A Rapid and Robust Gene Knock-in/Knock-out System in Geobacillus Thermoglucosidasius Based on the Pyre marker. 

The genus Geobacillus has increasingly attracted interests as thermophilic bioprocessing platforms, due to desirable properties such as catabolic versatility ^1,2. To fully utilize their potentials, both product yield and specificity needs to be improved via metabolic engineering, requiring gene knock-in/knock-out tools. Here, adopting the ACE strategy by heap et al ³ we developed a set of rapid strategies for gene knock-in/knock-out in Geobacillus thermoglucosidasius. The product of pyrE gene catalyzes an essential step in uracil biosynthesis but also metabolizes 5-fluororotic acid (5-FOA) to toxic metabolites, hence its disruption gives both positive and negative selectivity. A ΔpyrE strain, involving truncating an internal part of the gene leaving the first 300 bp and stop codon intact was obtained. For gene knock-in, by using a vector with homology arms that repaired the disrupted pyrE while simultaneous integrates DNA cargo, the trans-2-enoyl-CoA reductase gene from T. caldaria was successfully integrated at the pyrE locus, confirmed by both PCR and Western. The positive selectivity based on growth on uracil free medium resulted in 100% correct integrant with a turnaround time of just 4 days. For gene knock-out, the ΔpyrE phenotype was firstly complemented by using heterologous pyrE amplified from G. Kaustophilus and G. thermoleovorans fused to and co-expressed from the antibiotic selection marker. By co-utilizing uracil auxotrophy and the temperature sensitivity of the complementation vector we developed, single-crossover integrant was obtained at 100% efficiency when grown above 60 °C in the absence of uracil, avoiding the need for screening. Double crossover integrant, either corresponding to wild type or desired deletion mutants, were then selected based on the counter-selectivity of 5-FOA resistance. Turnaround time was only 6 days with high efficiency that screening of only 8-12 colonies was adequate. Lastly, the truncated pyrE was repaired back to wild type following the same procedure for gene knock-in. To exemplify the rapidity and robustness of the technique, the TMO production strain ², consisting of an ldh deletion, pfl deletion and pdh up-regulation via promoter replacement, were recreated in only 30 days. Although the intermediate strains (Δldh, Δldhpdhup) showed the expected fermentation profile, the final strain didn’t. While repeated construction of the strain led to no avail, Genome scale analysis revealed potential SNPs in both TM242 and our strain in contributing to the phenotype observed.

In summary, we established a full set of recombination based genetic tools, with very high efficiency and robustness, in the host G. thermoglucosidasius, offering new scopes for metabolic engineering in this organism.  

1.        Suzuki, H., Murakami, A. & Yoshida, K. Counterselection system for Geobacillus kaustophilus HTA426 through disruption of pyrF and pyrR. Appl. Environ. Microbiol. 78,7376–83 (2012).

2.        Cripps, R. E. et al. Metabolic engineering of Geobacillus thermoglucosidasius for high yield ethanol production. Metab. Eng. 11,398–408 (2009).

3.        Heap, J. T. et al. Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. Nucleic Acids Res. 40, e59 (2012).