Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems

Lal, Piyush Behari; Wells, Fritz; Myers, Kevin S.; Banerjee, Rajdeep; Guss, Adam M.; Kiley, Patricia J.; Kelly, ed., Robert M.

doi:10.1128/AEM.00808-21

Title: Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems

Journal Article · Fri Sep 10 00:00:00 EDT 2021 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/AEM.00808-21· OSTI ID:1819468

^[1]; Wells, Fritz ^[1];

^[2]; Banerjee, Rajdeep ^[3];

^[4];

^[1]; Kelly, ed., Robert M.

DOE Great Lakes Bioenergy Research Center, University of Wisconsin—Madison, Madison, Wisconsin, USA, Department of Biomolecular Chemistry, University of Wisconsin—Madison, Madison, Wisconsin, USA
DOE Great Lakes Bioenergy Research Center, University of Wisconsin—Madison, Madison, Wisconsin, USA, Wisconsin Energy Institute, University of Wisconsin—Madison, Madison, Wisconsin, USA
Department of Biomolecular Chemistry, University of Wisconsin—Madison, Madison, Wisconsin, USA
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA, Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

Zymomonas mobilis has emerged as a promising candidate for production of high-value bioproducts from plant biomass. However, a major limitation in equipping Z. mobilis with novel pathways to achieve this goal is restriction of heterologous DNA. Here, we characterized the contribution of several defense systems of Z. mobilis strain ZM4 to impeding heterologous gene transfer from an Escherichia coli donor. Bioinformatic analysis revealed that Z. mobilis ZM4 encodes a previously described mrr-like type IV restriction modification (RM) system, a type I-F CRISPR system, a chromosomal type I RM system (hsdMS_c), and a previously uncharacterized type I RM system, located on an endogenous plasmid (hsdRMS_p). The DNA recognition motif of HsdRMS_p was identified by comparing the methylated DNA sequence pattern of mutants lacking one or both of the hsdMS_c and hsdRMS_p systems to that of the parent strain. The conjugation efficiency of synthetic plasmids containing single or combinations of the HsdMS_c and HsdRMS_p recognition sites indicated that both systems are active and decrease uptake of foreign DNA. In contrast, deletions of mrr and cas3 led to no detectable improvement in conjugation efficiency for the exogenous DNA tested. Thus, the suite of markerless restriction-negative strains that we constructed and the knowledge of this new restriction system and its DNA recognition motif provide the necessary platform to flexibly engineer the next generation of Z. mobilis strains for synthesis of valuable products.

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Research Organization:: Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: FC-07ER64494; AC05-00OR22725

OSTI ID:: 1819468

Alternate ID(s):: OSTI ID: 1824970

Journal Information:: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Vol. 87 Journal Issue: 19; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
Zymomonas mobilis ZM4
restriction modification system
genome defense
conjugation efficiency of foreign genes
type I restriction enzymes

Title: Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems

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