Abstract
Methylobacterium sp. CLZ was isolated from soil contaminated with chemical wastewater. This strain simultaneously synthesizes Pyrroloquinoline quinone (PQQ), Coenzyme Q10 (CoQ10), and carotenoids by utilizing methanol as a carbon source. Comparative genomic analysis was performed for five Methylobacterium strains. As per the outcomes, the Methylobacterium CLZ strain showed the smallest genome size and the lowest number of proteins. Thus, it can serve as an ideal cell model for investigating the biological process of Methylobacterium and constructing genetically engineered Methylobacterium. The Methylobacterium CLZ strain’s pqqL gene, which does not occur in other Methylobacterium strains but plays a crucial role in PQQ synthesis. This was a surprising finding for the study of PQQ biosynthesis in Methylobacterium. Methylobacterium sp. NI91 strain was generated by random mutagenesis of CLZ strain, and NI91 strain showed a 72.44% increase in PQQ yield. The mutation in the mxaJ gene involved in the methanol dehydrogenase (MDH) synthesis was identified through comparative genomic analysis of the whole genome of mutant strain NI91 and wild-type strain CLZ. The mxaJ gene was found to be upregulated in the NI91 strain. Thus, the up-regulation of the mxaJ gene could be correlated with the high yield of PQQ, and it could provide valuable clues for strain engineering to improve PQQ production.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
Data is contained within the article or supplementary material.
References
Anthony C (1983) The biochemistry of methylotrophs, vol 160. Academic Press, London
Anthony C (2000) Methanol dehydrogenase, a PQQ-containing quinoprotein dehydrogenase. Subcell Biochem 35:73–117. https://doi.org/10.1007/0-306-46828-x_3
Anthony C (2004) The quinoprotein dehydrogenases for methanol and glucose. Arch Biochem Biophys 428:2–9. https://doi.org/10.1016/j.abb.2004.03.038
Ashburner M et al (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25:D25-29. https://doi.org/10.1038/75556
Carreto L, Eiriz MF, Gomes AC, Pereira PM, Schuller D, Santos MAS (2008) Comparative genomics of wild type yeast strains unveils important genome diversity. BMC Genomics 9:524–524. https://doi.org/10.1186/1471-2164-9-524
Chistoserdova L, Chen SW, Lapidus A, Lidstrom ME (2003) Methylotrophy in Methylobacterium extorquens AM1 from a genomic point of view. J Bacteriol 185:2980–2987. https://doi.org/10.1128/jb.185.10.2980-2987.2003
Ciufo S et al (2018) Using average nucleotide identity to improve taxonomic assignments in prokaryotic genomes at the NCBI. Int J Syst Evol Microbiol 68:2386–2392. https://doi.org/10.1099/ijsem.0.002809
Felton LM, Anthony C (2005) Biochemistry: role of PQQ as a mammalian enzyme cofactor? Nature 433:11–12. https://doi.org/10.1038/nature03322
Finn RD et al (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44:D279-285. https://doi.org/10.1093/nar/gkv1344
Galperin MY, Makarova KS, Wolf YI, Koonin EV (2015) Expanded microbial genome coverage and improved protein family annotation in the COG database. Nucleic Acids Res 43:D261-269. https://doi.org/10.1093/nar/gku1223
Green PN, Ardley JK (2018) Review of the genus Methylobacterium and closely related organisms: a proposal that some Methylobacterium species be reclassified into a new genus, Methylorubrum gen. nov. Int J Syst Evol Microbiol 68:2727–2748. https://doi.org/10.1099/ijsem.0.002856
Haft DH, Selengut JD, Richter RA, Harkins D, Basu MK, Beck E (2013) TIGRFAMs and genome properties in 2013. Nucleic Acids Res 41:D387-395. https://doi.org/10.1093/nar/gks1234
Kalvari I et al (2018) Rfam 13.0: shifting to a genome-centric resource for non-coding RNA families. Nucleic Acids Res 46:335–342. https://doi.org/10.1093/nar/gkx1038
Kanehisa M, Goto S, Sato Y, Kawashima M, Furumichi M, Tanabe M (2013) Data, information, knowledge and principle: back to metabolism in KEGG. Nuclc Acids Res 42:D199-205. https://doi.org/10.1093/nar/gkt1076
Keltjens JT, Pol A, Reimann J, Op den Camp HJ (2014) PQQ-dependent methanol dehydrogenases: rare-earth elements make a difference. Appl Microbiol Biotechnol 98:6163–6183. https://doi.org/10.1007/s00253-014-5766-8
Latham JA, Iavarone AT, Barr I, Juthani PV, Klinman JP (2015) PqqD is a novel peptide chaperone that forms a ternary complex with the radical S-adenosylmethionine protein PqqE in the pyrroloquinoline quinone biosynthetic pathway. J Biol Chem 290:12908–12918. https://doi.org/10.1074/jbc.M115.646521
Li H, Zeng W, Zhou J (2018) High-throughput screening of Methylobacterium extorquens for high production of pyrroloquinoline quinone. Chin J Biotechnol 34:794–802. https://doi.org/10.13345/j.cjb.170440
Martins A, Latham J, Martel P, Barr I, Iavarone A, Klinman J (2019) A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone. J Biol Chem 294:jbc.RA.119009684. https://doi.org/10.1074/jbc.RA119.009684
Misra HS, Khairnar NP, Barik A, Indira Priyadarsini K, Mohan H, Apte SK (2004) Pyrroloquinoline-quinone: a reactive oxygen species scavenger in bacteria. FEBS Lett 578:26–30. https://doi.org/10.1016/j.febslet.2004.10.061
Misra HS, Rajpurohit YS, Khairnar NP (2012) Pyrroloquinoline-quinone and its versatile roles in biological processes. J Biosci 37:313–325. https://doi.org/10.1007/s12038-012-9195-5
Myung Choi J, Cao TP, Wouk Kim S, Ho Lee K, Haeng Lee S (2017) MxaJ structure reveals a periplasmic binding protein-like architecture with unique secondary structural elements. Proteins 85:1379–1386. https://doi.org/10.1002/prot.25283
O’Leary NA et al (2016) Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res 44:D733-745. https://doi.org/10.1093/nar/gkv1189
Ramamoorthi R (1994) Genetics and regulation of pyrroloquinoline (PQQ) Biosynthesis in the Methylotrophic bacterium methylobacterium extorquens AM1. Dissertation, California Institute of Technology
Richter M, Rosselló-Móra R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci 106:19126–19131. https://doi.org/10.1073/pnas.0906412106
Richter M, Rosselló-Móra R, Oliver Glöckner F, Peplies J (2015) JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 32:929–931. https://doi.org/10.1093/bioinformatics/btv681
Shen Y-Q, Bonnot F, Imsand E, RoseFigura J, Sjölander K, Klinman J (2012) Distribution and properties of the genes encoding the biosynthesis of the bacterial cofactor, pyrroloquinoline quinone. Biochemistry 51:2265–2275. https://doi.org/10.1021/bi201763d
Singh VK, Mangalam AK, Dwivedi S, Naik S (1998) Primer premier: program for design of degenerate primers from a protein sequence. Biotechniques 24:318–319. https://doi.org/10.2144/98242pf02
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. https://doi.org/10.1093/molbev/mst197
Toyama H, Chistoserdova L, Lidstrom ME (1997) Sequence analysis of pqq genes required for biosynthesis of pyrroloquinoline quinone in Methylobacterium extorquens AM1 and the purification of a biosynthetic intermediate. Microbiology (reading) 143:595–602. https://doi.org/10.1099/00221287-143-2-595
Turlin E, Gasser F, Biville F (1996) Sequence and functional analysis of an Escherichia coli DNA fragment able to complement pqqE and pqqF mutants from Methylobacterium organophilum. Biochimie 78:823–831. https://doi.org/10.1016/s0300-9084(97)84334-9
Wang K, Li M, Hakonarson H (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38:e164. https://doi.org/10.1093/nar/gkq603
Whitaker WB, Sandoval NR, Bennett RK, Fast AG, Papoutsakis ET (2015) Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization. Curr Opin Biotechnol 33:165–175. https://doi.org/10.1016/j.copbio.2015.01.007
Xiong X, Yang L, Han X, Wang J, Zhang W (2010) Knockout and function analysis of pqqL gene in Escherichia coli. Wei sheng wu xue bao = Acta microbiologica Sinica 50:1380. https://doi.org/10.13343/j.cnki.wsxb.2010.10.009
Xu L et al (2019) OrthoVenn2: a web server for whole-genome comparison and annotation of orthologous clusters across multiple species. Nucleic Acids Res 47:W52–W58. https://doi.org/10.1093/nar/gkz333
Yongfang Z, Ning X, Yingshan W, Xiao H (1995) Determination of a new prothetic group PQQ using noenzymatic system. J Wuhan Univ (Nat Sci Ed) 41:777–780. https://doi.org/10.1186/1471-2105-10-421
Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J (2017) Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol 67:1613–1617. https://doi.org/10.1099/ijsem.0.001755
Zeng W, Fang F, Liu S, Du G, Chen J, Zhou J (2016) Comparative genomics analysis of a series of Yarrowia lipolytica WSH-Z06 mutants with varied capacity for alpha-ketoglutarate production. J Biotechnol 239:76–82. https://doi.org/10.1016/j.jbiotec.2016.10.008
Zhang M, Lidstrom ME (2003) Promoters and transcripts for genes involved in methanol oxidation in Methylobacterium extorquens AM1. Microbiology 149:1033–1040. https://doi.org/10.1099/mic.0.26105-0
Zheng YJ, Xia Z, Chen Z, Mathews FS, Bruice TC (2001) Catalytic mechanism of quinoprotein methanol dehydrogenase: a theoretical and x-ray crystallographic investigation. Proc Natl Acad Sci USA 98:432–434. https://doi.org/10.1073/pnas.021547498
Funding
This work was supported by the National Natural Science Foundation of China (Grant Number 21366028) and China Postdoctoral Science Foundation (Grant Number 2017M613301XB).
Author information
Authors and Affiliations
Contributions
HLZ and XB conceived and designed the research. CLZ and XJC and YPW conducted the experiments. HLZ and CLZ contributed new reagents or analytical tools. CLZ analyzed the data. HLZ and CLZ wrote the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
11274_2021_3068_MOESM3_ESM.eps
Supplementary file3 (EPS 1268 kb). Figure S1. Expression of internal reference genes. The median cycle threshold values for each gene in different samples and the values at the top of the histogram were obtained by NormFinder analysis of CT values of candidate internal reference genes at different culture periods of CLZ and NI91 samples
11274_2021_3068_MOESM5_ESM.eps
Supplementary file5 (EPS 799 kb). Figure S3. Global alignment between the genomes of CLZ and NI91. Alignment of the wild-type strain CLZ and the mutant strain NI91 by MUMmer software
Rights and permissions
About this article
Cite this article
Zhao, C., Wan, Y., Cao, X. et al. Comparative genomics and analysis of the mechanism of PQQ overproduction in Methylobacterium. World J Microbiol Biotechnol 37, 100 (2021). https://doi.org/10.1007/s11274-021-03068-5
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-021-03068-5