title: Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis.
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.
authors.affiliation: Central Laboratories for Frontier Technology, Kirin Holdings Co. Ltd., i-BIRD, Suematsu, Ishikawa 921-8836, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Harada
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Shindo
authors.forename: Kanoko
authors.initials: K
authors.lastname: Iki
authors.forename: Ayuko
authors.initials: A
authors.lastname: Teraoka
authors.forename: Sho
authors.initials: S
authors.lastname: Okamoto
authors.forename: Fengnian
authors.initials: F
authors.lastname: Yu
authors.forename: Jun-ichiro
authors.initials: J
authors.lastname: Hattan
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Utsumi
authors.forename: Norihiko
authors.initials: N
authors.lastname: Misawa
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005288:Ferredoxins / Q000378:metabolism; D015870:Gene Expression; D020939:Zingiber officinale / Q000201:enzymology* / Q000235:genetics; D008798:Mevalonic Acid / Q000378:metabolism; D005810:Multigene Family; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D046937:Nostoc / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D012717:Sesquiterpenes / Q000378:metabolism*
chemical_list: D005288:Ferredoxins; D012717:Sesquiterpenes; D003577:Cytochrome P-450 Enzyme System; C431677:germacrene A hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D008798:Mevalonic Acid