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首页> 外文期刊>Journal of Biotechnology >Investigation of cellular targeting of carotenoid pathway enzymes in Pichia pastoris
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Investigation of cellular targeting of carotenoid pathway enzymes in Pichia pastoris

机译：巴斯德毕赤酵母中类胡萝卜素途径酶的细胞靶向研究

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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.
机译：在毕赤酵母X-33中研究了番茄红素生物合成酶的细胞靶向。将三种番茄红素途径酶CrtE，CrtB和CrtI与带有或不带有过氧化物酶体靶向序列（PTS1）的荧光EGFP融合，然后在巴斯德毕赤酵母中表达。在YPD中生长巴斯德毕赤酵母时，发现PTS1融合酶位于过氧化物酶体中，而未与PTS1融合的酶在整个细胞中平均分布。在过氧化物酶体增殖培养基YPOT中生长的巴斯德毕赤酵母菌株中也观察到了类似的靶向模式。对分离的过氧化物酶体的荧光图像的分析表明，过氧化物酶体中主要存在PTS1融合酶，而少量未与PTS1融合的酶非特异性地传递给过氧化物酶体。这些结果表明PTS1特异性地将番茄红素途径的酶靶向到过氧化物酶体中，并且该靶向途径足够强大以克服其固有的靶向程序。总之，我们首先表明，可以将类胡萝卜素酶靶向到重组宿主的特定细胞位置，这种靶向策略可以作为后续开发微生物复杂途径工程的基础。

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