Increase in the astaxanthin synthase gene (crtS) dose by in vivo DNA fragment assembly in Xanthophyllomyces dendrorhous

Abstract

BackgroundXanthophyllomyces dendrorhous is a basidiomycetous yeast that is relevant to biotechnology, as it can synthesize the carotenoid astaxanthin. However, the astaxanthin levels produced by wild-type strains are low. Although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far. A promising alternative to facilitate the production of carotenoids in this yeast involves the use of genetic modification. However, a major limitation is the few available molecular tools to manipulate X. dendrorhous.ResultsIn this work, the DNA assembler methodology that was previously described in Saccharomyces cerevisiae was successfully applied to assemble DNA fragments in vivo and integrate these fragments into the genome of X. dendrorhous by homologous recombination in only one transformation event. Using this method, the gene encoding astaxanthin synthase (crtS) was overexpressed in X. dendrorhous and a higher level of astaxanthin was produced.ConclusionsThis methodology could be used to easily and rapidly overexpress individual genes or combinations of genes simultaneously in X. dendrorhous, eliminating numerous steps involved in conventional cloning methods.

Analysis of relationship between key genes and astaxanthin biosynthesis in Phaffia rhodozyma by transcript level and gene knock-out

“…The previous studies of Lodato, Miao and Marcoleta et al pointed that the expression of asy gene in high-yielding astaxanthin-producing strains was higher than that in low-yielding strains [17,26,27]. Contreras et al also found that asy gene was overexpressed in P. rhodozyma [28], the total carotenoid production of the strain did not change significantly, and the astaxanthin ratio increased. In this study, the asy gene expression showed high correlation with astaxanthin content and total carotene content.…”

Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides

“…Bands with same size indicate that the transformants maintain the introduced DNA in circular plasmids, but plasmid rearranges can be also presumed World J Microbiol Biotechnol Two genes are responsible for the conversion of b-carotene to astaxanthin in X. dendrorhous: the crtS encoding a cytochrome-P450 hydroxylase (astaxanthin synthase) is responsible for the addition of hydroxyl and keto groups to the b-ionone rings of b-carotene and the crtR encoding a cytochrome-P450 reductase, as electron donor, is required to the CrtS for addition of functional groups to the substrate (Á lvarez et al 2006;Ojima et al 2006;Alcaíno et al 2008). Overexpression of the crtS gene in X. dendrorhous resulted in higher level of astaxanthin production (Contreras et al 2013), while point mutations in the gene led to astaxanthin non-producing and b-carotene accumulating yellow mutants (Á lvarez et al 2006;Ojima et al 2006;Barbachano-Torres et al 2014). Deletion of the crtR gene was not lethal, suggesting an alternative electron donor in X. dendrorhous, but the transformants were not able to accumulate astaxanthin, indicating that the crtS and crtR genes are also necessary for the conversion of b-carotene to astaxanthin (Alcaíno et al 2008).…”

“…addition of two hydroxyl and two keto groups to the b-ionone rings of b-carotene (Hoshino et al 2000;Verdoes et al 2003;Á lvarez et al 2006;Ojima et al 2006;Lodato et al 2007;Contreras et al 2013). Overexpression of the crtS gene in X. dendrorhous resulted higher level of astaxanthin production (Contreras et al 2013), and complementation of the astaxanthin nonproducing and b-carotene accumulating X. dendrorhous ATCC 96815 mutant with crtS restored the astaxanthin biosynthesis (Á lvarez et al 2006). Although the b-carotene hydroxylase and ketolase activity of the enzyme was verified, Á lvarez et al (2006) found that CrtS had only bcarotene hydroxylase activity when it was expressed in M. circinelloides.…”

“…Gene named as ast, asy or crtS by the different authors, encoding a cytochrome-P450-type enzyme may be responsible for the formation of astaxanthin from bcarotene, i.e. addition of two hydroxyl and two keto groups to the b-ionone rings of b-carotene (Hoshino et al 2000;Verdoes et al 2003;Á lvarez et al 2006;Ojima et al 2006;Lodato et al 2007;Contreras et al 2013). Overexpression of the crtS gene in X. dendrorhous resulted higher level of astaxanthin production (Contreras et al 2013), and complementation of the astaxanthin nonproducing and b-carotene accumulating X. dendrorhous ATCC 96815 mutant with crtS restored the astaxanthin biosynthesis (Á lvarez et al 2006).…”

Microbiological Synthesis of Carotenoids: Pathways and Regulation

“…Despite the potential of X. dendrorhous, the specific production of astaxanthin by wild-type strains of this yeast is too low to be a commercially competitive source [5]. Due to the above, many researchers have tried to improve the production of astaxanthin using several methods as the optimization of culture conditions, classical random mutagenesis methods, genetic and metabolic engineering strategies [1,4,[6][7][8][9][10][11][12][13][14][15]. These studies have led to an extensive knowledge about the biology of this yeast and the carotenoid synthesis pathway.…”