(798c) Novel Variants of Self-Excising Beta-Recombinase/Six Cassettes Facilitating Efficient Sequential Repetitive Gene Deletions in Neurospora Crassa

For over 70 years, Neurospora crassa, a model filamentous ascomycete, has been studied to understand biological processes and phenomena relevant to higher eukaryotes.  It has received increasing interest because of its ability to convert lignocellulosic biomass to sugars, which can be converted into biofuels or platform chemicals.  Development of efficient genetic engineering tools including efficient targeted gene deletions methods are essential to meet the requirements of various tailor-made applications.

Recently we validated functionality of a bacterial recombination system employing β serine recombinase acting on six recognition sequences (β-rec/six) in the filamentous fungus Neurospora crassa. This self-excising resistance marker cassette allows repetitive deletions of genes with only one round of transformation per gene deletion. Moreover, the β serine recombinase acts in a strict cis-action manner, which enables only intramolecular recombination events and eliminates the risk of chromosomal rearrangements as brought by the Cre/loxP or FLP/FRT systems. Whereas the screening process is straightforward in Aspergilli, whose mononucleate conidia allow automatic homokaryon purification, the multinuclear nature of N. crassa macroconidia used for transformation makes the process more complicated. The promoter used for the induction of the cassette eviction is also of very low efficiency in N. crassa.

Here we report on the construction and validation of new variants of β-rec/six system cassettes that facilitate screening for self-excision events in N. crassa. Introduction of the tk (thymidine kinase) gene allows fast-forward, one-step, positive screening for the excision event and automatic homokaryon purification. Use of the regulatable promoter native to b-xylanase A of N. crassa, also significantly improves the efficiency of cassette eviction in this fungus.