Investigation of cellular targeting of carotenoid pathway enzymes in Pichia pastoris.
Cellular targeting of lycopene biosynthetic enzymes was investigated in Pichia pastoris X-33. Three lycopene pathway enzymes, CrtE, CrtB, and CrtI, were fused to fluorescent EGFPs with or without a peroxisomal targeting sequence (PTS1) and then expressed in P. pastoris. When P. pastoris was grown in YPD, the PTS1 fusion enzymes were found to be localized in peroxisomes, whereas the enzymes not fused with PTS1 were equally distributed throughout the entire cell. A similar targeting pattern was also observed in P. pastoris strains that were grown in peroxisome-proliferating medium, YPOT. Analysis of the fluorescent images of isolated peroxisomes showed that the PTS1 fused enzymes were dominantly present in peroxisomes whereas small amount of the enzymes not fused with PTS1 were non-specifically sent to peroxisomes. These results indicate that PTS1 specifically target lycopene pathway enzymes into peroxisomes and this targeting pathway was strong enough to overcome their inherent targeting program. In conclusion, we first showed that carotenogenic enzymes can be targeted into the specific cellular location of recombinant hosts and this targeting strategy can serve as the basis for the subsequent development of sophisticated pathway engineering in microorganisms.
Department of Molecular Science and Technology, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon 443-749, South Korea. pclee2000@gmail.com
Pyung Cheon
PC
Lee
Young geol
Yg
Yoon
Claudia
C
Schmidt-Dannert
D002338:Carotenoids / Q000378:metabolism*; D000077276:Lycopene; D008856:Microscopy, Fluorescence; D020675:Peroxisomes / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000378:metabolism*; D015398:Signal Transduction
D002338:Carotenoids; D000077276:Lycopene