Direct detection of recombinant gene expression by two genetically engineered yeasts in soil on the transcriptional and translational levels

The expression of a recombinant gene by yeasts seeded into soil samples was directly measured by analyzing transcripts and gene product occurrences in soil extracts. Two yeast species, Saccharomyces cerevisiae WHL292 and Hansenula polymorpha LR9-Apr4, both engineered by a synthetic gene sequence encoding the mammalian peptide aprotinin, produced and secreted this peptide in batch cultures at concentrations of 90 and 64 ng ml^-1, respectively. In S. cerevisiae, the aprotinin gene was located on plasmid p707 and expressed constitutively. H. polymorpha carried the gene chromosomally integrated, and its expression was inducible by methanol. To detect aprotinin transcripts, cells were directly lysed in the soil samples and the crude lysates were hybridized to oligo(dT)-coated magnetized polystyrene beads (Dynabeads). After separation and purification in a magnetic field, aprotinin mRNA was detected by reverse transcriptase PCR with aprotinin gene-specific primers. Transcripts from 10 cells g of soil^-1 were sufficient for detection. When 10⁷ cells of S. cerevisiae were inoculated into soil, aprotinin mRNA was detectable during the first 4 days. Addition of methanol and a combined nutrient solution was necessary to induce aprotinin gene expression of H. polymorpha in soil. Aprotinin could be detected directly in soil extracts by an indirect enzyme-linked immunosorbent assay with monoclonal aprotinin- specific antibodies. The detection threshold was 45 pg g of soil^-1. In presterilized soil inoculated with S. cerevisiae (10⁶ CFU g^-1), aprotinin accumulated during the first 10 days to 12 ng g of soil^-1 and then remained constant. In nonsterile soil, however, aprotinin formation was observed only during the first 4 days and was followed by a decrease below the level of detection after 2 weeks. Similar results were obtained with induced cells of H. polymorpha. Thus, disappearance of aprotinin was due to its degradation by the indigenous soil microorganisms.