Pathway engineering for efficient biosynthesis of violaxanthin in Escherichia coli
Carotenoids are naturally synthesized in some species of bacteria, archaea, and fungi (including yeasts) as well as all photosynthetic organisms. Escherichia coli has been the most popular bacterial host for the heterologous production of a variety of carotenoids, including even xanthophylls unique to photosynthetic eukaryotes such as lutein, antheraxanthin, and violaxanthin. However, conversion efficiency of these epoxy-xanthophylls (antheraxanthin and violaxanthin) from zeaxanthin remained substantially low. We here examined several factors affecting their productivity in E. coli. Two sorts of plasmids were introduced into the bacterial host, i.e., a plasmid to produce zeaxanthin due to the presence of the Pantoea ananatis crtE, crtB, crtI, crtY, and crtZ genes in addition to the Haematococcus pluvialis IDI gene, and one containing each of zeaxanthin epoxidase (ZEP) genes originated from nine photosynthetic eukaryotes. It was consequently found that paprika (Capsicum annuum) ZEP (CaZEP) showed the highest conversion activity. Next, using the CaZEP gene, we performed optimization experiments in relation to E. coli strains as the production hosts, expression vectors, and ribosome-binding site (RBS) sequences. As a result, the highest productivity of violaxanthin (231 μg/g dry weight) was observed, when the pUC18 vector was used with CaZEP preceded by a RBS sequence of score 5000 in strain JM101(DE3).
KeywordsPathway engineering Zeaxanthin epoxidase Violaxanthin Escherichia coli
We thank Dr. Changfu Zhu and Dr. Gerhard Sandmann for giving us the GlZEP cDNA clone. We also thank Dr. Hisashi Harada for giving us the Paeodactylum tricornutum genome DNA. The authentic samples violaxanthin and antheraxanthin are gifts from Dr. Kazutoshi Shindo.
This study was funded by the “Smart Cell Project” organized by the New Energy and Industrial Technology Development Organization (NEDO) (16100920-0).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with animals performed by any of the authors.
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