A novel mutagenesis system that enables the creation of unmarked and targeted deletions in Burkholderia strains.

Keywords: bioremediation, in-situ remediation, Burkholderia, mutagenesis, polychlorinated biphenyl compounds (PCB), polycyclic aromatic hydrocarbons (PAH), trichloroethylene (TCE)

DESCRIPTION OF THE INVENTION: To date the lack of molecular tools available to facilitate mutagenesis and functional analyses of Burkholderia has limited its exploitation in bioremediation. The present invention relates to a novel mutagenesis system that enables the creation of unmarked (i.e. no foreign material is integrated into the genome as a result of the mutagenesis procedure) and targeted deletions in Burkholderia strains. The technology presented allows for the unique feature of introducing multiple mutations to be made within the same strain. These new tools make it possible to engineer optimized strains of Burkholderia for use as bioremediation agents.

BACKGROUND: The international market for remediation products and services is valued at US $25-30 billion. It is estimated that there are over 1 billion contaminated sites in North America and Europe alone with hydrocarbons, chlorinated solvents, polychlorinated biphenyl compounds and nitroaromatics being the primary contaminants of concern. Strict environmental regulations and legislation enacted in Canada, the United States, Western Europe, Japan, and Australia have all focused on the need for remediation of contaminated sites. Federal funding programs have been established in most jurisdictions to provide financial assistance for remediation projects and have driven the growth of the remediation sector.

The use of microorganisms to degrade xenobiotic compounds has evolved as a cost-effective in situ strategy for the remediation of contaminated sites. Burkholderia is a genus of bacteria composed of approximately 30 species that are endogenous to soil and root rhizospheres. Burkholderia have been shown in laboratory and filed trials to degrade many xenobiotic compounds including: 1) Polycyclic aromatic hydrocarbons (e.g. naphthalene), 2) Halogenated hydrocarbons (e.g. trichloroethylene, polychlorinated biphenyl compounds), and 3) Pesticides (e.g. 2,4-D). The wide substrate diversity of these bacteria makes them attractive bioremediation agents. In contrast to this potential, several endogenous species of Burkholderia can be pathogenic in immunocompromised patients and those with Cystic Fibrosis. There is significant market potential for a non-pathogenic strain of Burkholderia.

POTENTIAL ADVANTAGES/USES:

• Allows altering of the genome (point changes, targeted deletion) without the introduction of foreign genetic material
• Can be used to engineer low-risk non-pathogenic strains of Burholderia for in situ bioremediation
• Opportunity in conjunction with a screen to identify lines with appropriate properties for specific compounds/toxins
• In conjunction with publicly available sequence maps, may be applied to specifically target genes of interest
• Can be used to introduce safeguards to limit release of bulk bioreactor preparations
• May be used to functionally screen for and investigate mechanisms of Burkholderia produced antinematodal and antimicrobial compounds or of Bukrholderia biofilm formation

 

Ref. #: UWO-AI-021

 

Preclinical

US provisional patent application filed.

Available for license and/or collaborative approach.