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      Whole genome sequencing of Rhodotorula mucilaginosa isolated from the chewing stick ( Distemonanthus benthamianus): insights into Rhodotorula phylogeny, mitogenome dynamics and carotenoid biosynthesis

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          Abstract

          In industry, the yeast Rhodotorula mucilaginosa is commonly used for the production of carotenoids. The production of carotenoids is important because they are used as natural colorants in food and some carotenoids are precursors of retinol (vitamin A). However, the identification and molecular characterization of the carotenoid pathway/s in species belonging to the genus Rhodotorula is scarce due to the lack of genomic information thus potentially impeding effective metabolic engineering of these yeast strains for improved carotenoid production. In this study, we report the isolation, identification, characterization and the whole nuclear genome and mitogenome sequence of the endophyte R. mucilaginosa RIT389 isolated from Distemonanthus benthamianus, a plant known for its anti-fungal and antibacterial properties and commonly used as chewing sticks. The assembled genome of R. mucilaginosa RIT389 is 19 Mbp in length with an estimated genomic heterozygosity of 9.29%. Whole genome phylogeny supports the species designation of strain RIT389 within the genus in addition to supporting the monophyly of the currently sequenced Rhodotorula species. Further, we report for the first time, the recovery of the complete mitochondrial genome of R. mucilaginosa using the genome skimming approach. The assembled mitogenome is at least 7,000 bases larger than that of Rhodotorula taiwanensis which is largely attributed to the presence of large intronic regions containing open reading frames coding for homing endonuclease from the LAGLIDADG and GIY-YIG families. Furthermore, genomic regions containing the key genes for carotenoid production were identified in R. mucilaginosa RIT389, revealing differences in gene synteny that may play a role in the regulation of the biotechnologically important carotenoid synthesis pathways in yeasts.

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          Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics.

          Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.
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            Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level.

            A carotenoid biosynthesis gene cluster for the production of astaxanthin was isolated from the marine bacterium Agrobacterium aurantiacum. This cluster contained five carotenogenic genes with the same orientation, which were designated crtW, crtZ, crtY, crtI, and crtB. The stop codons of individual crt genes except for crtB overlapped the start codons of the following crt genes. Escherichia coli transformants carrying the Erwinia uredovora carotenoid biosynthesis genes provide suitable substrates for carotenoid biosynthesis. The functions of the five crt genes of A. aurantiacum were determined through chromatographic and spectroscopic analyses of the pigments accumulated in some E. coli transformants carrying various combinations of the E. uredovora and A. aurantiacum carotenogenic genes. As a result, the astaxanthin biosynthetic pathway is proposed for the first time at the level of the biosynthesis genes. The crtW and crtZ gene products, which mediated the oxygenation reactions from beta-carotene to astaxanthin, were found to have low substrate specificity. This allowed the production of many presumed intermediates of astaxanthin, i.e., adonixanthin, phoenicoxanthin (adonirubin), canthaxanthin, 3'-hydroxyechinenone, and 3-hydroxyechinenone.
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              Biological roles of fungal carotenoids

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                Author and article information

                Contributors
                Journal
                PeerJ
                PeerJ
                peerj
                peerj
                PeerJ
                PeerJ Inc. (San Francisco, USA )
                2167-8359
                14 November 2017
                2017
                : 5
                : e4030
                Affiliations
                [1 ]Centre for Integrative Ecology-School of Life and Environmental Sciences, Deakin University , Victoria, Australia
                [2 ]Genomics Facility, Monash University , Selangor, Malaysia
                [3 ]School of Science, Monash University , Selangor, Malaysia
                [4 ]College of Health Science and Technology, Rochester Institute of Technology , Rochester, NY, United States of America
                [5 ]Thomas H. Gosnell School of School of Life Sciences, Rochester Institute of Technology , Rochester, NY, USA
                Article
                4030
                10.7717/peerj.4030
                5691792
                ad595932-7730-4815-9c55-472507ec6379
                ©2017 Gan et al.

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.

                History
                : 1 September 2017
                : 23 October 2017
                Funding
                Funded by: College of Science (COS)
                Funded by: The Gosnell School of Life Sciences (GSoLS)
                Funded by: College of Health Sciences and Technology (RIT)
                Funded by: Monash University Malaysia Tropical Medicine and Biology Multidisciplinary Platform
                Funding for this work was provided by the following: the College of Science (COS) and the Gosnell School of Life Sciences (GSoLS) at Rochester Institute of Technology (RIT), a Research Laboratory and Faculty Development Award from the College of Health Sciences and Technology (RIT) and the Monash University Malaysia Tropical Medicine and Biology Multidisciplinary Platform. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
                Categories
                Biotechnology
                Genomics
                Microbiology
                Mycology
                Plant Science

                rhodotorula mucilaginosa,carotenoid,phylogenomics,endophyte,next-generation sequencing,chew sticks mitogenome,distemonanthus benthamianus

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