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

Abstract

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′.

In silico analysis of antidiabetic potential of phenolic compounds from blue corn (Zea mays L.) and black bean (Phaseolus vulgaris L.)

“…(Anguiano-Sevilla et al, 2018). The ESI conditions were a positive ion mode capillary voltage 32 Kv; SAMPLIng cone 42; source offset 80; and N2 cone gas flows: cone gas 50 1/h: desolvation gas 5000 MS/MS cone voltage: 35V and energy collision: 25V dry gas (Carballo-Uicab et al, 2019).…”

Carotenoids and Apocarotenoids in Planta: Their Role in Plant Development, Contribution to the Flavour and Aroma of Fruits and Flowers, and Their Nutraceutical Benefits

“…Furthermore, Ilg et al [208], using both in vitro and in vivo assays, demonstrated that the activity of OsCCD1 converts lycopene into pseudoionone (Figure 4) and MHO (Figure 5), suggesting that the 7,8(7 ,8 ) double bonds of acyclic carotenoid ends constitute a novel cleavage site for the CCD1 plant subfamily. Carballo-Conejo et al [222] also identified a CCD1 lycopene-specific 5,6(5 ,6 )-cleavage dioxygenase (BoCCD1-1) from Bixa orellana, responsible for the formation of bixin dialdehyde and MHO [223,224]. Bixin dialdehyde is the precursor for the formation of the dye bixin/annatto (Figure 5; see Section 3.6.1).…”

“…BoCCD1-1 gene expression correlated with bixin accumulation in B. orellana [224], suggesting that BoCCD1-1 and its homologue BoCCD1-2 could be involved in the cleavage of lycopene in seed to form bixin. However, data from a study by Cárdenas-Conejo et al [222,223] indicated that although BoCCD1-1 is expressed in immature seed, it is also expressed in green tissue (leaf), and BoCCD1-2 was preferentially expressed in leaf. These authors also identified two additional CCD1 s, BoCCD1-3 and BoCCD1-4, which were shown to be expressed in immature seeds at 1.5-fold and 10-fold the levels found in leaf, respectively suggesting that BoCCD1-3 and BoCCD1-4 are more likely involved in the cleavage of lycopene to form bixin dialdehyde in the seed (Figure 5).…”

Identifying Bixa orellana L. New Carotenoid Cleavage Dioxygenases 1 and 4 Potentially Involved in Bixin Biosynthesis

“…Derived from achiote transcriptomes analysis, Cárdenas-Conejo et al (2015) identified eight candidate BoCCDs genes involved in bixin biosynthesis, distributed in hypothetical cytosolic and plastidic pathways, four of them belonging to the BoCCD1 subfamily: BoCCD1-1, BoCCD1-2, BoCCD1-3, and BoCCD1-4; and four of them to the BoCCD4 subfamily: BoCCD4-1, BoCCD4-2, BoCCD4-3 and, BoCCD4-4 (Cárdenas-Conejo et al, 2015). Recently, Carballo-Uicab et al (2019) demonstrated that the recombinant proteins BoCCD1-1 and BoCCD4-3 exhibited cleavage activity at the 5,6/5 ,6 positions of lycopene producing bixin aldehyde, indicating their involvement in the first step of bixin biosynthesis (Carballo-Uicab et al, 2019). Other BoCCD1 and BoCCD4 genes have been isolated from different B. orellana cultivars, but their role in bixin biosynthesis has not been unveiled (Rodríguez-Ávila et al, 2011b;Soares et al, 2011;Sankari et al, 2016).…”

“…Other BoCCD1 and BoCCD4 genes have been isolated from different B. orellana cultivars, but their role in bixin biosynthesis has not been unveiled (Rodríguez-Ávila et al, 2011b;Soares et al, 2011;Sankari et al, 2016). In general, the expression levels of all BoCCD genes identified in the transcriptomes increased in the immature stages of the seed (S3 and S4) when bixin reached its maximum levels and decreased in the mature stages of the seed (S5) when bixin levels dropped dramatically in the two accessions that produce contrasting levels of bixin (N4P and P13W) (Carballo-Uicab et al, 2019). Therefore, we hypothesize spatio-temporal participation in bixin biosynthesis of all the BoCCD1 and BoCCD4 enzymes identified in the transcriptomes of B. orellana.…”