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.

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. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 334
  Illegal NgoMIV site found at 726
  Illegal NgoMIV site found at 793
  
• 1000
  COMPATIBLE WITH RFC[1000]