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

Abstract

Carotene cleavage dioxygenases (CCDs) are a large family of Fe2+ dependent enzymes responsible for the production of a wide variety of apocarotenoids, such as bixin. Among the natural apocarotenoids, bixin is second in economic importance. It has a red-orange color and is produced mainly in the seeds of B. orellana. The biosynthesis of bixin aldehyde from the oxidative cleavage of lycopene at 5,6/5′,6′ bonds by a CCD is considered the first step of bixin biosynthesis. Eight BoCCD (BoCCD1-1, BoCCD1-3, BoCCD1-4, CCD4-1, BoCCD4-2, BoCCD4-3 and BoCCD4-4) genes potentially involved in the first step of B. orellana bixin biosynthesis have been identified. However, the cleavage activity upon lycopene to produce bixin aldehyde has only been demonstrated for BoCCD1-1 and BoCCD4-3. Using in vivo (Escherichia coli) and in vitro approaches, we determined that the other identified BoCCDs enzymes (BoCCD1-3, BoCCD1-4, BoCCD4-1, BoCCD4-2, and BoCCD4-4) also participate in the biosynthesis of bixin aldehyde from lycopene. The LC-ESI-QTOF-MS/MS analysis showed a peak corresponding to bixin aldehyde (m/z 349.1) in pACCRT-EIB E. coli cells that express the BoCCD1 and BoCCD4 proteins, which was confirmed by in vitro enzymatic assay. Interestingly, in the in vivo assay of BoCCD1-4, BoCCD4-1, BoCCD4-2, and BoCCD4-4, bixin aldehyde was oxidized to norbixin (m/z 380.2), the second product of the bixin biosynthesis pathway. In silico analysis also showed that BoCCD1 and BoCCD4 proteins encode functional dioxygenases that can use lycopene as substrate. The production of bixin aldehyde and norbixin was corroborated based on their ion fragmentation pattern, as well as by Fourier transform infrared (FTIR) spectroscopy. This work made it possible to clarify at the same time the first and second steps of the bixin biosynthesis pathway that had not been evaluated for a long time.

Gene expression profile during seed development of Bixa orellana accessions varying in bixin pigment

“…These genes are closely connected to lycopene production in plastids ( Simkin, 2021 ). Lycopene is also considered the precursor of bixin through the selective cleavage of BoCCDs enzymes ( Bouvier et al., 2003 ; Carballo-Uicab et al., 2019 ; Us-Camas et al., 2022 ) to form bixin aldehyde, and then this molecule is oxidized by an BoALDH to produce norbixin, and finally, the norbixin product is methylated by METs to produce bixin ( Jako et al., 2002 ; Bouvier et al., 2003 ). Only BoCCDs involved with the bixin production were included in this analysis; those belonging to the group of the 9-cis epoxycarotenoid dioxygenases involved with the biosynthesis of ABA were discarded.…”

“…In the current study, BoCCD1-4 and several BoCCDs of the subfamily 4 ( BoCCD4-2 , BoCCD4-3 , and BoCCD4-4 ) show a high correlation to bixin, particularly in S3 and S5, suggesting an active role in the apocarotenoid biosynthesis. In general, diverse BoCCD gene members of both subfamilies have demonstrated involvement with the bixin biosynthesis ( Cárdenas-Conejo et al., 2015 ; Carballo-Uicab et al., 2019 ; Us-Camas et al., 2022 ), and its subcellular compartmentalization has been hypothesized ( Cárdenas-Conejo et al., 2015 ). BoCCD4-3 gene expression has also been correlated with the increase in bixin in the leaves of another accession ( Faria et al., 2020 ).…”

“…It was suggested that lycopene is converted to bixin in sequential steps by three types of enzymes, carotenoid dioxygenase ( CCDs ), aldehyde dehydrogenase ( ALDHs ) and methyltransferases ( METs ) ( Jako et al., 2002 ). By using a heterologous system, different BoCCDs gene members ( BoCCD1-1 , BoCCD1-3 , BoCCD1-4 , BoCCD4-1 , BoCCD4-2 , BoCCD4-3 , and BoCCD4-4 ) can produce bixin aldehyde, a key precursor in bixin production ( Cárdenas-Conejo et al., 2015 ; Carballo-Uicab et al., 2019 ; Us-Camas et al., 2022 ). Other genes related to apocarotenoids have also been studied, such as the MET members of the SABATH family ( Wei et al., 2021 ).…”

Identification of the carotenoid cleavage dioxygenase genes and functional analysis reveal DoCCD1 is potentially involved in beta-ionone formation in Dendrobium officinale

“…( Auldridge et al, 2006a ; Vogel et al, 2008 ; Ahrazem et al, 2016b ; Zheng et al, 2021 ; Figure 1 ). The plastid-located CCD4 has higher substrate specificity ( Us-Camas et al, 2022 ). CCD1 and CCD4 cleave carotenoids to increase the content of apocarotenoids and therefore have important influences on plant quality, such as the carotenoid contents in crops ( Ko et al, 2018 ; Thakur et al, 2021 ), the color and flavors in vegetables and fruits ( Campbell et al, 2010 ; Fukamatsu et al, 2013 ; Ilg et al, 2014 ), the flower color and fragrance ( Ohmiya et al, 2006 ; Huang et al, 2009 ; Chiou et al, 2010 ; Zhang et al, 2015 ; Gao et al, 2021 ) and tea quality ( Wang et al, 2020a , b ).…”

“…CCD1 in tomato was responsible for the formation of volatile isoprenes including beta-ionone, pseudoionone, and geranylacetone ( Simkin et al, 2004a ). CCD1 and CCD4 in Bixa orellana were recently found to be involved in the bixin biosynthesis ( Us-Camas et al, 2022 ). Moreover, CCD1 have great effects on the emission of beta-ionone.…”