Abstract
This study reports the discovery of a polyhydroxyalkanoate (PHA) synthase (PhaC) possessing very wide substrate specificity from a mangrove soil metagenome. For the first time, putative PhaCs were identified from a metagenome using next-generation sequencing (NGS) and bioinformatic approaches. High-throughput shotgun metagenomic sequencing was conducted using the Illumina HiSeq 2000 platform. Sequence annotation and bioinformatic analyses were performed using the MG-RAST metagenomic pipeline. Reads annotated as PhaC against the NCBI RefSeq database were retrieved using the MG-RAST RESTful API (Application Programming Interface). PhaC gene sequence assembly was accomplished using the SPAdes assembler. A total of two de novo assembled contigs were subjected to sequence verification. A putative PhaC sequence, “BP-M-CPF4”, was selected for functional assessment by in vivo PHA biosynthesis in a PHA-negative mutant. An artificial stop codon was added at the 3′-end of the incomplete coding gene sequence. This novel PhaC showed very broad substrate specificity with the ability to incorporate six types of PHA monomers, 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 3-hydroxy-4-methylvalerate (3H4MV), 5-hydroxyvalerate (5HV) and 3-hydroxyhexanoate (3HHx) in the presence of suitable precursors. This PHA synthase is suitable for the biosynthesis of PHAs that can be used in various biomedical applications due to its ability to incorporate the lipase-degradable monomer sequences of 4HB and 5HV. This study demonstrates that a functional metagenomic approach using next-generation sequencing can be used to mine novel PHA synthases with interesting substrate specificities from unculturable microorganisms.
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References
Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54(4):450–472
Madison LL, Huisman GW (1999) Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 63(1):21–53
Ayub ND, Tribelli PM, Lopez NI (2009) Polyhydroxyalkanoates are essential for maintenance of redox state in the Antarctic bacterium Pseudomonas sp 14-3 during low temperature adaptation. Extremophiles 13(1):59–66. https://doi.org/10.1007/s00792-008-0197-z
Obruca S, Sedlacek P, Krzyzanek V, Mravec F, Hrubanova K, Samek O, Kucera D, Benesova P, Marova I (2016) Accumulation of poly(3-hydroxybutyrate) helps bacterial cells to survive freezing. PLoS One 11(6):e0157778. https://doi.org/10.1371/journal.pone.0157778
Zinn M, Witholt B, Egli T (2001) Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev 53(1):5–21. https://doi.org/10.1016/S0169-409X(01)00218-6
Jendrossek D, Handrick R (2002) Microbial degradation of polyhydroxyalkanoates. Annu Rev Microbiol 56:403–432. https://doi.org/10.1146/annurev.micro.56.012302.160838
Sudesh K, Iwata T (2008) Sustainability of biobased and biodegradable plastics. Clean (Weinh) 36(5–6):433–442. https://doi.org/10.1002/clen.200700183
Wittenborn EC, Jost M, Wei Y, Stubbe J, Drennan CL (2016) Structure of the catalytic domain of the class I polyhydroxybutyrate synthase from Cupriavidus necator. J Biol Chem 291(48):25264–25277
Kim YJ, Choi SY, Kim J, ** KS, Lee SY, Kim KJ (2017) Structure and function of the N-terminal domain of Ralstonia eutropha polyhydroxyalkanoate synthase, and the proposed structure and mechanisms of the whole enzyme. Biotechnol J 12 (1):1600649. https://doi.org/10.1002/biot.201600649
Kim J, Kim YJ, Choi SY, Lee SY, Kim KJ (2017) Crystal structure of Ralstonia eutropha polyhydroxyalkanoate synthase C-terminal domain and reaction mechanisms. Biotechnol J 12 (1):1600648. https://doi.org/10.1002/biot.201600648
Chek MF, Kim S-Y, Mori T, Arsad H, Samian MR, Sudesh K, Hakoshima T (2017) Structure of polyhydroxyalkanoate (PHA) synthase PhaC from Chromobacterium sp. USM2, producing biodegradable plastics. Sci Rep 7:5312. https://doi.org/10.1038/s41598-017-05509-4
Hyakutake M, Tomizawa S, Mizuno K, Hisano T, Abe H, Tsuge T (2014) A common active site of polyhydroxyalkanoate synthase from Bacillus cereus YB-4 is involved in polymerization and alcoholysis reactions. Appl Microbiol Biotechnol 99(11):4701–4711. https://doi.org/10.1007/s00253-014-6276-4
Rehm BH (2003) Polyester synthases: natural catalysts for plastics. Biochem J 376(Pt 1):15–33. https://doi.org/10.1042/BJ20031254
Koller M, Salerno A, Braunegg G (2013) Polyhydroxyalkanoates: basics, production and applications of microbial biopolyesters. In: Kabasci S (ed) Bio-based plastics: materials and applications. Wiley, Chichester, pp 137–170. https://doi.org/10.1002/9781118676646.ch7
Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59(1):143–169
Lok C (2015) Mining the microbial dark matter. Nature 522(7556):270–273. https://doi.org/10.1038/522270a
Schallmey M, Ly A, Wang C, Meglei G, Voget S, Streit WR, Driscoll BT, Charles TC (2011) Harvesting of novel polyhydroxyalkanaote (PHA) synthase encoding genes from a soil metagenome library using phenotypic screening. FEMS Microbiol Lett 321(2):150–156. https://doi.org/10.1111/j.1574-6968.2011.02324.x
Kapardar RK, Ranjan R, Grover A, Puri M, Sharma R (2010) Identification and characterization of genes conferring salt tolerance to Escherichia coli from pond water metagenome. Bioresour Technol 101(11):3917–3924. https://doi.org/10.1016/j.biortech.2010.01.017
Cheema S, Bassas-Galia M, Sarma PM, Lal B, Arias S (2012) Exploiting metagenomic diversity for novel polyhydroxyalkanoate synthases: production of a terpolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) with a recombinant Pseudomonas putida strain. Bioresour Technol 103(1):322–328. https://doi.org/10.1016/j.biortech.2011.09.098
Cheng JJ, Charles TC (2016) Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases. Appl Microbiol Biotechnol 100(17):7611–7627. https://doi.org/10.1007/s00253-016-7666-6
Michinaka A, Arou J, Onuki M, Satoh H, Mino T (2007) Analysis of polyhydroxyalkanoate (PHA) synthase gene in activated sludge that produces PHA containing 3-hydroxy-2-methylvalerate. Biotechnol Bioeng 96(5):871–880. https://doi.org/10.1002/bit.21085
Ciesielski S, Pokoj T, Klimiuk E (2008) Molecular insight into activated sludge producing polyhydroxyalkanoates under aerobic–anaerobic conditions. J Ind Microbiol Biotechnol 35(8):805–814. https://doi.org/10.1007/s10295-008-0352-7
Foong CP, Lau NS, Deguchi S, Toyofuku T, Taylor TD, Sudesh K, Matsui M (2014) Whole genome amplification approach reveals novel polyhydroxyalkanoate synthases (PhaCs) from Japan trench and Nankai trough seawater. BMC Microbiol 14. https://doi.org/10.1186/s12866-014-0318-z
Parnanen K, Karkman A, Virta M, Eronen-Rasimus E, Kaartokallio H (2015) Discovery of bacterial polyhydroxyalkanoate synthase (PhaC)-encoding genes from seasonal Baltic Sea ice and cold estuarine waters. Extremophiles 19(1):197–206. https://doi.org/10.1007/s00792-014-0699-9
Tai YT, Foong CP, Najimudin N, Sudesh K (2016) Discovery of a new polyhydroxyalkanoate synthase from limestone soil through metagenomic approach. J Biosci Bioeng 121(4):355–364. https://doi.org/10.1016/j.jbiosc.2015.08.008
Meyer F, Paarmann D, D'Souza M, Olson R, Glass EM, Kubal M, Paczian T, Rodriguez A, Stevens R, Wilke A, Wilkening J, Edwards RA (2008) The metagenomics RAST server - a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 9:386. https://doi.org/10.1186/1471-2105-9-386
Scholz MB, Lo CC, Chain PSG (2012) Next generation sequencing and bioinformatic bottlenecks: the current state of metagenomic data analysis. Curr Opin Biotechnol 23(1):9–15. https://doi.org/10.1016/j.copbio.2011.11.013
Sahoo K, Dhal N (2009) Potential microbial diversity in mangrove ecosystems: a review. Indian. J Mar Sci 38(2):249–256
Holguin G, Vazquez P, Bashan Y (2001) The role of sediment microorganisms in the productivity, conservation, and rehabilitation of mangrove ecosystems: an overview. Biol Fertil Soils 33(4):265–278
Gomes NC, Cleary DF, Calado R, Costa R (2011) Mangrove bacterial richness. Commun Integr Biol 4(4):419–423
Rawte T, Padte M, Mavinkurve S (2002) Incidence of marine and mangrove bacteria accumulating polyhydroxyalkanoates on the mid-west coast of India. World J Microbiol Biotechnol 18(7):655–659. https://doi.org/10.1023/A:1016872631403
Doan TV, Nguyen BT (2012) Polyhydroxyalkanoates production by a bacterium isolated from mangrove soil samples collected from Quang Ninh province. J Viet Env 3(2):4
Lau NS, Sam KK, Amirul AA (2017) Genome features of moderately halophilic polyhydroxyalkanoate-producing Yangia sp. CCB-MM3. Stand Genomic Sci 12:12. https://doi.org/10.1186/s40793-017-0232-8
Wilke A, Bischof J, Harrison T, Brettin T, D'Souza M, Gerlach W, Matthews H, Paczian T, Wilkening J, Glass EM, Desai N, Meyer F (2015) A RESTful API for accessing microbial community data for MG-RAST. PLoS Comput Biol 11(1):e1004008. https://doi.org/10.1371/journal.pcbi.1004008
Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19(5):455–477. https://doi.org/10.1089/cmb.2012.0021
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402
Vallone PM, Butler JM (2004) AutoDimer: a screening tool for primer-dimer and hairpin structures. BioTechniques 37(2):226–231
Espah Borujeni A, Channarasappa AS, Salis HM (2013) Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res 42(4):2646–2659
Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166(1):175–176. https://doi.org/10.1016/0378-1119(95)00584-1
Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nat Biotechnol 1(9):784–791
Friedrich B, Hogrefe C, Schlegel HG (1981) Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus. J Bacteriol 147(1):198–205
Ch’ng DH-E, Lee W-H, Sudesh K (2012) Biosynthesis and lipase-catalysed hydrolysis of 4-hydroxybutyrate-containing polyhydroxyalkanoates from Delftia acidovorans. Malays J Microbiol 9(1):33–42
Chuah J-A, Yamada M, Taguchi S, Sudesh K, Doi Y, Numata K (2013) Biosynthesis and characterization of polyhydroxyalkanoate containing 5-hydroxyvalerate units: effects of 5HV units on biodegradability, cytotoxicity, mechanical and thermal properties. Polym Degrad Stab 98(1):331–338. https://doi.org/10.1016/j.polymdegradstab.2012.09.008
Kunasundari B, Sudesh K (2011) Isolation and recovery of microbial polyhydroxyalkanoates. Express Polym Lett 5(7):620–634. https://doi.org/10.3144/expresspolymlett.2011.60
Murugan P, Han L, Gan CY, Maurer FH, Sudesh K (2016) A new biological recovery approach for PHA using mealworm, Tenebrio Molitor. J Biotechnol 239:98–105. https://doi.org/10.1016/j.jbiotec.2016.10.012
Rehm BHA, Steinbüchel A (1999) Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. Int J Biol Macromol 25(1–3):3–19. https://doi.org/10.1016/s0141-8130(99)00010-0
Reddy CSK, Ghai R, Rashmi KVC (2003) Polyhydroxyalkanoates: an overview. Bioresour Technol 87(2):137–146. https://doi.org/10.1016/s0960-8524(02)00212-2
Verlinden RA, Hill DJ, Kenward MA, Williams CD, Radecka I (2007) Bacterial synthesis of biodegradable polyhydroxyalkanoates. J Appl Microbiol 102(6):1437–1449. https://doi.org/10.1111/j.1365-2672.2007.03335.x
Alma'abadi AD, Gojobori T, Mineta K (2015) Marine metagenome as a resource for novel enzymes. Genomics Proteomics Bioinformatics 13(5):290–295. https://doi.org/10.1016/j.gpb.2015.10.001
Zarafeta D, Moschidi D, Ladoukakis E, Gavrilov S, Chrysina ED, Chatziioannou A, Kublanov I, Skretas G, Kolisis FN (2016) Metagenomic mining for thermostable esterolytic enzymes uncovers a new family of bacterial esterases. Sci Rep 6:38886. https://doi.org/10.1038/srep38886
Baud D, Jeffries JWE, Moody TS, Ward JM, Hailes HC (2017) A metagenomics approach for new biocatalyst discovery: application to transaminases and the synthesis of allylic amines. Green Chem 19(4):1134–1143. https://doi.org/10.1039/c6gc02769e
Beck DA, Kalyuzhnaya MG, Malfatti S, Tringe SG, Glavina Del Rio T, Ivanova N, Lidstrom ME, Chistoserdova L (2013) A metagenomic insight into freshwater methane-utilizing communities and evidence for cooperation between the Methylococcaceae and the Methylophilaceae. PeerJ 1:e23. https://doi.org/10.7717/peerj.23
Ying J, Wang H, Bao B, Zhang Y, Zhang J, Zhang C, Li A, Lu J, Li P, Ying J, Liu Q, Xu T, Yi H, Li J, Zhou L, Zhou T, Xu Z, Ni L, Bao Q (2015) Molecular variation and horizontal gene transfer of the homocysteine methyltransferase gene mmuM and its distribution in clinical pathogens. Int J Biol Sci 11(1):11–21. https://doi.org/10.7150/ijbs.10320
Luo C, Rodriguez RL, Konstantinidis KT (2014) MyTaxa: an advanced taxonomic classifier for genomic and metagenomic sequences. Nucleic Acids Res 42(8):e73. https://doi.org/10.1093/nar/gku169
Wang YX, Liu JH, Zhang XX, Chen YG, Wang ZG, Chen Y, Li QY, Peng Q, Cui XL (2009) Fodinicurvata sediminis gen. nov., sp. nov. and Fodinicurvata fenggangensis sp. nov., poly-ß-hydroxybutyrate-producing bacteria in the family Rhodospirillaceae. Int J Syst Evol Microbiol 59(Pt 10):2575–2581. https://doi.org/10.1099/ijs.0.009340-0
Fukui T, Kichise T, Yoshida Y, Doi Y (1997) Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxy-heptanoate) terpolymers by recombinant Alcaligenes eutrophus. Biotechnol Lett 19(11):1093–1097. https://doi.org/10.1023/a:1018436426032
Bhubalan K, Kam YC, Yong KH, Sudesh K (2010) Cloning and expression of the PHA synthase gene from a locally isolated Chromobacterium sp. USM2. Malays. J Microbiol 6(1):81–90. 10.21161/mjm.21809
Chen JY, Song G, Chen GQ (2006) A lower specificity PhaC2 synthase from Pseudomonas stutzeri catalyses the production of copolyesters consisting of short-chain-length and medium-chain-length 3-hydroxyalkanoates. Anton Leeuw Int J G 89(1):157–167. https://doi.org/10.1007/s10482-005-9019-9
Acknowledgements
This study was supported by the Long-Term Research Grant Scheme (USM) and the USM-RIKEN Centre of Aging Sciences (URICAS). We are also grateful to Dr. Kovach ME for his kind gift of the pBBR1MCS broad-host-range vector derivatives used in this study. CPF gratefully acknowledges the MyPhD scholarship program from the Ministry of Higher Education Malaysia.
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Foong, C.P., Lakshmanan, M., Abe, H. et al. A novel and wide substrate specific polyhydroxyalkanoate (PHA) synthase from unculturable bacteria found in mangrove soil. J Polym Res 25, 23 (2018). https://doi.org/10.1007/s10965-017-1403-4
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DOI: https://doi.org/10.1007/s10965-017-1403-4