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      Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779

        1 , 2 , 3 , 4 , 1 , 1 , 1 , 4 , 1 , 5 , 1 , 2 , 6 , 4 , 3 , 7 , 3 , 8 , 9 , 10 , 2 , 1 , 3 , 4 , 1 , 1 , 3 , 4 , 1 , 1 , 3 , 1 , 3 , 3 , 1 , 3 , 1 , 10 , 1 , 7 , 8 , 9 , 3 , 3 , 3 , 3 , 5 , 1 , 6 , 2 , 3 , * , 1 , *

      PLoS Genetics

      Public Library of Science

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          Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.

          Author Summary

          Algae are a highly diverse group of organisms that have become the focus of renewed interest due to their potential for producing biofuel feedstocks, nutraceuticals, and biomaterials. Their high photosynthetic yields and ability to grow in areas unsuitable for agriculture provide a potential sustainable alternative to using traditional agricultural crops for biofuels. Because none of the algae currently in use have a history of domestication, and bioengineering of algae is still in its infancy, there is a need to develop algal strains adapted to cultivation for industrial large-scale production of desired compounds. Model organisms ranging from mice to baker's yeast have been instrumental in providing insights into fundamental biological structures and functions. The algal field needs versatile models to develop a fundamental understanding of photosynthetic production of biomass and valuable compounds in unicellular, marine, oleaginous algal species. To contribute to the development of such an algal model system for basic discovery, we sequenced the genome and two sets of transcriptomes of N. oceanica CCMP1779, assembled the genomic sequence, identified putative genes, and began to interpret the function of selected genes. This species was chosen because it is readily transformable with foreign DNA and grows well in culture.

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          Most cited references 112

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          Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes.

          The completion of the Arabidopsis thaliana genome sequence allows a comparative analysis of transcriptional regulators across the three eukaryotic kingdoms. Arabidopsis dedicates over 5% of its genome to code for more than 1500 transcription factors, about 45% of which are from families specific to plants. Arabidopsis transcription factors that belong to families common to all eukaryotes do not share significant similarity with those of the other kingdoms beyond the conserved DNA binding domains, many of which have been arranged in combinations specific to each lineage. The genome-wide comparison reveals the evolutionary generation of diversity in the regulation of transcription.
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            Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor.

            Thirty microalgal strains were screened in the laboratory for their biomass productivity and lipid content. Four strains (two marine and two freshwater), selected because robust, highly productive and with a relatively high lipid content, were cultivated under nitrogen deprivation in 0.6-L bubbled tubes. Only the two marine microalgae accumulated lipid under such conditions. One of them, the eustigmatophyte Nannochloropsis sp. F&M-M24, which attained 60% lipid content after nitrogen starvation, was grown in a 20-L Flat Alveolar Panel photobioreactor to study the influence of irradiance and nutrient (nitrogen or phosphorus) deprivation on fatty acid accumulation. Fatty acid content increased with high irradiances (up to 32.5% of dry biomass) and following both nitrogen and phosphorus deprivation (up to about 50%). To evaluate its lipid production potential under natural sunlight, the strain was grown outdoors in 110-L Green Wall Panel photobioreactors under nutrient sufficient and deficient conditions. Lipid productivity increased from 117 mg/L/day in nutrient sufficient media (with an average biomass productivity of 0.36 g/L/day and 32% lipid content) to 204 mg/L/day (with an average biomass productivity of 0.30 g/L/day and more than 60% final lipid content) in nitrogen deprived media. In a two-phase cultivation process (a nutrient sufficient phase to produce the inoculum followed by a nitrogen deprived phase to boost lipid synthesis) the oil production potential could be projected to be more than 90 kg per hectare per day. This is the first report of an increase of both lipid content and areal lipid productivity attained through nutrient deprivation in an outdoor algal culture. The experiments showed that this marine eustigmatophyte has the potential for an annual production of 20 tons of lipid per hectare in the Mediterranean climate and of more than 30 tons of lipid per hectare in sunny tropical areas.
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              Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis.

              Draft genome sequences have been determined for the soybean pathogen Phytophthora sojae and the sudden oak death pathogen Phytophthora ramorum. Oömycetes such as these Phytophthora species share the kingdom Stramenopila with photosynthetic algae such as diatoms, and the presence of many Phytophthora genes of probable phototroph origin supports a photosynthetic ancestry for the stramenopiles. Comparison of the two species' genomes reveals a rapid expansion and diversification of many protein families associated with plant infection such as hydrolases, ABC transporters, protein toxins, proteinase inhibitors, and, in particular, a superfamily of 700 proteins with similarity to known oömycete avirulence genes.

                Author and article information

                Role: Editor
                PLoS Genet
                PLoS Genet
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                November 2012
                November 2012
                15 November 2012
                : 8
                : 11
                [1 ]Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, United States of America
                [2 ]Cell and Molecular Biology Program, Michigan State University, East Lansing, Michigan, United States of America
                [3 ]Department of Plant Biology, Michigan State University, East Lansing, Michigan, United States of America
                [4 ]DOE–Plant Research Laboratory, Michigan State University, East Lansing, Michigan, United States of America
                [5 ]Deptartment of Computer Science and Engineering, Michigan State University, East Lansing, Michigan, United States of America
                [6 ]Department of Human Genetics, University of Utah, Salt Lake City, Utah, United States of America
                [7 ]Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan, United States of America
                [8 ]Howard Hughes Medical Institute, Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, California, United States of America
                [9 ]Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America
                [10 ]Department of Horticulture, Michigan State University, East Lansing, Michigan, United States of America
                Rutgers University, United States of America
                Author notes

                CB and KKN are on the advisory board of Aurora Alga. AV was supported in part by a grant from Aurora Alga.

                Conceived and designed the experiments: AV GW S-HS CB BBS. Performed the experiments: AV BB C-HT CH CT I-BR AJC GW. Analyzed the data: AV GW RA CB BB MSC KLC TJC AJC RD EE EMF AAF WH ELH NJ QK M-HK XL BL YL SL KKN JO KWO CP I-BR RLR S YS-H BBS S-HS JPS YS HT AT C-HT JW MY SZ CJB. Contributed reagents/materials/analysis tools: MY. Wrote the paper: CB AV GW DC AJC RD EE EMF ELH NJ QK BL YL KKN JO I-BR RLR S YS-H BBS S-HS JPS YS HT AT C-HT.


                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.

                Page count
                Pages: 25
                Sequencing and bioinformatics was supported by a Strategic Partnership grant from the Michigan State University Foundation and Michigan State University AgBioResearch. MAKER is supported by NIH R01-HG004694 and NSF IOS-1126998 to MY. Annotation of ncRNAs was supported by NSF CAREER Grant DBI-0953738 to YS. Annotation of photosynthetic genes was supported by a National Science Foundation Graduate Research Fellowship to EE and by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through FWP number 449B to KKN. Algal biofuels research in the Benning lab is supported by a grant from the Air Force Office of Scientific Research (FA9550-08-1-0165 to CB). Lipid gene annotation was supported in part by a grant from Aurora Algae to CB. Cell wall analysis was funded in part by the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494). Annotation of the organelle division genes was supported by National Science Foundation grant MCB1121943 to KWO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Model Organisms
                Plant Science
                Systems Biology



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