Transcription profiling of yeast grown in gastric or duodenal medium to identify promoters that could be used to down-regulate genes used in the release of therapeutic proteins

The use of yeast as a delivery system is an attractive option for the oral administration of therapeutic agents. We recently developed mutants of Saccharomyces cerevisiae capable of lysis upon conditional down-regulation of the expression of the cell wall genes PKC1 and SRB1. The lysis mechanism of the mutant is based on the use of the MET3 promoter, which, upon addition of methionine and cysteine, blocks transcription of SRB1 and PKC1. This strain has the potential to be an integral part of an oral yeast delivery system, in which there is lysis of yeasts in the human gut, followed by release of recombinant proteins for therapeutic use. In order to provide proof-of-principle, the system was evaluated testing the cells viability and lysis performance under conditions, which simulate those found in the human stomach and the duodenum. Upon incubation of yeast cells in these conditions, lysis could be induced and was accompanied by release of GFP reporter protein into the medium. However, the conditional lysis mechanism based on the MET3 promoter is not applicable in vivo. Therefore, alternative promoters suitable for in-vivo down-regulation of SRB1 and PKC1 were identified by a microarray experiments. The transcripts of genes ANB1, TIR1, and MF(ALPHA)2 were significantly reduced upon exposure of the yeast cells to conditions of the two gut compartments. Their promoters could be used to down-regulate SRB1/VIG9 and PKC1 in vivo to achieve lysis of the yeast in the gut to release cargo therapeutic proteins.

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Omara WA, Rash BM, Hayes A, Wickham MS, Oliver SG, Stateva LI