An efficient Agrobacterium tumefaciens-mediated transformation method for Simplicillium subtropicum (Hypocreales: Cordycipitaceae)

Abstract

Filamentous fungi are the organisms of choice for most industrial biotechnology. Some species can produce a variety of secondary metabolites and enzymes of commercial interest, and the production of valuable molecules has been enhanced through different molecular tools. Methods for genetic manipulation and transformation have been essential for the optimization of these organisms. The genus Simplicillium has attracted increased attention given several potential biotechnological applications. The Simplicillium genus harbors several entomopathogenic species and some isolates have been explored for bioremediation of heavy metal contaminants. Furthermore, the myriad of secondary metabolites isolated from Simplicillium spp. render these organisms as ideal targets for deep exploration and further biotechnological mining possibilities. However, the lack of molecular tools hampered the exploration of this genus. Thus, an Agrobacterium tumefaciens -mediated transformation method was established for Simplicillium subtropicum , employing the far-red fluorescent protein TURBOFP635/Katushka, as a visual marker, and the selection marker SUR gene, that confers resistance to chlorimuron ethyl. Notably, one round of transformation using the established method yielded almost 400 chlorimuron resistant isolates. Furthermore, these transformants displayed mitotic stability for, at least, five generations. We anticipate that this method can be useful for deep molecular exploration and improvement of strains in the Simplicillium genus.

Development of a Versatile Toolbox for Genetic Manipulation of Sporothrix brasiliensis

“…4B ). We have also constructed an S. brasiliensis strain expressing both TURBOFP635/Katushka ( 22 ) and H2A::sGFP fluorescent proteins, revealing that strains with multiple fluorescent tags are feasible (Fig. S2).…”

“…All plasmids constructed were confirmed by Sanger DNA sequencing (Stab Vida, Portugal). The pPZP201BK::SUR::gpdA::zKat::TrpC, kindly provided by Dr. Augusto Schrank ( 22 ), was used to express the far-red fluorescent protein TURBOFP635/Katushka.…”

“…Because the HUPE 114158 strain did not grow properly in YPD, the BHI medium was used with the same concentration of hygromycin B. For the selective purpose, yeast nitrogen base (YNB) solid medium (0.16% BD Difco YNB, 2% glucose, 0.5% ammonium sulfate, 25 μg/mL of leucine, 25 μg/mL of histidine, 25 μg/mL of methionine, and 25 μg/mL of uracil), supplemented with Chlorimuron ethyl (30 μg/mL, Santa Cruz Biotechnology, USA) was used for selection of S. brasiliensis transformed with A. tumefaciens carrying the plasmid described elsewhere ( 22 ).…”