期刊
JOURNAL OF BIOSCIENCE AND BIOENGINEERING
卷 128, 期 6, 页码 755-765出版社
SOC BIOSCIENCE BIOENGINEERING JAPAN
DOI: 10.1016/j.jbiosc.2019.06.008
关键词
Cytochrome P450; Carotenoids hydroxylase; Phaeodactylum tricornutum; Cloning; Identification; Functional characterization
资金
- China Postdoctoral Science Foundation [2018M631768]
- Science and Technology Planning Project of Liaoning Province [20170540047]
- Applying Basic Research Planning Project of Shanxi Province (Science and Technology Foundation for Youths) [201801D221250]
- Key Research and Development (RAMP
- D) Planning Project of Shanxi Province [201803D31063]
- State Ministry of Agriculture 948 Project [2014-Z39]
- Shanxi Province Key Projects of Coal-based Science and Technology [FT-2014-01]
- Shanxi Scholarship Council of China [2015-064]
- Key Project of the Key Research and Development Program of Shanxi Province, China [201603D312005]
The diatom microalgal Phaeodactylum tricornutum accumulates a large amount of fucoxanthin. Carotenoids hydroxylases (CHYs) play key roles in fucoxanthin biosynthesis in diatoms. However, not any type of CHYs had been identified in P. tricornutum. In this study, two genes (designated Ptrcyp97b1 and Ptrcyp97b2) were cloned, identified and functionally characterized. They shared high sequence identity (50-94 %) with lutein deficient 1-like proteins from other eukaryotes. The typical catalytic active motifs of cytochrome P450s (CYP) were detected in the amino acid sequences of PtrCYP97B1 and PtrCYP97B2. The two genes were probably due to gene duplication. Ptrcyp97b1 and Ptrcyp97b2 transcriptional expression was up-regulated with distinct patterns under high light conditions. The metabolic profiles of the major carotenoids (beta-carotene, zeaxanthin, diadinoxanthin, diatoxanthin and fucoxanthin) were determined based on the high performance liquid chromatography method. The fucoxanthin and diatoxanthin contents were increased, while the beta-carotene content was decreased. By truncation of the N-terminal trans-membrane anchor or chloroplast transit peptide and addition of a 6 x His-tag, PtrCYP97B1 and PtrCYP97B2 were separately heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. Functional analysis showed that PrtCYP97B2 was able to catalyze the hydroxylation of the beta-rings of beta-carotene to produce zeaxanthin in beta-carotene-accumulating E. coli BL21(DE3) cells. PtrCYP97B1 might have the ability to catalyze the hydroxylation of other substrates other than beta-carotene. These results contribute to the further elucidation of xanthophyll biosynthesis in diatoms. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.
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