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The genus Geobacillus is abundant in ecological diversity and is also well-known as an authoritative source for producing various thermostable enzymes. Although it is clear now that Geobacillus evolved from Bacillus, relatively little knowledge has been obtained regarding its evolutionary mechanism, which might also contribute to its ecological diversity and biotechnology potential. Here, a statistical comparison of thirty-two Geobacillus genomes was performed with a specific focus on pan- and core genomes. The pan-genome of this set of Geobacillus strains contained 14,913 genes, and the core genome contained 940 genes. The Clusters of Orthologous Groups (COG) and Carbohydrate-Active Enzymes (CAZymes) analysis revealed that the Geobacillus strains had huge potential industrial application in composting for agricultural waste management. Detailed comparative analyses showed that basic functional classes and housekeeping genes were conserved in the core genome, while genes associated with environmental interaction or energy metabolism were more enriched in the pan-genome. Therefore, the evolution of Geobacillus seems to be guided by environmental parameters. In addition, horizontal gene transfer (HGT) events among different Geobacillus species were detected. Altogether, pan-genome analysis was a useful method for detecting the evolutionary mechanism, and Geobacillus’ evolution was directed by the environment and HGT events.

Deciphering the complex cellular behaviours and advancing the biotechnology applications of filamentous fungi increase the requirement for genetically manipulating a large number of target genes. The current strategies cannot cyclically coedit multiple genes simultaneously. In this study, we firstly revealed the existence of diverse homologous recombination (HR) types in marker-free editing of filamentous fungi, and then, demonstrated that sgRNA efficiency-mediated competitive inhibition resulted in the low integration of multiple genetic sites during coediting, which are the two major obstacles to limit the efficiency of cyclically coediting of multiple genes. To overcome these obstacles, we developed a biased cutting strategy by Cas9 to greatly enhance the desired HR type and applied a new selection marker labelling strategy for multiple donor DNAs, in which only the donor DNA with the lowest sgRNA efficiency was labelled. Combined with these strategies, we successfully developed a convenient method for cyclically coediting multiple genes in different filamentous fungi. In addition, diverse HRs resulted in a useful and convenient one-step approach for gene functional study combining both gene disruption and complementation. This research provided both a useful one-step approach for gene functional study and an efficient strategy for cyclically coediting multiple genes in filamentous fungi.

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