Transposase-Mediated Chromosomal Integration of Exogenous Genes in Acidithiobacillus ferrooxidans
Abstract
The development of Acidithiobacillus ferrooxidans as a non-model host organism for synthetic biology is hampered by a lack of genetic tools and techniques. New plating and liquid-based selection methods were developed to improve the identification of transformed cell lines. Enabled by these methods, a hyperactive transposase was used to generate mutants with integrated genes for the expression of the superfolder green fluorescent protein (sfGFP) gene or a 2-keto decarboxylase (KDC) gene, which enabled the production and secretion of isobutyric acid (IBA). An inverse PCR method was used to identify the insertion sites of the KDC gene in several mutants, leading to the identification of a region on the chromosome that may be suitable for future genetic insertions. These results demonstrate that functional exogenous metabolic genes have been chromosomally integrated into A. ferrooxidans, and this advance will facilitate the future development of these cells for new biotechnology applications.IMPORTANCEAcidithiobacillus ferrooxidans is an iron- and sulfur-oxidizing chemolithoautotroph and is a key member of the microbial consortia used in industrial biomining applications. There is interest in exploiting these cells for other metal...Continue Reading
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