Projects per year
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.
Original language | English |
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Article number | 58 |
Number of pages | 8 |
Journal | Microbial Cell Factories |
Volume | 16 |
Issue number | 1 |
DOIs | |
Publication status | Published - 5 Apr 2017 |
Keywords
- Journal Article
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- 2 Finished
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David Leak - Development of Geobacillus Thermoglucosidasis as a Robust Platform for Production of Chemicals from Renewables
Leak, D. (PI)
Biotechnology and Biological Sciences Research Council
16/04/12 → 15/10/15
Project: Research council