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Title: A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4

Abstract

Metabolic engineering of the biofuel-producing Zymomonas mobilis is necessary if we are to unlock the metabolic potential present in this non-model microbe. Manipulation of such organisms can be challenging because of the limited genetic tools for iterative genome modification. Here, we have developed an efficient method for generating markerless genomic deletions or additions in Z. mobilis . This is a two-step process that involves homologous recombination of an engineered suicide plasmid bearing Z. mobilis targeting sequences and a subsequent recombination event that leads to loss of the suicide plasmid and a genome modification. A key feature of this strategy is that GFP expressed from the suicide plasmid allows easy identification of cells that have lost the plasmid by using a fluorescence activated cell sorter. Using this method, we demonstrated deletion of the gene encoding lactate dehydrogenase ( ldh ) and the operon for cellulose synthase ( bcsABC ). In addition, by modifying the plasmid design, we demonstrated targeted insertion of the crtIBE operon encoding a neurosporene biosynthetic pathway into the Z. mobilis genome without addition of any antibiotic resistance genes. We propose this approach will provide an efficient and flexible platform for improved genetic engineering of Z. mobilis .

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Univ. of Wisconsin, Madison, WI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1570044
Alternate Identifier(s):
OSTI ID: 1799692
Grant/Contract Number:  
SC0018409; FC02-07ER64494
Resource Type:
Published Article
Journal Name:
Frontiers in Microbiology
Additional Journal Information:
Journal Name: Frontiers in Microbiology Journal Volume: 10; Journal ID: ISSN 1664-302X
Publisher:
Frontiers Media SA
Country of Publication:
Switzerland
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Microbiology; genome engineering of a non-model bacterium; green fluorescent protein; fluorescence activated cell sorting; recombineering suicide plasmid; biofuels; Zymomonas mobilis

Citation Formats

Lal, Piyush Behari, Wells, Fritz M., Lyu, Yucai, Ghosh, Indro N., Landick, Robert, and Kiley, Patricia J. A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4. Switzerland: N. p., 2019. Web. doi:10.3389/fmicb.2019.02216.
Lal, Piyush Behari, Wells, Fritz M., Lyu, Yucai, Ghosh, Indro N., Landick, Robert, & Kiley, Patricia J. A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4. Switzerland. https://doi.org/10.3389/fmicb.2019.02216
Lal, Piyush Behari, Wells, Fritz M., Lyu, Yucai, Ghosh, Indro N., Landick, Robert, and Kiley, Patricia J. Fri . "A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4". Switzerland. https://doi.org/10.3389/fmicb.2019.02216.
@article{osti_1570044,
title = {A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4},
author = {Lal, Piyush Behari and Wells, Fritz M. and Lyu, Yucai and Ghosh, Indro N. and Landick, Robert and Kiley, Patricia J.},
abstractNote = {Metabolic engineering of the biofuel-producing Zymomonas mobilis is necessary if we are to unlock the metabolic potential present in this non-model microbe. Manipulation of such organisms can be challenging because of the limited genetic tools for iterative genome modification. Here, we have developed an efficient method for generating markerless genomic deletions or additions in Z. mobilis . This is a two-step process that involves homologous recombination of an engineered suicide plasmid bearing Z. mobilis targeting sequences and a subsequent recombination event that leads to loss of the suicide plasmid and a genome modification. A key feature of this strategy is that GFP expressed from the suicide plasmid allows easy identification of cells that have lost the plasmid by using a fluorescence activated cell sorter. Using this method, we demonstrated deletion of the gene encoding lactate dehydrogenase ( ldh ) and the operon for cellulose synthase ( bcsABC ). In addition, by modifying the plasmid design, we demonstrated targeted insertion of the crtIBE operon encoding a neurosporene biosynthetic pathway into the Z. mobilis genome without addition of any antibiotic resistance genes. We propose this approach will provide an efficient and flexible platform for improved genetic engineering of Z. mobilis .},
doi = {10.3389/fmicb.2019.02216},
journal = {Frontiers in Microbiology},
number = ,
volume = 10,
place = {Switzerland},
year = {2019},
month = {10}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.3389/fmicb.2019.02216

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Cited by: 1 work
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