Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.

Leann F Bacon, Charlotte Hamley-Bennett, Michael J Danson, David J Leak

Research output: Contribution to journalArticle

Abstract

BACKGROUND: Geobacillus thermoglucosidasius is a thermophilic, natural ethanol producer and a potential candidate for commercial bioethanol production. Previously, G. thermoglucosidasius has been genetically modified to create an industrially-relevant ethanol production strain. However, creating chromosomal integrations and deletions in Geobacillus spp. is laborious. Here we describe a new technique to create marker-less mutations in Geobacillus utilising a novel homologous recombination process.

RESULTS: Our technique incorporates counter-selection using β-glucosidase and the synthetic substrate X-Glu, in combination with a two-step homologous recombination process where the first step is a selectable double-crossover event that deletes the target gene. We demonstrate how we have utilised this technique to delete two components of the proteinaceous shell of the Geobacillus propanediol-utilization microcompartment, and simultaneously introduce an oxygen-sensitive promoter in front of the remaining shell-component genes and confirm its functional incorporation.

CONCLUSION: The selectable deletion of the target gene in the first step of our process prevents re-creation of wild-type which can occur in most homologous recombination techniques, circumventing the need for PCR screening to identify mutants. Our new technique therefore offers a faster, more efficient method of creating mutants in Geobacillus.

LanguageEnglish
Article number58
Number of pages8
JournalMicrobial Cell Factories
Volume16
Issue number1
DOIs
StatusPublished - 5 Apr 2017

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Geobacillus
Gene Deletion
Genes
Homologous Recombination
Ethanol
Propylene Glycols
Bioethanol
beta-Glucosidase
Screening
Gene Components
Oxygen
Substrates
Polymerase Chain Reaction
Mutation

Keywords

  • Journal Article

Cite this

Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp. / Bacon, Leann F; Hamley-Bennett, Charlotte; Danson, Michael J; Leak, David J.

In: Microbial Cell Factories, Vol. 16, No. 1, 58, 05.04.2017.

Research output: Contribution to journalArticle

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