title: Coordinate expression of multiple bacterial carotenoid genes in canola leading to altered carotenoid production.
abstract: Carotenoids have drawn much attention recently because of their potentially positive benefits to human health as well as their utility in both food and animal feed. Previous work in canola (Brassica napus) seed over-expressing the bacterial phytoene synthase gene (crtB) demonstrated a change in carotenoid content, such that the total levels of carotenoids, including phytoene and downstream metabolites like beta-carotene, were elevated 50-fold, with the ratio of beta- to alpha-carotene being 2:1. This result raised the possibility that the composition of metabolites in this pathway could be modified further in conjunction with the increased flux obtained with crtB. Here we report on the expression of additional bacterial genes for the enzymes geranylgeranyl diphosphate synthase (crtE), phytoene desaturase (crtI) and lycopene cyclase (crtY and the plant B. napus lycopene beta-cyclase) engineered in conjunction with phytoene synthase (crtB) in transgenic canola seed. Analysis of the carotenoid levels by HPLC revealed a 90% decrease in phytoene levels for the double construct expressing crtB in conjunction with crtI. The transgenic seed from all the double constructs, including the one expressing the bacterial crtB and the plant lycopene beta-cyclase showed an increase in the levels of total carotenoid similar to that previously observed by expressing crtB alone but minimal effects were observed with respect to the ratio of beta- to alpha-carotene compared to the original construct. However, the beta- to alpha-carotene ratio was increased from 2:1 to 3:1 when a triple construct consisting of the bacterial phytoene synthase, phytoene desaturase and lycopene cyclase genes were expressed together. This result suggests that the bacterial genes may form an aggregate complex that allows in vivo activity of all three proteins through substrate channeling. This finding should allow further manipulation of the carotenoid biosynthetic pathway for downstream products with enhanced agronomic, animal feed and human nutritional values.
authors.affiliation: Monsanto Company, Calgene Campus, 1920 Fifth St, Davis, CA 95616, USA. monica.p.ravello@monsanto.com
authors.forename: Monica P
authors.initials: MP
authors.lastname: Ravanello
authors.forename: Dangyang
authors.initials: D
authors.lastname: Ke
authors.forename: Julie
authors.initials: J
authors.lastname: Alvarez
authors.forename: Bihua
authors.initials: B
authors.lastname: Huang
authors.forename: Christine K
authors.initials: CK
authors.lastname: Shewmaker
mesh_terms: D029688:Brassica napus / Q000235:genetics* / Q000378:metabolism*; D002338:Carotenoids / Q000096:biosynthesis* / Q000235:genetics*; D004885:Erwinia / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D018014:Gene Transfer Techniques; D024861:Genetic Enhancement / Q000379:methods*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D002338:Carotenoids