bioRxiv preprint doi: https://doi.org/10.1101/349191; this version posted June 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Genome-scale metabolic reconstruction and metabolic versatility of 2 an obligate methanotroph Methylococcus capsulatus str. Bath 3 4 Authors : Ankit Gupta1†, Ahmad Ahmad2†, Dipesh Chothwe1†, Midhun K. Madhu1, 5 Shireesh Srivastava2*, Vineet K. Sharma1* 6 7 Affiliation 8 1 Indian Institute of Science Education and Research, Bhopal, Madhya Pradesh, INDIA 9 2 International Centre For Genetic Engineering And Biotechnology, New Delhi, INDIA 10 †These authors have contributed equally to this work 11 *Corresponding Authors 12 13 Correspondence to be addressed: 14 Dr. Vineet K. Sharma 15 Address: IISER Bhopal, Bhopal By-Pass Road, Bhauri, Bhopal, Madhya Pradesh - 462066, 16 INDIA 17 Telephone: +91-755-6691401 18 Email-id: [email protected] 19 20 Dr. Shireesh Srivastava, Systems Biology for Biofuels Group, ICGEB Campus, Aruna Asaf 21 Ali Marg, New Delhi 110067 22 Telephone: +91-11-26741361 x 450 23 Email: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/349191; this version posted June 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 Abstract 25 The increase in greenhouse gases with high global warming potential such as methane is a 26 matter of concern and requires multifaceted efforts to reduce its emission and increase its 27 mitigation from the environment. Microbes such as methanotrophs can assist in methane 28 mitigation. To understand the metabolic capabilities of methanotrophs, a complete genome- 29 scale metabolic model of an obligate methanotroph, Methylococcus capsulatus str. Bath was 30 reconstructed. The model contains 535 genes, 898 reactions and 784 unique metabolites and 31 is named iMC535. The predictive potential of the model was validated using previously- 32 reported experimental data. The model predicted the Entner-Duodoroff (ED) pathway to be 33 essential for the growth of this bacterium, whereas the Embden-Meyerhof-Parnas (EMP) 34 pathway was found non-essential. The performance of the model was simulated on various 35 carbon and nitrogen sources and found that M. capsulatus can grow on amino acids. The 36 analysis of network topology of the model identified that six amino acids were in the top- 37 ranked metabolic hubs. Using flux balance analysis (FBA), 29% of the metabolic genes were 38 predicted to be essential, and 76 double knockout combinations involving 92 unique genes 39 were predicted to be lethal. In conclusion, we have reconstructed a genome-scale metabolic 40 model of a unique methanotroph Methylococcus capsulatus str. Bath. The model will serve as 41 a knowledge-base for deeper understanding, as a platform for exploring the metabolic 42 potential, and as a tool to engineer this bacterium for methane mitigation and industrial 43 applications. 44 45 Keywords 46 Methanotrophs, Methylococcus capsulatus, Methane mitigation, Metabolic model, 47 Constraint-based model, Metabolic network, Genome-scale 2 bioRxiv preprint doi: https://doi.org/10.1101/349191; this version posted June 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 48 1 Introduction 49 The problem of global warming needs urgent attention and requires a multifaceted approach 50 including the control on the emission of greenhouse gases. Methane is among the most potent 51 greenhouse gases and has 21 times higher global warming potential than CO2 over a 100 year 52 period (MacFarling Meure et al., 2006). Atmospheric methane value has increased 53 significantly from 722 ppb in the preindustrial era (in 1750) to 1,834 ppb in 2015 54 (MacFarling Meure et al., 2006), primarily due to anthropogenic activities such as wastewater 55 treatment plants, manure management, landfills, etc. Efforts are underway to reduce methane 56 emission into the atmosphere and to synthesize other useful chemical commodities from it. 57 However, the conversion of natural methane to desired liquid products has been a 58 technological challenge (Conrado and Gonzalez, 2014;Clomburg et al., 2017). In this 59 scenario, the use of 'methanotrophs', microbes for methane mitigation, seems to be an 60 appealing approach. Methanotrophs are organisms capable of surviving on methane as the 61 sole source of carbon and energy. In addition, methanotrophs can also act as biocatalysts to 62 convert methane to various value-added products such as high-protein feed and liquid 63 biofuels. Furthermore, methanotrophs can also produce industrially important products and 64 biopolymers such as polyhydroxybutarate (PHB), vitamins, carboxylic acids, single cell 65 protein (SCP), and antibiotics using methane as the carbon source (Fei et al., 2014;Strong et 66 al., 2015). Despite these positives, bioconversion through methanotrophs faces multiple 67 challenges of low energy and carbon efficiencies, and cultures with low productivity (Shima 68 et al., 2012;Conrado and Gonzalez, 2014). However, with the advancements in synthetic 69 biology, there is a potential to address these challenges as shown in some recent studies 70 (Shima et al., 2012;Bogorad et al., 2013;de la Torre et al., 2015). 71 72 Methanotrophs perform the task of methane bioconversion by expressing membrane- 73 associated (pMMO) or soluble (sMMO) forms of methane monooxygenase, which can 74 activate methane by oxidizing it to methanol. The methanol is converted to formaldehyde, via 75 the ribulose monophosphate RuMP in type I methanotrophs, or serine or CBB cycle in type II 76 methanotrophs (Shapiro, 2009;Haynes and Gonzalez, 2014). Methylococcus capsulatus Bath 77 (henceforth referred as Mcap), is a gram-negative type-I gammaproteobacterium with ability 78 to grow optimally at high temperatures of around 450C and even in very low oxygen 79 concentrations (Walters et al., 1999). All these properties make this bacterium a unique 80 candidate to study methanotrophs, and to exploit its metabolic potential as cell factories for 81 synthesis of bioproducts. Furthermore, Mcap is among the few known methanotrophs with 82 available genome sequence, which is essential for identifying the metabolic potential of an 83 organism. 84 85 A genome-scale metabolic model (GSMM) is a collection of most of the annotated metabolic 86 reactions in form of a model which makes it possible to simulate cellular metabolic behavior 87 under different conditions. The GSMM are reconstructed and analyzed in Flux Balance 88 Analysis (FBA) using tools such as COBRA (Constraint-Based Reconstruction and Analysis) 89 Toolbox (Becker et al., 2007;Schellenberger et al., 2011). At present, only one validated 90 genome-scale metabolic model for a methanotroph Methylomicrobium buryatense strain 91 5G(B1) is available (de la Torre et al., 2015). For Mcap, the genome-scale biochemical 92 network reconstructions based on automatic annotation pipelines are available in BioCyc and 93 ModelSeed. However, the network information in these automated reconstructions is not 94 curated and contains general reactions which are not specific to Mcap. More importantly, 95 these automated models do not contain information on the reactions utilizing methane as the 96 carbon source, which is one of the most significant properties of methanotrophs. Therefore, 97 these models are insufficient to explore the metabolic landscape of this organism. In this 3 bioRxiv preprint doi: https://doi.org/10.1101/349191; this version posted June 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 98 study, we present a detailed genome-scale metabolic reconstruction ‘iMC535’ for the type I 99 methanotroph Methylococcus capsulatus Bath, constructed using a systematic standard 100 approach (Thiele and Palsson, 2010;Ahmad et al., 2017;Shah, 2017) and was validated using 101 flux balance analysis. 102 103 2 Material And Methods 104 2.1 Model Reconstruction 105 The metabolic reconstruction of Methylococcus capsulatus Bath (Mcap) was carried out 106 using its annotated genome sequence obtained from NCBI. The complete reaction network 107 was built using primary and review literature, biochemical databases such as KEGG 108 (http://www.genome.jp/kegg/) and MetaCyc (http://metacyc.org/), and automated GSMM 109 pipelines such as ModelSEED (Henry et al., 2010) and BioModels (Chelliah et al., 2013). 110 The model was manually constructed on a pathway-by-pathway basis, beginning from the 111 core set of reactions (central metabolic pathways) and expanding it manually to include the 112 annotated reactions and pathways associated with Mcap. Annotations for reaction IDs and 113 compound IDs used in the constructed model were obtained from the KEGG database. The 114 neutral formula for the compounds was obtained from KEGG database or EMBL-ChEBI 115 database. The charge on the metabolites and charged formula were calculated at pH 7.2. All 116 of the reactions included in the network were both elementally and charged balanced and 117 were categorized into either reversible or irreversible. If available, the directionality for each 118 of the reactions was determined from primary literature data,
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