Isolation and functional characterization of two dioxygenases putatively involved in bixin biosynthesis in annatto ( Bixa orellana L.)

PeerJ

article

Victor Manuel Carballo-Uicab¹  Yair Cárdenas-Conejo²  Alba Adriana Vallejo-Cardona³  Margarita Aguilar-Espinosa¹  Jacobo Rodríguez-Campos⁴  Hugo Serrano-Posada²  José Alberto Narváez-Zapata⁵  Felipe Vázquez-Flota¹  Renata Rivera-Madrid¹ 

[1] Unidad de Bioquímica y Biología Molecular de Plantas;Laboratorio de Agrobiotecnología. CONACYT, Universidad de Colima;Unidad de Biotecnología Médica y Farmacéutica;Unidad de Servicios Analíticos y Metrológicos;Instituto Politécnico Nacional-Centro de Biotecnología Genómica

关键词: Annatto;    Carotenoid dioxygenase;    Bixin synthesis;    Bixa orellana;    Apocarotenoids;    Bixin aldehyde;   

DOI  :  10.7717/peerj.7064

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

Carotenoid cleavage dioxygenases (CCDs) are enzymes that have been implicated in the biosynthesis of a wide diversity of secondary metabolites with important economic value, including bixin. Bixin is the second most used pigment in the world’s food industry worldwide, and its main source is the aril of achiote (Bixa orellana L.) seeds. A recent transcriptome analysis of B. orellana identified a new set of eight CCD members (BoCCD4s and BoCCD1s) potentially involved in bixin synthesis. We used several approaches in order to discriminate the best candidates with CCDs genes. A reverse transcription-PCR (RT-qPCR) expression analysis was carried out in five developmental stages of two accessions of B. orellana seeds with different bixin contents: (P13W, low bixin producer and N4P, high bixin producer). The results showed that three BoCCDs (BoCCD4-1, BoCCD4-3, and BoCCD1-1) had an expression pattern consistent with bixin accumulation during seed development. Additionally, an alignment of the CCD enzyme family and homology models of proteins were generated to verify whether the newly proposed CCD enzymes were bona fide CCDs. The study confirmed that these three enzymes were well-preserved and belonged to the CCD family. In a second selection round, the three CCD genes were analyzed by in situ RT-qPCR in seed tissue. Results indicated that BoCCD4-3 and BoCCD1-1 exhibited tissue-specific expressions in the seed aril. To test whether the two selected CCDs had enzymatic activity, they were expressed in Escherichia coli; activity was determined by identifying their products in the crude extract using UHPLC-ESI-QTOF-MS/MS. The cleavage product (bixin aldehyde) was also analyzed by Fourier transform infrared. The results indicated that both BoCCD4-3 and BoCCD1-1 cleave lycopene in vitro at 5,6-5′,6′.

【 授权许可】

CC BY