Proteomic Analysis Of The Tolerance Of D-limonene InSaccharomyces Cerevisiae

D-limonene is a kind of important monoterpene and has been widely applied in foodindustry, chemical industry and make-up products industry, for its nature quality, such asantibacterial, antifungal, insecticidal activities and special odour. Its short metabolic pathwayfrom mevalonate pathway and its typical molecular structure made it to be the mostlyinvestigated model monoterpene for both chemical and biochemical research. As theapplication of monoterpenes becoming widely, biosynthesis of these products is obtainingmore and more attentions than ever before However, most of the monoterpenes, includingD-limonene can inhibit the cell growth of common prokaryotic and eukaryoticmicroorganisms. Therefore, the antibiotic property of restricted final yields of D-limonenesynthetize by microorganisms or the utilization of D-limonene containing citrus peel waste forbiofuel production. Different physical methods could be used to remove D-limonene prior tofermentation, but none of them can eliminate D-limonene with affordable cost. Therefore, theenhancement of D-limonene tolerance by Saccharomyces cerevisiae is significant both inbiosynthesis of D-limonene and in facilitating the utilization of citrus peel waste. S. cerevisiaeis the most commonly used host for the monoterpene production by metabolic engineering.Therefore its resistance to D-limonene should be improved to achieve high yield ofD-limonene.ATP-binding cassette (ABC) transporters are a group of important detoxification proteinsto endow the enhanced resistance to different natural products to microorganisms. In thiswork, the transcriptional level of typical ABC transporters of the S. cerevisiae CEN.PK2under85mg/L D-limonene stress was tested by real time PCR. The result showed that only thetranscriptional levels of PDR5, PDR15and YOR1were significantly improved. Besides,overexpression of these typical ABC transporters in S. cerevisiae failed to improve thetolerance of S. cerevisiae to D-limonene. The results suggested that to enhance the toleranceof S. cerevisiae to D-limonene could not be improved by the overexpression of the ABCtransporter genes.2DE and MALDI-TOF/TOF were used to study the mechanism of S. cerevisiaeCEN.PK2under85mg/L D-limonene stress.74different points in the2DE mapcorresponding to56different proteins were identified and analyzed by Gene Ontology (GO).The result of GO analysis showed that D-limonene could influence S. cerevisiae CEN.PK2ondifferent aspects: small molecule metabolic process, response to stress, protein process,carbohydrate metabolic process, DNA metabolic process, sulfur compound metabolic process,membrane organization, cytoskeleton organization, RNA processing, cell death, cell cycle andso on. Three main hypothesis were obtained:(1) the energy demand of S. cerevisiae underD-limonene stress was increased;(2) the protein regulating cell proliferation was destroyed byD-limonene, which directly result in the inhibition of growth;(3) D-limonene influenced themembrane, and damaged the lipid raft, which was severe damage to the cell.