Transposase-Mediated Chromosomal Integration of Exogenous Genes in Acidithiobacillus ferrooxidans

Abstract

The development of 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, and this advance will facilitate the future development of these cells for new biotechnology applications. 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 recovery applications as well as in developing them as unique nonmodel microbial cell factories. Plasmid-driven expression of exogenous genes has been reported, and homologous recombination has been used to knock out some gene expression. Here, new selection protocols facilitated the development of a transposition method for chromosomal integration of exogenous genes into This greatly expands the available genetic toolbox, which will open the door to greater metabolic engineering efforts for these cells.

Identification and Unusual Properties of the Master Regulator FNR in the Extreme Acidophile Acidithiobacillus ferrooxidans

“…It is difficult to generate a Δ fnr mutant of At. ferrooxidans as the organism is challenging to manipulate genetically (Inaba et al, 2018) as reviewed in (Gumulya et al, 2018). Therefore, we chose to complement an E. coli strain RZ8480 lacking fnr (Δ fnr ).…”

Microbially Influenced Corrosion of Stainless Steel by Acidithiobacillus ferrooxidans Supplemented with Pyrite: Importance of Thiosulfate

“…ferrooxidans is an important microorganism in bioleaching operations due to its ability to interact directly with sulfidic ores through ferric iron (15,35). There have been some efforts to develop these cells as a platform for biofuel production (36)(37)(38)(39)(40). However, its potential role in MIC processes has received less attention.…”

“…Two hundred-mesh pyrite was provided as a kind gift from Freeport-McMoRan (Phoenix, AZ). Acidithiobacillus ferrooxidans ATCC 23270 was obtained as described previously (37). All A. ferrooxidans cultures were initiated with a starting optical density at 600 nm (OD 600 ) of 0.001, corresponding to a cell density of 8.3 ϫ 10 6 cells/ml (53).…”

“…for materials that are difficult to leach. Should the genetic tools that are being developed to modify the bacterium become implemented to improve various leaching processes to be more efficient, engineering the genes related to sulfur oxidation, metal tolerances, and salt tolerance of these acidophiles will allow for more development of A. ferrooxidans for new applications (23,36,37,51,52).…”

Enhanced microbial corrosion of stainless steel by Acidithiobacillus ferrooxidans through the manipulation of substrate oxidation and overexpression of rus

“…The plasmid maps of pYI11 and pYI37 are provided in Plasmid pYI37 was conjugated into A. ferrooxidans using the mating and conjugation protocol previously described using the donor strain E. coli S17-1. Three single colonies of A. ferrooxidans transconjugants on S204 solid medium plates were screened in the liquid selection SM4 medium (Table S2; Inaba et al, 2018).…”