RRC ID 12328
Author Harada H, Shindo K, Iki K, Teraoka A, Okamoto S, Yu F, Hattan J, Utsumi R, Misawa N.
Title Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis.
Journal Appl. Microbiol. Biotechnol.
Abstract Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB and NsFER), which are derived from Pseudomonas putida and cyanobacterium Nostoc sp. strain PCC 7120, respectively, as well as three higher-plant NADPH-P450 reductases, the Arabidopsis thaliana ATR2 and two corresponding enzymes derived from ginger (Zingiber officinale), named ZoRED1 and ZoRED2. We also constructed plasmids for functional analysis of two P450s, α-humulene-8-hydroxylase (CYP71BA1) from shampoo ginger (Zingiber zerumbet) and germacrene A hydroxylase (P450NS; CYP110C1) from Nostoc sp. PCC 7120, and co-transformed E. coli with each of the pAC-Mv-based plasmids. Production levels of 8-hydroxy-α-humulene with recombinant E. coli cells (for CYP71BA1) were 1.5- to 2.3-fold higher than that of a control strain without the mevalonate-pathway genes. Level of the P450NS product with the combination of NsRED and NsFER was 2.9-fold higher than that of the CamA and CamB. The predominant product of P450NS was identified as 1,2,3,5,6,7,8,8a-octahydro-6-isopropenyl-4,8a-dimethylnaphth-1-ol with NMR analyses.
Volume 90(2)
Pages 467-76
Published 2011-4
DOI 10.1007/s00253-010-3062-9
PMID 21229242
MeSH Arabidopsis / enzymology Arabidopsis / genetics Cloning, Molecular Cytochrome P-450 Enzyme System / analysis* Cytochrome P-450 Enzyme System / metabolism* Escherichia coli / enzymology Escherichia coli / genetics Ferredoxins / metabolism Gene Expression Ginger / enzymology* Ginger / genetics Mevalonic Acid / metabolism Multigene Family NADPH-Ferrihemoprotein Reductase / metabolism Nostoc / enzymology* Nostoc / genetics Oxidation-Reduction Plasmids / genetics Plasmids / metabolism Pseudomonas putida / enzymology Pseudomonas putida / genetics Sesquiterpenes / metabolism*
IF 3.34
Times Cited 15
Arabidopsis / Cultured plant cells, genes rpc00008