Comparative Analysis of beta-Carotene Hydroxylase Genes for Astaxanthin Biosynthesis
ABSTRACT Astaxanthin (3,3'-dihydroxy-4,4'-diketo-β-carotene) (1) is a carotenoid of significant commercial value due to its superior antioxidant potential, application as a component of animal feeds, and ongoing research that links its application to the treatment and prevention of human pathologies. The high commercial cost of 1 is also based upon its complex synthesis. Chemical synthesis has been demonstrated, but produces a mixture of stereoisomers with limited applications. Production from biological sources is limited to natural producers with complex culture requirements. The biosynthetic pathway for 1 is well studied; however, questions remain that prevent optimized production in heterologous systems. Presented is a direct comparison of 12 β-carotene (2) hydroxylases derived from archaea, bacteria, cyanobacteria, and plants. Expression in Escherichia coli enables a comparison of catalytic activity with respect to zeaxanthin (3) and 1 biosynthesis. The most suitable β-carotene hydroxylases were subsequently expressed from an efficient dual expression vector, enabling 1 biosynthesis at levels up to 84% of total carotenoids. This supports efficient 1 biosynthesis by balanced expression of β-carotene ketolase and β-carotene hydroxylase genes. Moreover, our work suggests that the most efficient route for astaxanthin biosynthesis proceeds by hydroxylation of β-carotene to zeaxanthin, followed by ketolation.
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ABSTRACT: L. barbarum contains high contents of zeaxanthin, which is produced by conversion of β-carotene into zeaxanthin. β-Carotene hydroxylase catalyzes this reaction. We cloned a cDNA (chyb) encoding β-carotene hydroxylase (Chyb) from L. barbarum leaf. A 939-bp full-length cDNA sequence was determined with 3’-Rapid Amplification of cDNA End (3’-RACE) assay encoding a deduced Chyb protein (34.8 kDa) with a theoretical pI 8.36. Bioinformatics analysis showed that the L. barbarum Chyb located in the chloroplast. Further, to investigate the catalytic activity of L. barbarum Chyb, complementation analysis was conducted in Escherichia coli. The results strongly demonstrated that Chyb chould catalyze β-carotene to produce zeaxanthin. Thus, this study suggests that L. barbarum β-carotene hydroxylase could be a means of zeaxanthin production by genetic manipulation in Escherichia coli.This article is protected by copyright. All rights reservedBiotechnology and Applied Biochemistry 03/2014; 61(6). DOI:10.1002/bab.1224
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ABSTRACT: Astaxanthin, a high value ketocarotenoid used in the pharmaceutical and nutraceutical industries is mainly produced from green alga, Haematococcus pluvialis. It is biosynthesized by the action of key enzyme, β-carotene ketolase (BKT) on β-carotene through intermediates echinenone and canthaxanthin. In this study, the (-carotene ketolase (bkt) gene was isolated from H. pluvialis and cloned in a vector pRT100 and further mobilized to a binary vector pCAMBIA 1304. The T-DNA of pCAMBIA 1304, which consists of cloned bkt was successfully transformed to H. pluvialis through Agrobacterium mediation. The cloning and transformation of bkt in H. pluvialis was confirmed by southern blotting and also by PCR analysis. Total carotenoids and astaxanthin content in the transformed cells were found to be two to three fold higher while the intermediates like echinenone and canthaxanthin were found to be eight to ten fold higher than in the control cells. The expression level of carotenogenic genes like phytoene synthase (psy), phytoene desaturase (pds), lycopene cyclase (lcy), bkt, and β-carotene hydroxylase (bkh) were found to be higher in transformed cells compared to the non-transformed (NT) H. pluvialis. Copyright © 2015. Published by Elsevier B.V.Journal of Biotechnology 01/2015; 196-197. DOI:10.1016/j.jbiotec.2015.01.006