title: Genetic and biochemical basis for alternative routes of tocotrienol biosynthesis for enhanced vitamin E antioxidant production.
abstract: Vitamin E tocotrienol synthesis in monocots requires homogentisate geranylgeranyl transferase (HGGT), which catalyzes the condensation of homogentisate and the unsaturated C20 isoprenoid geranylgeranyl diphosphate (GGDP). By contrast, vitamin E tocopherol synthesis is mediated by homogentisate phytyltransferase (HPT), which condenses homogentisate and the saturated C20 isoprenoid phytyl diphosphate (PDP). An HGGT-independent pathway for tocotrienol synthesis has also been shown to occur by de-regulation of homogentisate synthesis. In this paper, the basis for this pathway and its impact on vitamin E production when combined with HGGT are explored. An Arabidopsis line was initially developed that accumulates tocotrienols and homogentisate by co-expression of Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) and Escherichia coli bi-functional chorismate mutase/prephenate dehydrogenase (TyrA). When crossed into the vte2-1 HPT null mutant, tocotrienol production was lost, indicating that HPT catalyzes tocotrienol synthesis in HPPD/TyrA-expressing plants by atypical use of GGDP as a substrate. Consistent with this, recombinant Arabidopsis HPT preferentially catalyzed in vitro production of the tocotrienol precursor geranylgeranyl benzoquinol only when presented with high molar ratios of GGDP:PDP. In addition, tocotrienol levels were highest in early growth stages in HPPD/TyrA lines, but decreased strongly relative to tocopherols during later growth stages when PDP is known to accumulate. Collectively, these results indicate that HPPD/TyrA-induced tocotrienol production requires HPT and occurs upon enrichment of GGDP relative to PDP in prenyl diphosphate pools. Finally, combined expression of HPPD/TyrA and HGGT in Arabidopsis leaves and seeds resulted in large additive increases in vitamin E production, indicating that homogentisate concentrations limit HGGT-catalyzed tocotrienol synthesis.
authors.affiliation: National Key Laboratory of Crop Genetic Improvement and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
authors.forename: Chunyu
authors.initials: C
authors.lastname: Zhang
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: Cahoon
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Hunter
authors.forename: Ming
authors.initials: M
authors.lastname: Chen
authors.forename: Jixiang
authors.initials: J
authors.lastname: Han
authors.forename: Edgar B
authors.initials: EB
authors.lastname: Cahoon
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000737:chemistry*; D017360:Arabidopsis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors / Q000235:genetics; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames; D018515:Plant Leaves / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D024508:Tocotrienols / Q000378:metabolism*; D014170:Transformation, Genetic; D019076:Transgenes; D014810:Vitamin E / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000975:Antioxidants; D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D011106:Polyisoprenyl Phosphates; D024508:Tocotrienols; C076398:TyrA protein, Bacteria; D014810:Vitamin E; D049308:Dioxygenases; D019883:Alkyl and Aryl Transferases; C446531:HPT1 protein, Arabidopsis; C569419:homogentisate geranylgeranyl transferase, Hordeum vulgare; C002963:geranylgeranyl pyrophosphate