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Title: Electroporation-Based Genetic Manipulation in Type I Methanotrophs

Abstract

Methane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. In order to facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs: Methylomicrobium buryatense 5GB1C, Methylomonas sp. strain LW13, and Methylobacter tundripaludum 21/22. We further developed this technique in M. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency of M. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) and sacB counterselection systems were employed to performmore » marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation of M. buryatense and other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains.« less

Authors:
 [1];  [2];  [2];  [2];  [3]
  1. Univ. of Washington, Seattle, WA (United States). Dept. of Chemical Engineering; Nanjing Agricultural Univ. (China). College of Life Sciences and Dept. of Microbiology
  2. Univ. of Washington, Seattle, WA (United States). Dept. of Chemical Engineering
  3. Univ. of Washington, Seattle, WA (United States). Dept. of Chemical Engineering and Dept. of Microbiology
Publication Date:
Research Org.:
Univ. of Washington, Seattle, WA (United States)
Sponsoring Org.:
USDOE Advanced Research Projects Agency - Energy (ARPA-E); National Basic Research Program of China; China Scholarship Council (CSC)
OSTI Identifier:
1470733
Grant/Contract Number:  
AR0000350; 2015CB150505; 201306855020
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 82; Journal Issue: 7; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Yan, Xin, Chu, Frances, Puri, Aaron W., Fu, Yanfen, and Lidstrom, Mary E. Electroporation-Based Genetic Manipulation in Type I Methanotrophs. United States: N. p., 2016. Web. doi:10.1128/AEM.03724-15.
Yan, Xin, Chu, Frances, Puri, Aaron W., Fu, Yanfen, & Lidstrom, Mary E. Electroporation-Based Genetic Manipulation in Type I Methanotrophs. United States. doi:10.1128/AEM.03724-15.
Yan, Xin, Chu, Frances, Puri, Aaron W., Fu, Yanfen, and Lidstrom, Mary E. Fri . "Electroporation-Based Genetic Manipulation in Type I Methanotrophs". United States. doi:10.1128/AEM.03724-15. https://www.osti.gov/servlets/purl/1470733.
@article{osti_1470733,
title = {Electroporation-Based Genetic Manipulation in Type I Methanotrophs},
author = {Yan, Xin and Chu, Frances and Puri, Aaron W. and Fu, Yanfen and Lidstrom, Mary E.},
abstractNote = {Methane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. In order to facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs: Methylomicrobium buryatense 5GB1C, Methylomonas sp. strain LW13, and Methylobacter tundripaludum 21/22. We further developed this technique in M. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency of M. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) and sacB counterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation of M. buryatense and other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains.},
doi = {10.1128/AEM.03724-15},
journal = {Applied and Environmental Microbiology},
number = 7,
volume = 82,
place = {United States},
year = {2016},
month = {1}
}

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Works referenced in this record:

Metabolic engineering in methanotrophic bacteria
journal, May 2015


REBASE—a database for DNA restriction and modification: enzymes, genes and genomes
journal, November 2014

  • Roberts, Richard J.; Vincze, Tamas; Posfai, Janos
  • Nucleic Acids Research, Vol. 43, Issue D1, p. D298-D299
  • DOI: 10.1093/nar/gku1046

One-Step Cloning and Chromosomal Integration of DNA
journal, May 2013

  • St-Pierre, François; Cui, Lun; Priest, David G.
  • ACS Synthetic Biology, Vol. 2, Issue 9
  • DOI: 10.1021/sb400021j

Draft Genome Sequence of Methylomicrobium buryatense Strain 5G, a Haloalkaline-Tolerant Methanotrophic Bacterium
journal, June 2013


Enrichment, Isolation and Some Properties of Methane-utilizing Bacteria
journal, May 1970

  • Whittenbury, R.; Phillips, K. C.; Wilkinson, J. F.
  • Journal of General Microbiology, Vol. 61, Issue 2
  • DOI: 10.1099/00221287-61-2-205

Taxonomic Characterization of New Alkaliphilic and Alkalitolerant Methanotrophs from Soda Lakes of the Southeastern Transbaikal Region and description of Methylomicrobium buryatense sp.nov.
journal, January 2001

  • Kaluzhnaya, Marina; Khmelenina, Valentina; Eshinimaev, Bulat
  • Systematic and Applied Microbiology, Vol. 24, Issue 2
  • DOI: 10.1078/0723-2020-00028

REBASE—a database for DNA restriction and modification: enzymes, genes and genomes
journal, October 2009

  • Roberts, Richard J.; Vincze, Tamas; Posfai, Janos
  • Nucleic Acids Research, Vol. 38, Issue suppl_1
  • DOI: 10.1093/nar/gkp874

Bioconversion of natural gas to liquid fuel: Opportunities and challenges
journal, May 2014


Methanobactin and MmoD work in concert to act as the ‘copper-switch’ in methanotrophs: Copper-switch in methanotrophy
journal, May 2013


Replication and recombination functions associated with the yeast plasmid, 2μ circle
journal, September 1980


Methane as a Resource: Can the Methanotrophs Add Value?
journal, March 2015

  • Strong, P. J.; Xie, S.; Clarke, W. P.
  • Environmental Science & Technology, Vol. 49, Issue 7
  • DOI: 10.1021/es504242n

Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously
journal, January 2004


Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria
journal, August 2001


Draft Genome Sequences of Gammaproteobacterial Methanotrophs Isolated from Lake Washington Sediment: TABLE 1 
journal, March 2015

  • Kalyuzhnaya, Marina G.; Lamb, Andrew E.; McTaggart, Tami L.
  • Genome Announcements, Vol. 3, Issue 2
  • DOI: 10.1128/genomeA.00103-15

Broad-Host-Range cre-lox System for Antibiotic Marker Recycling in Gram-Negative Bacteria
journal, November 2002

  • Marx, Christopher J.; Lidstrom, Mary E.
  • BioTechniques, Vol. 33, Issue 5
  • DOI: 10.2144/02335rr01

Two isozymes of particulate methane monooxygenase with different methane oxidation kinetics are found in Methylocystis sp. strain SC2
journal, July 2008

  • Baani, M.; Liesack, W.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 29
  • DOI: 10.1073/pnas.0702643105

Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

Genetic Tools for the Industrially Promising Methanotroph Methylomicrobium buryatense
journal, December 2014

  • Puri, Aaron W.; Owen, Sarah; Chu, Frances
  • Applied and Environmental Microbiology, Vol. 81, Issue 5
  • DOI: 10.1128/AEM.03795-14

In Vivo Assessment of Drug Efficacy against Mycobacterium abscessus Using the Embryonic Zebrafish Test System
journal, May 2014

  • Bernut, Audrey; Le Moigne, Vincent; Lesne, Tiffany
  • Antimicrobial Agents and Chemotherapy, Vol. 58, Issue 7
  • DOI: 10.1128/AAC.00142-14

Cre/lox System and PCR-Based Genome Engineering in Bacillus subtilis
journal, July 2008

  • Yan, X.; Yu, H. -J.; Hong, Q.
  • Applied and Environmental Microbiology, Vol. 74, Issue 17
  • DOI: 10.1128/AEM.01156-08

Methanotrophic bacteria.
journal, January 1996


Applications of a colorimetric plate assay for soluble methane monooxygenase activity.
journal, January 1992


    Works referencing / citing this record:

    Biological conversion of methane to chemicals and fuels: technical challenges and issues
    journal, February 2018

    • Hwang, In Yeub; Nguyen, Anh Duc; Nguyen, Thu Thi
    • Applied Microbiology and Biotechnology, Vol. 102, Issue 7
    • DOI: 10.1007/s00253-018-8842-7

    Physiological Effect of XoxG(4) on Lanthanide-Dependent Methanotrophy
    journal, March 2018


    A Mutagenic Screen Identifies a TonB-Dependent Receptor Required for the Lanthanide Metal Switch in the Type I Methanotroph “ Methylotuvimicrobium buryatense ” 5GB1C
    journal, May 2019

    • Groom, Joseph D.; Ford, Stephanie M.; Pesesky, Mitchell W.
    • Journal of Bacteriology, Vol. 201, Issue 15
    • DOI: 10.1128/jb.00120-19

    MxaY regulates the lanthanide-mediated methanol dehydrogenase switch in Methylomicrobium buryatense
    journal, January 2016

    • Chu, Frances; Beck, David A. C.; Lidstrom, Mary E.
    • PeerJ, Vol. 4
    • DOI: 10.7717/peerj.2435

    Efficient production of d-lactate from methane in a lactate-tolerant strain of Methylomonas sp. DH-1 generated by adaptive laboratory evolution
    journal, September 2019


    XoxF Acts as the Predominant Methanol Dehydrogenase in the Type I Methanotroph Methylomicrobium buryatense
    journal, February 2016

    • Chu, Frances; Lidstrom, Mary E.
    • Journal of Bacteriology, Vol. 198, Issue 8
    • DOI: 10.1128/jb.00959-15

    A modular approach for high-flux lactic acid production from methane in an industrial medium using engineered Methylomicrobium buryatense 5GB1
    journal, April 2018

    • Garg, Shivani; Clomburg, James M.; Gonzalez, Ramon
    • Journal of Industrial Microbiology & Biotechnology, Vol. 45, Issue 6
    • DOI: 10.1007/s10295-018-2035-3

    Oxygen-limited metabolism in the methanotroph Methylomicrobium buryatense 5GB1C
    journal, January 2017


    Industrial biomanufacturing: The future of chemical production
    journal, January 2017

    • Clomburg, James M.; Crumbley, Anna M.; Gonzalez, Ramon
    • Science, Vol. 355, Issue 6320
    • DOI: 10.1126/science.aag0804

    Metals and Methanotrophy
    journal, January 2018

    • Semrau, Jeremy D.; DiSpirito, Alan A.; Gu, Wenyu
    • Applied and Environmental Microbiology, Vol. 84, Issue 6
    • DOI: 10.1128/aem.02289-17

    Efficient Counterselection for Methylococcus capsulatus (Bath) by Using a Mutated pheS Gene
    journal, September 2018

    • Ishikawa, Masahito; Yokoe, Sho; Kato, Souichiro
    • Applied and Environmental Microbiology, Vol. 84, Issue 23
    • DOI: 10.1128/aem.01875-18

    Efficient production of d-lactate from methane in a lactate-tolerant strain of Methylomonas sp. DH-1 generated by adaptive laboratory evolution
    journal, September 2019