Cloning and Characterization of Genes Involved in Nostoxanthin Biosynthesis of Sphingomonas elodea ATCC 31461 Liang Zhu., Xuechang Wu*., Ou Li, Chaodong Qian, Haichun Gao Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China Abstract Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a b-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,29-b-hydroxylase and the crtZ gene encoding the b-carotene hydroxylase, both responsible for hydroxylation of b-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and b- carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,29-dihydroxy-b-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed. Citation: Zhu L, Wu X, Li O, Qian C, Gao H (2012) Cloning and Characterization of Genes Involved in Nostoxanthin Biosynthesis of Sphingomonas elodea ATCC 31461. PLoS ONE 7(4): e35099. doi:10.1371/journal.pone.0035099 Editor: Eric A. Johnson, University of Wisconsin, Food Research Institute, United States of America Received December 7, 2011; Accepted March 8, 2012; Published April 11, 2012 Copyright: ß 2012 Zhu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction utive steps to produce the red pigment lycopene. Various further modifications by cyclases, hydroxylases, ketolases and other Carotenoids are isoprenoid pigments that are widely distributed enzymes lead to the formation of different carotenoids [7–9]. in nature [1]. They can be synthesized by all known phototrophic Sphingomonas elodea ATCC 31461 (originally designated as organisms and by some non-phototrophic fungi, bacteria, and Pseudomonas elodea, also referred to as Sphingomonas paucimobilis) was archaea [1,2]. Due to their unique physiochemical properties, they isolated as a Gram-negative bacterium capable of producing gellan have diverse biological functions in different organisms that either gum [10]. It synthesizes a yellow carotenoid identified as produce or consume them. These functions include their nostoxanthin ((2R,3R,29R,39R)-b,b-Carotene-2,3,29,39-tetrol) [11]. anticarcinogenic and antioxidant activity, protection against Nostoxanthin is a poly-hydroxy derivative of b-carotene isolated photo-oxidative damage, contribution to the light-harvesting only from some prokaryotes, including some species of cyanobac- process in photosynthesis, provitamin A property of b-carotene teria [12], the novel bacteriochlorophyll a-containing bacterium and as nutritional factors important for chronic disease prevention Sandarakinorhabdus limnophila [13], the moderately thermophilic [3–5]. The interesting properties and beneficial effects on human aerobic photosynthetic bacterium Porphyrobacter tepidarius [14], the health have drawn much attention. Over recent years, some marine bacterium Brevundimonas sp. strain SD212 [15], the strictly identified carotenoids have been used as colorants, nutritional aerobic photosynthetic bacterium Erythrobacter longus [16], and most supplements and nutraceuticals for food, cosmetic and pharma- Sphingomonas species [11]. Although all the necessary genes ceutical purposes [6]. required to synthesize nostoxanthin have been identified from Carotenoid biosynthetic pathway has been extensively studied Brevundimonas sp. strain SD212, Brevundimonas vesicularis strain in various organisms and remarkable progress has been made. All DC263 and Thermosynechococcus elongatus strain BP-1 [12,15,17], carotenoids are derived from the isoprenoids pathway. The first genetic data on nostoxanthin biosynthesis are limited and the step in the carotenoid biosynthetic pathway is the formation of carotenoid biosynthetic pathway of Sphingomonas species remains geranylgeranyl pyrophosphate (GGPP) from farnesyl pyrophos- unclear. phate (FPP) by GGPP synthase. Then two GGPP molecules are We previously cloned and identified the crtI gene encoding condensed head to head by phytoene synthase, resulting in the phytoene desaturase in S. elodea ATCC 31461 [18]. In the present formation of the first carotene phytoene. After phytoene formation study, we describe the cloning and characterization of the other the biosynthetic pathways vary in different organisms resulting in a genes involved in the nostoxanthin biosynthetic pathway of this wide carotenoid diversity. In most bacteria, the colorless phytoene organism. Using gene inactivation together with chromatographic is desaturased by the phytoene desaturase through four consec- and spectroscopic analysis of the pigments, we determined the PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35099 Nostoxanthin Biosynthesis of Sphingomonas functions of four carotenoid biosynthesis genes. In particular, the Carotenoid identification of knockout mutants crtG gene encoding the 2,29-b-hydroxylase, was also found in the HPLC analysis of the carotenoids isolated from the cells of carotenoid biosynthesis gene cluster of S. elodea ATCC 31461. ATCC 31461 showed several peaks at 475 nm. On the basis of Moreover, the functions of the two hydroxylase genes, crtZ and previous studies as well as mass spectrometic analysis, peaks 1 crtG, both responsible for hydroxylation of b-carotene were through 4 were identified as nostoxanthin, caloxanthin, zeaxan- confirmed by complementation studies using Escherichia coli thin and b-carotene, respectively (Fig. 2A). The other peaks might producing different carotenoids. As a result, the nostoxanthin be impurities of the carotenoid extract. biosynthetic pathway has been proposed. Targeted deletions of the candidate carotenogenic genes in S. elodea ATCC 31461 were performed by double-crossover recom- Results bination. All knockout mutants were analyzed for carotenoid identification by LC-APCI-MS. Compared to the wild-type strain, Cloning of the nostoxanthin biosynthetic genes DcrtZ did not produce 3-hydroxy carotenoids, and the pigments From SiteFinding-PCR [19], a 9.6-kb fragment containing a were separated into three major peaks. Peaks 5 and 6 were carotenoid biosynthesis gene cluster was obtained by assembly of determined to be 2, 29-dihydroxy-b-carotene and b-carotene the PCR products. However, the crtZ gene is not contained as a respectively (Fig. 2B). These results demonstrated that an inactive member of this cluster. Although we performed several rounds of crtZ gene inhibited 3,39-hydroxylation of b-carotene synthesis. In SiteFinding-PCR (obtained about 22 kb sequence data), we could mutant DcrtG, nostoxanthin and caloxanthin were not detected. not amplify the crtZ gene. Because the crtZ gene is not linked to the The major pigment was identified as zeaxanthin (peak 8, Fig. 2C), carotenoid biosynthesis gene cluster, the CODEHOP strategy was and a small amount of its precursor b-carotene was present (peak used to generate PCR primers for partial crtZ fragment 9, Fig. 2C). These data indicate that in this mutant the 2,29- amplification. An internal crtZ fragment of 266 bp was isolated hydroxylation step was missing. The HPLC elution profile of the by PCR amplification using primers deduced from conserved carotenoids accumulated by double knockout mutant DcrtZG internal domains of CrtZs of sphingomonadales (see Materials and showed a single major peak. This peak correspond to non- Methods), providing sequence information for designing specific hydroxylated b-carotene (peak 11, Fig. 2D), indicating that CrtZ primers for SiteFinding-PCR that generated full-length crtZ. and CrtG were responsible for hydroxylation of the b-ionone rings Sequences have been deposited in GenBank under accession to produce nostoxanthin. The crtY knockout mutant exhibited a number JN224892 for the carotenoid biosynthesis gene cluster and light red pigmentation, distinct from the yellow one of the wild- JN224893 for crtZ. type strain. This mutant, called DcrtY, accumulated lycopene that was absent from the wild-type strain (peak 13, Fig. 2E), suggesting Sequence analysis of the nostoxanthin biosynthetic that lycopene cyclization was impaired in this mutant. In addition, genes a knockout mutant of crtI accumulating phytoene instead of the final nostoxanthin has been described earlier [18]. Because The carotenoid biosynthesis gene cluster is 8,412 bp long and phytoene is colorless, this mutant forms white colonies. Similarly, contains 7 putative ORFs. Based on the alignments of the deduced the colonies formed by the mutant DcrtB were also white, which