Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.
Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.
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DOI：
10.1186/s12934-017-0670-4
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发表时间：
2017-04-05
影响因子：
6.4
通讯作者：
Leak DJ
中科院分区：
文献类型：
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作者：
Bacon LF;Hamley-Bennett C;Danson MJ;Leak DJ
热葡萄糖苷地质杆菌是一种嗜热的天然乙醇生产菌，是商业生物乙醇生产的潜在候选者。此前，热葡萄糖苷菌已经经过基因改造，产生了一种与工业相关的乙醇生产菌株。然而，在地质杆菌中产生染色体整合和缺失。是很费力的。在这里，我们描述了一种新的技术，利用一种新的同源重组过程在地质杆菌中创建无标记突变。我们的技术结合了使用β-葡萄糖苷酶和合成底物X-Glu的反选择，以及两步同源重组过程，其中第一步是删除目标基因的可选择双交换事件。我们展示了我们如何利用这项技术删除地质杆菌丙二醇利用微隔室蛋白质壳的两个组分，并同时在剩余的壳组分基因前面引入一个氧敏感启动子，并确认其功能整合。在我们过程的第一步，目标基因的选择性删除防止了野生型的重新产生，这在大多数同源重组技术中都可能发生，从而避免了通过PCR筛选来识别突变体的需要。因此，我们的新技术提供了一种更快、更有效的方法来在地质杆菌中创建突变。
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. 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. 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.