해양 미생물 Kocuria gwangalliensis서 유래된 lycopene 생합성 유전자의 클로닝 및 특성 연구

Abstract

Carotenoids are the most widely distributed class of pigments in nature. They typically consist of a C40 hydrocarbon backbone often modified by different oxygen-containing functional group. Previously, we reported the isolation of a new marine bacterium, Kocuria gwangalliensis, which produces pink-orange pigment. A genes encoding a novel geranylgeranyl pyrophosphate (GGPP) synthase (crtE), phytoene synthase (crtB) and phytoene desaturase (crtI) from Kocuria gwangalliensis has been sequenced and expressed in Escherichia coli. The deduced amino acid sequences showed 59.6%, 70.9% and 74.1% identity with a putative GGPP synthase (crtE), phytoene synthase (crtB) and phytoene desaturase (crtI) from K. rhizophila. An expression plasmid containing the crtE, crtB and crtI genes were constructed, and Escherichia coli cells containing these plasmids produced the recombinant proteins of approximately 41 kDa, 34 kDa and 57 kDa, corresponding to the molecular weight of GGPP synthase, phytoene synthase and phytoene desaturase, respectively Biosynthesis of lycopene was obtained when the plasmid pCcrtE was co-transformed into E. coli containing the pRScrtBI carrying crtB, and crtI genes required for lycopene biosynthesis. pCcrtB was co-transformed into E. coli containing the pRScrtEI carrying crtE, and crtI genes, pCcrtI was co-transformed into E.coli containing the pRScrtEB carrying crtE, and crtB. The biochemical studies on the expressed proteins were performed by HPLC. The results obtained from this study will provide a wider base of knowledge on the primary structure of the K. gwangalliensis CrtE, CrtB and CrtI at the molecular level.