Methylocystis suflitae as a promising industrial workhorse for methane-based polyhydroxyalkanoate production

Abstract

Growing interest in the abatement of greenhouse gases has spurred a surge in research within the field of methanotrophy in recent decades. Certain methanotrophic organisms exhibit the unique ability for the production of biodegradable polymers known as polyhydroxyalkanoates (PHAs) using methane as their solitary carbon source. In this study, we harnessed the capability of Methylocystis suflitae, a Type II methanotroph, for the production of PHA from methane. The genome analysis unveiled the presence of four paralogs of PHA synthase gene in Methylocystis suflitae. Subsequently, we elucidated the catalytic sites of each PHA synthase using protein modeling and molecular docking. Both hydroxybutyrate and hydroxydodecanoate demonstrated the highest docking energies among all the tested substrates, recording at − 7.5, and − 7.8 kcal/mol, respectively. The capability of Methylocystis suflitae to synthesize polyhydroxybutyrate (PHB) was evaluated by analyzing the FTIR spectrum, revealing the characteristic carbonyl (C=O) peak at 1723 cm⁻¹. The study included the optimization of the substrate-to-electron acceptor ratio to optimize PHA productivity. Notably, the organism exhibited a productivity value of 11.90±1.34 mg PHA/L/hr. This higher productivity holds significant promise for industrial PHA production, particularly in scenarios where achieving sufficiently high dissolved methane concentrations in industrial fermenters is inherently challenging, potentially enabling more efficient PHA production. Additionally, we determined the melting temperature for PHB produced by Methylocystis suflitae which closely aligns with the standards of commercial-grade PHB, at around 188 °C.

Keywords

Polyhydroxybutyrate / Methanotroph / Docking / Biopolymer / PHA synthase / Characterization

References

[1] Raza ZA, Abid S, Banat IM. Polyhydroxyalkanoates: characteristics, production, recent developments and applications. Int Biodeterior Biodegrad. 2018, 126: 45-56

[2] Singh AK, et al. . Biomedical applications of microbially engineered polyhydroxyalkanoates: An insight into recent advances, bottlenecks, and solutions. Appl Microbiol Biotechnol. 2019, 103: 2007-2032

[3] López JC, et al. . CH 4-based polyhydroxyalkanoate production: a step further towards a sustainable bioeconomy. Biotechnological Applications of Polyhydroxyalkanoates. 2019, Singapore, Springer Singapore: 283321

[4] Samanta D, et al. . Methane monooxygenases: their regulations and applications in biofuel production. Microbes for sustainable development and bioremediation. 2019CRC Press: 187206

[5] Samanta D, et al. . Integrating metabolomics and whole genome sequencing to elucidate the metabolic pathways in Methylosinus trichosporium OB3b. Syst Microbiol Biomanufacturing. 2023

[6] Dalal J, Lal B. Microbial polyhydroxyalkanoates: Current status and future prospects. High Value Fermentation Products: Human Welfare II; Saran, S., Babu, V., Chaubey, A., Eds, 2019; pp. 351–387.

[7] Yadav B, et al. . Polyhydroxyalkanoate production from feedstocks: technological advancements and techno-economic analysis in reference to circular bioeconomy. Biomass, biofuels, biochemicals. 2021Elsevier: 477513

[8] Rai PK, et al. . Micro-and nano-plastic pollution: Behavior, microbial ecology, and remediation technologies. J Clean Prod. 2021, 291: 125240

[9] Levett I, et al. . Techno-economic assessment of poly-3-hydroxybutyrate (PHB) production from methane—the case for thermophilic bioprocessing. J Environ Chem Eng. 2016, 4(4): 3724-3733

[10] Liu L-Y, et al. . Biological conversion of methane to polyhydroxyalkanoates: current advances, challenges, and perspectives. Environ Sci Ecotechnol. 2020, 2: 100029

[11] Samanta D, Sani RK. Methane oxidation via chemical and biological methods: challenges and solutions. Methane. 2023, 2(3): 279-303

[12] Bhola S, et al. . Established and emerging producers of PHA: rSedefining the possibility. Appl Biochem Biotechnol. 2021, 193: 3812-3854

[13] Zheng Y, et al. . Engineering biosynthesis of polyhydroxyalkanoates (PHA) for diversity and cost reduction. Metab Eng. 2020, 58: 82-93

[14] Zúñiga C, et al. . Production of poly-β-hydroxybutyrate (PHB) by Methylobacterium organophilum isolated from a methanotrophic consortium in a two-phase partition bioreactor. J Hazard Mater. 2011, 190(1–3): 876-882

[15] Sundstrom ER. Selection and optimization strategies for production of polyhydroxybutyrate (phb) in methanotrophic bacteria. 2013: Stanford University.

[16] Gupta D, et al. . iTRAQ-based quantitative proteomics reveals insights into metabolic and molecular responses of glucose-grown cells of Rubrivivax benzoatilyticus JA2. J Proteomics. 2019, 194: 49-59

[17] Mifune J, Nakamura S, Fukui T. Engineering of pha operon on Cupriavidus necator chromosome for efficient biosynthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Polym Degrad Stab. 2010, 95(8): 1305-1312

[18] Zhang Z. A novel RNA extraction method for cyanobacteria. 2012: Louisiana State University and Agricultural & Mechanical College.

[19] Zhang S, et al. . Mechanism of the polymerization reaction initiated and catalyzed by the polyhydroxybutyrate synthase of Ralstonia e utropha. Biomacromol. 2003, 4(3): 504-509

[20] Mezzolla V, D’Urso OF, Poltronieri P. Role of PhaC type I and type II enzymes during PHA biosynthesis. Polymers. 2018, 10(8): 910

[21] Nguyen DTN, et al. . Type II methanotrophs: a promising microbial cell-factory platform for bioconversion of methane to chemicals. Biotechnol Adv. 2021, 47: 107700

[22] Zhang T, et al. . Poly-β-hydroxybutyrate production by Methylosinus trichosporium OB3b at different gas-phase conditions. Iran J Biotechnol. 2019, 17(1): 10-16

[23] Garner CT, et al. . Methylocystis suflitae sp. nov., a novel type II methanotrophic bacterium isolated from landfill cover soil. Int J Syst Evol Microbiol. 2024, 74(1): 006239

[24] Aziz RK, et al. . The RAST Server: rapid annotations using subsystems technology. BMC Genom. 2008, 9: 1-15

[25] Fiser A, Šali A. Modeller: generation and refinement of homology-based protein structure models. Methods in enzymology. 2003Elsevier: 461-491

[26] Mahato D, et al. . A systems biology approach for elucidating the interaction of curcumin with Fanconi anemia FANC G protein and the key disease targets of leukemia. J Recept Signal Transduct. 2017, 37(3): 276-282

[27] Johnson M, et al. . NCBI BLAST: a better web interface. Nucleic Acids Res. 2008, 36(suppl_2): W5-W9

[28] Tripathi AK, et al. . Identification of AHL Synthase in Desulfovibrio vulgaris Hildenborough using an in-silico methodology. Catalysts. 2023, 13(2): 364

[29] Samanta D, et al. . Enhancement of methane catalysis rates in Methylosinus trichosporium OB3b. Biomolecules. 2022, 12(4): 560

[30] Fan H, Mark AE. Refinement of homology-based protein structures by molecular dynamics simulation techniques. Protein Sci. 2004, 13(1): 211-220

[31] Williams CJ, et al. . MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 2018, 27(1): 293-315

[32] Krishnaraj RN, et al. . Bioprospecting of thermostable cellulolytic enzymes through modeling and virtual screening method. Can J Biotechnol. 2017, 1(1): 19

[33] Navanietha Krishnaraj R, Samanta D, Sani RK. Computational Nanotechnology: a tool for screening therapeutic nanomaterials against Alzheimer’s disease. Computational modeling of drugs against Alzheimer’s disease. 2018, NY, Springer: 613-635

[34] Vina A. Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading Trott, Oleg; Olson. Arthur J J Comput Chem. 2010, 31(2): 455-461

[35] Dallakyan S, Olson AJ. Small-molecule library screening by docking with PyRx. Chemical biology methods and protocols. 2015, New York, Springer: 243250

[36] Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021, 38(7): 3022-3027

[37] Rai R, et al. . Biochemical unravelling of the endoxylanase activity in a bifunctional GH39 enzyme cloned and expressed from thermophilic Geobacillus sp. WSUCF1. Int J Biol Macromol. 2024, 257: 128679

[38] Samanta D, et al. . Genetical and biochemical basis of methane monooxygenases of Methylosinus trichosporium OB3b in response to copper. Methane. 2024, 3(1): 103-121

[39] Tripathi AK, et al. . Transcriptomics and functional analysis of Copper stress response in the sulfate-reducing bacterium Desulfovibrio alaskensis G20. Int J Mol Sci. 2022, 23(3): 1396

[40] Paul BG, et al. . Methane-oxidizing bacteria shunt carbon to microbial mats at a marine hydrocarbon seep. Front Microbiol. 2017, 8: 186

[41] Govil T, et al. . Lignocellulosic feedstock: a review of a sustainable platform for cleaner production of nature’s plastics. J Clean Prod. 2020, 270: 122521

[42] Fitch CA, et al. . Arginine: Its pKa value revisited. Protein Sci. 2015, 24(5): 752-761

[43] Padermshoke A, et al. . Melting behavior of poly (3-hydroxybutyrate) investigated by two-dimensional infrared correlation spectroscopy. Spectrochim Acta Part A Mol Biomol Spectrosc. 2005, 61(4): 541-550

[44] Bhagowati P, et al. . Production, optimization and characterization of polyhydroxybutyrate, a biodegradable plastic by Bacillus spp. Biosci Biotechnol Biochem. 2015, 79(9): 1454-1463

[45] Addison M. Identification and functional characterisation of novel viral pseudomurein ensopeptidase proteins from methanogens. University of Otago. 2023;

[46] Alvarez-Santullano N, et al. . Genome-wide metabolic reconstruction of the synthesis of polyhydroxyalkanoates from sugars and fatty acids by Burkholderia sensu lato species. Microorganisms. 2021, 9(6): 1290

[47] Jin H, Nikolau BJ. Role of genetic redundancy in polyhydroxyalkanoate (PHA) polymerases in PHA biosynthesis in Rhodospirillum rubrum. J Bacteriol. 2012, 194(20): 5522-5529

[48] Quelas JI, et al. . Analysis of two polyhydroxyalkanoate synthases in Bradyrhizobium japonicum USDA 110. J Bacteriol. 2013, 195(14): 3145-3155

[49] Pohlmann A, et al. . Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nat Biotechnol. 2006, 24(10): 1257-1262

[50] Yin J, et al. . Effects of chromosomal gene copy number and locations on polyhydroxyalkanoate synthesis by Escherichia coli and Halomonas sp.. Appl Microbiol Biotechnol. 2015, 99: 5523-5534

[51] Rodge SP, et al. . A polyhydroxyalkanoate synthesised by halophilic archaeon Natrialba swarupiae. Environ Sci: Adv. 2023, 2(7): 990-1000

[52] Zhang W, et al. . Inhibitors of polyhydroxyalkanoate (PHA) synthases: synthesis, molecular docking, and implications. ChemBioChem. 2015, 16(1): 156-166

[53] Nawaz M, et al. . Structural elucidation, molecular docking, α-amylase and α-glucosidase inhibition studies of 5-amino-nicotinic acid derivatives. Bmc Chem. 2020, 14: 1-11

[54] Yamin R, et al. . Identifying plant-derived antiviral alkaloids as dual inhibitors of SARS-CoV-2 main protease and spike glycoprotein through computational screening. Front Pharmacol. 2024, 15: 1369659

[55] Neoh SZ, et al. . Polyhydroxyalkanoate synthase (PhaC): The key enzyme for biopolyester synthesis. Curr Res Biotechnol. 2022, 4: 87-101

[56] Lim H, et al. . Identification of regions affecting enzyme activity, substrate binding, dimer stabilization and polyhydroxyalkanoate (PHA) granule morphology in the PHA synthase of Aquitalea sp. USM4. Int J Biol Macromol. 2021, 186: 414-423

[57] Vuong P, et al. . Developing bioprospecting strategies for bioplastics through the large-scale mining of microbial genomes. Front Microbiol. 2021, 12: 697309

[58] Wittenborn EC, et al. . Structure of the catalytic domain of the class I polyhydroxybutyrate synthase from Cupriavidus necator. J Biol Chem. 2016, 291(48): 25264-25277

[59] Chek MF, et al. . Structure of polyhydroxyalkanoate (PHA) synthase PhaC from Chromobacterium sp. USM2, producing biodegradable plastics. Sci Rep. 2017, 7(1): 5312

[60] Satoh K, et al. . Versatile aliphatic polyester biosynthesis system for producing random and block copolymers composed of 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates using the sequence-regulating polyhydroxyalkanoate synthase PhaCAR. Microb Cell Fact. 2022, 21(1): 84

[61] Al Temimi AH, et al. . The nucleophilic amino group of lysine is central for histone lysine methyltransferase catalysis. Commun Chem. 2019, 2(1): 112

[62] Samanta D, et al. . From genome to evolution: investigating type II methylotrophs using a pangenomic analysis. Msystems. 2024

[63] Huang G, et al. . The hydride transfer process in NADP-dependent methylene-tetrahydromethanopterin dehydrogenase. J Mol Biol. 2020, 432(7): 2042-2054

[64] Vorholt JA, et al. . The NADP-dependent methylene tetrahydromethanopterin dehydrogenase in Methylobacterium extorquens AM1. J Bacteriol. 1998, 180(20): 5351-5356

[65] Fergala AM. Development of methane-utilizing mixed cultures for sustainable biopolymers production from wastewater treatment streams. 2019;

[66] Myung J, et al. . Expanding the range of polyhydroxyalkanoates synthesized by methanotrophic bacteria through the utilization of omega-hydroxyalkanoate co-substrates. AMB Express. 2017, 7(1): 1-10

[67] Amabile C, et al. . Sustainable process for the production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) from renewable resources: a simulation study. ACS Sustain Chem Eng. 2022, 10(43): 14230-14239

[68] Cal AJ, et al. . Methanotrophic production of polyhydroxybutyrate-co-hydroxyvalerate with high hydroxyvalerate content. Int J Biol Macromol. 2016, 87: 302-307

Funding

National Science Foundation(1736255)

RIGHTS & PERMISSIONS

Jiangnan University