Regulation of interrelated aromatic amino acid and vitamin biosyntheses in Coprinopsis cinerea

Chorismate from the shikimate pathway acts as common precursor in the biosyntheses of aromatic amino acids and PABA. Their production is regulated by feedback and cross-pathway controls. Tryptophan biosynthesis in Coprinopsis cinerea is mediated by four enzymes anthranilate synthase Trp3, the tri-functional Trp2 (with TrpG, TrpF and TrpC domains), Trp4, and the bi-functional tryptophan synthase Trp1 (with TrpA and TrpB domains). Ectopic transformation of trp3 and trp1 copies into the fungal genome causes a paradoxical phenomenon: Transformation with either trp⁺ gene halves the numbers of viable prototrophic transformants as compared to cotransformations with a non-trp⁺ vector. Cotransformation of both trp⁺ genes reduces number of transformants further. Loss of transformants reflects feedback inhibition above a critical level that irreversibly shuts off the Trp production with growth inhibition as consequence. Lethal effects can be further enhanced by addition of Trp precursors and also through cross-pathway control by addition of other aromatic amino acids. We split Trp1 into its units TrpA and TrpB. TrpA and TrpB can complement each one of two mutations the defective allele trp1-1,1-6. Their cotransformation into trp1-1,1-6 mutants gives prototrophic clones. Thus, indole produced by TrpA is transferred to TrpB for Trp production. Depending on specific mutations and resulting steric effects on the lockable 2.5 Å tunnel between TrpA and TrpB as pathway for indole transfer, such transfer is not always possible from TrpA unit to a TrpA-mutated full-length Trp1 protein or from a TrpB-defective Trp1 protein to TrpB. Now, we plan to uncouple the domains of Trp2 and of the PABA-synthase whose TrpG-like domains might be interchangeable. Trp2 with a defective TrpF domain is expected to accumulate the intermediate PRA which in bacteria can serve in synthesis of the HMP precursor of thiamin. C. cinerea lacks a respective thi5 gene which we introduce from a foreign host.