Cloning and identification of a novel β-carotene hydroxylase gene from Haematococcus pluvialis and its function in Escherichia coli

Haematococcus pluvialis is a freshwater microalga distributed worldwide and is considered as the best natural source of astaxanthin for human absorption. In this study, a novel beta-carotene hydroxylase (HpCrtR-b2) was discovered from an Iso-Seq database of H. pluvialis and was cloned by RT-PCR using designed specific primers. As a comparison, HpCrtR-b1 gene previously reported by other researchers was also cloned. Sequencing results suggested that HpCrtR-b2 gene was 1177 bp in length encoding 297 amino acids, which only has 73% identities with HpCrtR-b1 gene encoding 292 amino acids. Further analysis of deduced amino acids revealed that HpCrtR-b2 contains the signature FA_hydroxylase domain and histidine-rich motifs as HpCrtR-b1. It is interesting that a chloroplast transit peptide was predicted in HpCrtR-b2 while a mitochondrial transit peptide was in HpCrtR-b1, indicating two HpCrtR-b genes might have different specific function. The hetero-expression analysis suggested that HpCrtR-b2 gene has the enzyme activity to convert beta-carotene into zeaxanthin. The HPLC quantification experiments showed that E. coli transformants harboring HpCrtR-b2 produced significant higher amount of zeaxanthin than that harboring HpCrtR-b1 and even produced 1.5 times of zeaxanthin than that harboring CrtZ (a bacterium derived beta-carotene hydroxylase). Therefore, the novel beta-carotene hydroxylase gene found in this study provides a more efficient pathway for zeaxanthin biosynthesis and might contribute to other carotenoids biosynthesis with higher productivity.

Automated summary

A novel beta-carotene hydroxylase gene, HpCrtR-b2, from Haematococcus pluvialis was discovered in this study, showing higher enzyme activity and zeaxanthin production compared to the previously reported HpCrtR-b1 gene. The two genes may have different specific functions, with HpCrtR-b2 providing a more efficient pathway for carotenoid biosynthesis with higher productivity.