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:

Lal, Piyush Behari; Wells, Fritz M.; Lyu, Yucai; Ghosh, Indro N.; Landick, Robert; Kiley, Patricia J.

Publication Date:

2019-10-11

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