In vivo assembly of genetic constructs in filamentous fungus Talaromyces cellulolyticus.

In the filamentous fungus Talaromyces cellulolyticus, similar to other filamentous fungi, non-homologous recombination predominates over homologous recombination. For instance, to achieve an acceptable integration frequency of a genetic construct into a target site on the intact chromosome, the flanking sequences directing this integration should be approximately 2.5 kb in length. However, despite the requirement of long flanks for integration into the intact chromosome, we found that homologous recombination between linear DNA fragments in T. cellulolyticus effectively occurs when these fragments overlap by just 50 bp. This allows for the assembly of full-sized genetic constructs in vivo from relatively small blocks, eliminating the need for in vitro assembly, similar to the approach previously developed for the yeast Saccharomyces cerevisiae. To validate this possibility, we replaced the native promoter of the target gene by transforming the recipient strain with five DNA fragments: two flanks for recombination with the target locus, two parts of the marker gene, and a donor promoter. This discovery significantly expedites the genetic engineering of T. cellulolyticus and potentially other fungi.

Improving the selection efficiency of the counter-selection marker pheS* for the genetic engineering of Bacillus amyloliquefaciens.

Bacillus subtilis pheS was genetically modified to obtain a counter-selection marker with high selection efficiency in Bacillus amyloliquefaciens. The application of the new replication-thermosensitive integrative vector pNZTM1, containing this marker, pheSBsT255S/A309G, with a two-step replacement recombination procedure provides an effective tool for the genetic engineering of industrially important Bacillus species.