Split trp1+ Gene Markers for Selection in Sequential Transformations of the Agaricomycete Coprinopsis cinerea

Title:Split trp1+ Gene Markers for Selection in Sequential Transformations of the Agaricomycete Coprinopsis cinerea

Volume: 6 Issue: 2

Author(s): Bastian Dornte and Ursula Kues*

Affiliation:

• Section of Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, Faculty of Forest Sciences and Forest Ecology, Georg-August-Universitat Gottingen, Gottingen,Germany

Keywords: Transformation, basidiomycete, selection marker, auxotrophy, tryptophan synthase, indole, eGFP, laccase.

Abstract: Background: A major bottleneck in DNA-mediated transformation of fungi is the limited number of available selective marker genes, especially when sequential transformations of the same strain are necessary. In the model basidiomycete Coprinopsis cinerea, the trp1+ gene is a commonly used dominant selection marker for transformation to complement trp1 auxotrophies. The trp1+ encoded tryptophan synthase is a bifunctional enzyme that catalyzes the final two reactions in the biosynthetic pathway of tryptophan. The Nterminal A-domain is responsible for the conversion of indole-3-glycerol-phosphate into indole, while the Cterminal B-domain catalyzes the subsequent production of tryptophan from serine and indole. The trp1-1,1-6 mutant allele used in C. cinerea hosts in transformation carries a mutation in each gene half, both of which prevent the strain from completing tryptophan biosynthesis.

Methods: Taking advantage of this situation, we developed a new set of vectors containing either just the A- or the B-domain encoding trp1+ sequences (referred to as trpA+ and trpB+). The constructs were used in single-vector transformations and in co-transformations with other vectors containing genes which are not directly selectable (C. cinerea lcc1+ for laccase, egfp for green fluorescent protein). The trpB+-construct pYBdom and the trpA+-construct pYAdom can be applied in series in subsequent rounds of transformations. This allows successive introduction of different genes into the same genetic background in succeeding rounds of co-transformations.

Results: For the first time in fungi, the functionality of independent tryptophan synthase domains TrpA and TrpB were documented. The new marker set enables the independent complementation of the distinct trp1 mutations in separate transformations. First, trpB+ needs to be transformed and indole must be added to the regeneration medium for production of tryptophan. The subsequent transformation with trpA+ results in the production of indole which is then used by TrpB in tryptophan synthesis. Double transformants are prototrophic but differ in growth rates as compared to transformants with the complete trp1+ gene. The two new dominant selection markers have successfully been used in sequential cotransformations with C. cinerea lcc1+ and egfp as additional genes. Plate assays helped in the detection of co-transformants expressing these additional genes. Co-transformation frequencies of 21-23% were obtained.

Conclusions: Application of vectors pYBdom and pYAdom opens novel possibilities to add different genes in series into a same genetic background to manipulate metabolic or developmental processes in the fungus.

Cite this article as:

Dornte Bastian and Kues Ursula*, Split trp1+ Gene Markers for Selection in Sequential Transformations of the Agaricomycete Coprinopsis cinerea, Current Biotechnology 2017; 6 (2) . https://dx.doi.org/10.2174/2211550105666160517142815

DOI https://dx.doi.org/10.2174/2211550105666160517142815	Print ISSN 2211-5501
Publisher Name Bentham Science Publisher	Online ISSN 2211-551X