Cargando…
A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4
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 develop...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2019
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797605/ https://www.ncbi.nlm.nih.gov/pubmed/31681183 http://dx.doi.org/10.3389/fmicb.2019.02216 |
_version_ | 1783459865541214208 |
---|---|
author | Lal, Piyush Behari Wells, Fritz M. Lyu, Yucai Ghosh, Indro N. Landick, Robert Kiley, Patricia J. |
author_facet | Lal, Piyush Behari Wells, Fritz M. Lyu, Yucai Ghosh, Indro N. Landick, Robert Kiley, Patricia J. |
author_sort | Lal, Piyush Behari |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6797605 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-67976052019-11-01 A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4 Lal, Piyush Behari Wells, Fritz M. Lyu, Yucai Ghosh, Indro N. Landick, Robert Kiley, Patricia J. Front Microbiol Microbiology 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. Frontiers Media S.A. 2019-10-11 /pmc/articles/PMC6797605/ /pubmed/31681183 http://dx.doi.org/10.3389/fmicb.2019.02216 Text en Copyright © 2019 Lal, Wells, Lyu, Ghosh, Landick and Kiley. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology 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 |
title | A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4 |
title_full | A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4 |
title_fullStr | A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4 |
title_full_unstemmed | A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4 |
title_short | A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4 |
title_sort | markerless method for genome engineering in zymomonas mobilis zm4 |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797605/ https://www.ncbi.nlm.nih.gov/pubmed/31681183 http://dx.doi.org/10.3389/fmicb.2019.02216 |
work_keys_str_mv | AT lalpiyushbehari amarkerlessmethodforgenomeengineeringinzymomonasmobiliszm4 AT wellsfritzm amarkerlessmethodforgenomeengineeringinzymomonasmobiliszm4 AT lyuyucai amarkerlessmethodforgenomeengineeringinzymomonasmobiliszm4 AT ghoshindron amarkerlessmethodforgenomeengineeringinzymomonasmobiliszm4 AT landickrobert amarkerlessmethodforgenomeengineeringinzymomonasmobiliszm4 AT kileypatriciaj amarkerlessmethodforgenomeengineeringinzymomonasmobiliszm4 AT lalpiyushbehari markerlessmethodforgenomeengineeringinzymomonasmobiliszm4 AT wellsfritzm markerlessmethodforgenomeengineeringinzymomonasmobiliszm4 AT lyuyucai markerlessmethodforgenomeengineeringinzymomonasmobiliszm4 AT ghoshindron markerlessmethodforgenomeengineeringinzymomonasmobiliszm4 AT landickrobert markerlessmethodforgenomeengineeringinzymomonasmobiliszm4 AT kileypatriciaj markerlessmethodforgenomeengineeringinzymomonasmobiliszm4 |