Yeast as a prototype expression system for lytic peptides

Proof of concept for lytic peptide expression and secretion by a microbial shuttle system was achieved using a commercially available yeast-based expression system in Kluyvero-myces lactis from New England BioLabs Inc . (Ipswich, MA) . Successful ingestion of K. lactis by termites and survival in the hindgut was established by feeding yeast that was stained with nonlethal fluorescent yeast vacuole stain (Sigma-Aldrich, St Louis, MO) to termite workers: presence of labeled yeast in the hindgut was visually confirmed under a fluorescence microscope

The genes for L-Hecate and L-Hecate fused to green fluorescent protein (GFP) were codon-optimized for expression in K. lactis and chemically synthesized (Genscript Corp , Piscataway, NJ) . The genes were cloned into the yeast's chromosome using an integration vector (pKLACI) . Untransformed yeast and a strain of K. lactis containing the integration vector but no lytic peptide encoding gene served as controls . Secretion of active lytic peptide by the transformed K. lactis into the growth media was verified in bioassays using in vitro cultures of the free-living laboratory ciliate Tetrahymena pyriformis. Live ciliates were counted after 24, 48, and 72 hours using a hemocytometer. Supernatants of growth media of two strains of K. lactis, one engineered to secrete Hecate and the other Hecate-GFP, produced high enough levels of the peptide to result in mortality of T. pyriformis that was significantly higher than that of the controls at 72 hours (Figure 17.4) .

Subsequently, control strains and Kluyveromyces lactis strains that showed toxicity toward T. pyriformis were grown for three days at 30°C . Termites were fed with medium containing live yeast strains expressing Hecate or, Hecate-GFP or with control strains for one day and then kept on damp filter paper. Workers that were fed with the yeast strains expressing lytic peptide were defaunated at four weeks, whereas the control continued to contain active protozoa of all three species in high population density in their guts .

Aliquots of the yeast strains containing lytic peptide genes were maintained in culture and tested for loss of activity over time in bioassays with Tetrahymena . Approximately eight weeks after the initial confirmation of their protozoacidal activity, the yeast strains did not produce sufficient active lytic peptide to cause mortality in T. pyriformis This suggests that expression of lytic peptide inflicts negative selection pressure on the yeast itself, resulting in genomic recombination and the loss of lytic peptide expression, which would increase

Oplan Termites

Oplan Termites

You Might Start Missing Your Termites After Kickin'em Out. After All, They Have Been Your Roommates For Quite A While. Enraged With How The Termites Have Eaten Up Your Antique Furniture? Can't Wait To Have Them Exterminated Completely From The Face Of The Earth? Fret Not. We Will Tell You How To Get Rid Of Them From Your House At Least. If Not From The Face The Earth.

Get My Free Ebook


Post a comment