Part:BBa_K1959002

Modification of β-carotene hydroxylase (BHY)

This part codes for beta-carotene hydroxylase(BHY), which can catalyze the convertion of beta-carotene to zeaxanthin. A Pea transit peptide has been fused to the coding sequenceand and codon optimization has been made in order to efficiently express BHY in Orazy sativa.

Usage and Biology

Astaxanthin is a high-value keto-carotenoid which processes a powerful antioxidant activity which board health implications.Given by its medical value,astaxanthin syhthesis, especialy, its biosynthesis, attracts considerable interest of the scientists. Haematococcus pluvialis is thought to be the promosing bioresource of astaxanthin bioproduct. Beta-carotene hydroxylase (BHY, EC 1.14.13.129) is one the key enzymes of the astaxanthin biosynthesis in Haematococcus pluvialis. It catalyzes the conversion of beta-carotene to zeaxanthin, the precusor of astaxanthin(fig.1). Under the catalyzation of BHY, β-ionone of β-carotene converts into a 3-hydroxy-β-ionone of zeaxanthin, with the concomitant oxidation of NADH and two molecules oxygen to NAD+ and two molecules H20. In our project, we use β-carotene hydroxylase to convertβ-carotene into zeaxanthin and subsequently complete the overall astaxanthin biosynthetic reaction. To better express BHY into rice (Orazy sativa), the coding sequence was codon optimized. In additon, a Pea transit peptide was fused to the coding sequence in order to avoid degradation.

Transciptional activity

Semi-quantitative RT-PCR was performed to detect the expression level of BHY involed 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 electrophoretic gel, indicated that BHY gene was capable to transcript 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]