Part:BBa_K1959002

Modification of β-carotene hydroxylase (BHY)

This part contains the coding sequence (CDS) of β-carotene hydroxylase of algae (BHY, EC 1.14.13.129), which catalyzes the conversion of β-carotene to zeaxanthin. A Pea transit peptide of RUBISCO small subunit has been fused to BHY and the codon has been optimized for rice.

Usage and Biology

　Astaxanthin is a keto-carotenoid which processes a powerful antioxidant activity with board health implications. Given its high value, astaxanthin synthesis, especially, its biosynthesis attracts considerable interest of the scientists. Haematococcus pluvialis is a promising bioresource of astaxanthin. β-carotene hydroxylase (BHY, EC 1.14.13.129) is one key enzymes of the astaxanthin biosynthesis in Haematococcus pluvialis. It catalyzes the conversion of β-carotene to zeaxanthin, the precursor of astaxanthin (Figure.1). In this reaction, β-ionone of β-carotene converts into a 3-hydroxy-β-ionone of zeaxanthin, with the concomitant oxidation of NADH and two oxygen to NAD⁺ and two H₂0 (Figure.2).
　In our project, we used BHY to convert β-carotene into zeaxanthin and subsequently complete the overall astaxanthin biosynthetic reaction. The CDS of BHY was codon-optimized for a better expression in rice. In addition, a Pea transit peptide was fused to the BHY for correct sorting of BHY into the plastid.

Figure.1　The reconstructed biosynthesis pathway of astaxanthin in the endosperm of aSTARice.
The dotted arrows indicate pathway is absent in rice endosperm. The solid arrows indicate the existence of carotenogenic reactions. The red arrows indicate the reactions catalyzed by four exogenous transgenic PSY, CrtI, BHY and BKT.

Figure.2　Enzymatic reaction of BHY

Transciptional activity

　Semi-quantitative RT-PCR was performed to detect the expression level of BHY involved in astaxanthin biosynthesis, total RNA of transgenic rice seeds were extracted and cDNA was synthesized from 1μg DNase-treated RNA.

Figure.3　RT-PCR analyses of expression levels of BHY genes in several transgenic rice.
Rice OsActin1 was as an internal control. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).

　Expected bands of the BHY gene were observed on the gel, indicated that BHY gene was transcribed in endosperm.

aSTRice Phenotype

　BHY is the key enzyme of astaxanthin biosynthesis. Rice without BHY expression are unable to accumulate astaxanthin, in other words, rice would not appear in orange-red color (Rice marked as “Wild Type” and “Golden Rice” in Figure.4). Orange-red-color rice is the consequence of the cooperation of BHY gene and other astaxanthin-producing genes. Therefore, the phenotype of aSTARice demonstrates that the BHY gene is capable to express in the rice cell.

Figure.4 The polished rice phenotype of aSTARice.
Wild type rice with transgenic PSY and Crt1 produces β-carotene resulting in Golden Rice; wild type rice with transgenic PSY, Crt1, BHY and BKT produces astaxanthin resulting in aSRARice.

　BHY is the key enzyme of astaxanthin biosynthesis. Rice without BHY fails to accumulate astaxanthin, appearing white or gold color in “wild type” and “Golden Rice”, respectively. aSTARice contains astaxanthin and appears orange-red-color because of the coordinated expression of BHY gene and other key astaxanthin biosynthetic genes (Figure. 3). Therefore, the phenotype of aSTARice indicated that the BHY gene is a functional gene in rice.

Validation of astaxanthin by HPLC analysis

　To further confirm the synthetic astaxanthin in aSTARice, HPLC was performed to analyze the pigment composition. Astaxanthin was identified on the basis of retention times related to standard sample.

Figure 5 Validation of astaxanthin by HPLC.
HPLC chromatogram of methanol extracts from the seeds of transgenic aSTARice (red line) and wild-type rice (blue line). HPLC analysis recorded at 480 nm of extracts.

　According to the retention time of standard astaxanthin sample, astaxanthin compound of extracts from aSTARice can be confirmed. In addition, astaxanthin possessed the biggest peak area in the carotenoids profile, indicated that astaxanthin was the predominant carotenoid in aSTARice.

BHY is Essential for Biosynthesis of Astaxanthin

　Seeds of T₂ generation were subjected to HPLC and qRT-PCR analysis. The transcriptional level of BHY was low in Lines #13, which also accumulate low level of astaxanthin. These results proved that BHY is essential for biosynthesis of astaxanthin. Thus, coordinated expression of functional BHY gene together with other key genes enables a high astaxanthin accumulation.

Figure.6 Relative expression levels of four foreign astaxanthin biosynthetic genes and astaxanthin content in seeds of wild-type and aSTARice T₂ generation.
Expression levels are normalized to OsActin transcript level. All reactions were carried out in triplicate, and each experiment was repeated at least twice. Error bars indicate ± SEM. DW, dry weight.

Sequence and Features

Assembly Compatibility: