CoIN: co-inducible nitrate expression system for secondary metabolites in .

BACKGROUND

Sequencing of fungal species has demonstrated the existence of thousands of putative secondary metabolite gene clusters, the majority of them harboring a unique set of genes thought to participate in production of distinct small molecules. Despite the ready identification of key enzymes and potential cluster genes by bioinformatics techniques in sequenced genomes, the expression and identification of fungal secondary metabolites in the native host is often hampered as the genes might not be expressed under laboratory conditions and the species might not be amenable to genetic manipulation. To overcome these restrictions, we developed an inducible expression system in the genetic model .

RESULTS

We genetically engineered a strain of devoid of producing eight of the most abundant endogenous secondary metabolites to express the sterigmatocystin Zn(II)Cys transcription factor-encoding gene and its cofactor under control of the nitrate inducible / promoter. Furthermore, we identified a subset of promoters from the sterigmatocystin gene cluster that are under nitrate-inducible AflR/S control in our production strain in order to yield coordinated expression without the risks from reusing a single inducible promoter. As proof of concept, we used this system to produce β-carotene from the carotenoid gene cluster of .

CONCLUSION

Utilizing one-step yeast recombinational cloning, we developed an inducible expression system in the genetic model and show that it can be successfully used to produce commercially valuable metabolites.