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 Peatransit 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.
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.
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.3). 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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 334
Illegal NgoMIV site found at 726
Illegal NgoMIV site found at 793 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
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