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Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana.

Radakovits R, Jinkerson RE, Fuerstenberg SI, Tae H, Settlage RE, Boore JL, Posewitz MC - Nat Commun (2012)

Bottom Line: The potential use of algae in biofuels applications is receiving significant attention.We define the genes required for glycerolipid biogenesis and detail the differential regulation of genes during nitrogen-limited lipid biosynthesis.Phylogenomic analysis identifies genetic attributes of this organism, including unique stramenopile photosynthesis genes and gene expansions that may explain the distinguishing photoautotrophic phenotypes observed.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, Colorado 80401, USA.

ABSTRACT
The potential use of algae in biofuels applications is receiving significant attention. However, none of the current algal model species are competitive production strains. Here we present a draft genome sequence and a genetic transformation method for the marine microalga Nannochloropsis gaditana CCMP526. We show that N. gaditana has highly favourable lipid yields, and is a promising production organism. The genome assembly includes nuclear (~29 Mb) and organellar genomes, and contains 9,052 gene models. We define the genes required for glycerolipid biogenesis and detail the differential regulation of genes during nitrogen-limited lipid biosynthesis. Phylogenomic analysis identifies genetic attributes of this organism, including unique stramenopile photosynthesis genes and gene expansions that may explain the distinguishing photoautotrophic phenotypes observed. The availability of a genome sequence and transformation methods will facilitate investigations into N. gaditana lipid biosynthesis and permit genetic engineering strategies to further improve this naturally productive alga.

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Biomass production by N. gaditana.(a) N. gaditana production of biomass, lipids, protein and sugars quantified during continuous growth over a period of 3 months in 50% salinity seawater medium supplemented with nitrate, phosphate and CO2 with continuous 1,000 μE light. Every week, half of the culture was collected and replaced with fresh medium. Inset values show the yield in mg l−1 per day. Values are from 12 measurements and error bars show the standard deviation. (b) Chart illustrating collected biomass compositions, the majority of which consists of lipids even under nutrient replete conditions. Inset values show percentage of total biomass. (c) Comparison of N. gaditana lipid production rates with other algae examined in this work. Values are from at least three separate experiments and error bars show the standard deviation. (d) Comparison of N. gaditana large-scale production rates with other biofuel production platforms. Bars in green indicate our estimations; bars in grey indicate estimations by Atsumi et al. 30 The values for N. gaditana have been extrapolated from 1 l cultures and adjusted for our observed productivity in 12 h/12 h light/dark cycles. The S. elongatus production values are for 24 h light and would presumably be lower in 12 h/12 h light/dark cycles.
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f1: Biomass production by N. gaditana.(a) N. gaditana production of biomass, lipids, protein and sugars quantified during continuous growth over a period of 3 months in 50% salinity seawater medium supplemented with nitrate, phosphate and CO2 with continuous 1,000 μE light. Every week, half of the culture was collected and replaced with fresh medium. Inset values show the yield in mg l−1 per day. Values are from 12 measurements and error bars show the standard deviation. (b) Chart illustrating collected biomass compositions, the majority of which consists of lipids even under nutrient replete conditions. Inset values show percentage of total biomass. (c) Comparison of N. gaditana lipid production rates with other algae examined in this work. Values are from at least three separate experiments and error bars show the standard deviation. (d) Comparison of N. gaditana large-scale production rates with other biofuel production platforms. Bars in green indicate our estimations; bars in grey indicate estimations by Atsumi et al. 30 The values for N. gaditana have been extrapolated from 1 l cultures and adjusted for our observed productivity in 12 h/12 h light/dark cycles. The S. elongatus production values are for 24 h light and would presumably be lower in 12 h/12 h light/dark cycles.

Mentions: N. gaditana is a robust producer of both biomass and lipids under a wide array of culture conditions, including minimal f/2 seawater medium and artificial seawater (10–120% seawater salinity, pH 7–10) supplemented with nitrate, phosphate and CO2. The yields from N. gaditana cultures grown in f/2 medium at 50% seawater salinity are shown in Figure 1a,b. Yields of 0.65 g l−1 d−1 biomass and 0.31 g l−1 d−1 total lipids were achieved over a period of 3 months in 1 l Roux Flasks sparged with air/2% CO2, when half the cultures were exchanged for fresh medium every week. Lipid body accumulation can be triggered/enhanced in most algae by nitrogen deprivation or other stress conditions27, and the high lipid content (47.5%) in actively growing cultures of N. gaditana is likely facilitated by the rapid depletion of nitrate in dense cultures (3–8 g l−1) during growth. Optimal lipid yields were obtained with a starting culture density of ~3.6 g l−1. It is likely that lack of light penetration due to self-shading is the main limiting factor for cultures at higher starting densities. Low-density cultures (<0.5 g l−1) can be growth inhibited by high light (>200 μE) but the higher density cultures have good production between 1,000 μE and 2,000 μE. For medium to high-density cultures (3–10 g l−1), no substantial increase in productivity is observed on increasing the light from 1,000 μE to 2,000 μE, supporting the hypothesis that self shading becomes the limiting factor at these densities. The laboratory productivity numbers have been extrapolated to calculate potential lipid yields in comparison with other algae (Fig. 1c) and to other biofuel production platforms (Fig. 1d). In Figure 1d, the green bars indicate our extrapolations based on data from Chisti et al.28 and Chen et al.29, whereas grey bars indicate estimations originally provided by Atsumi et al.30 It is important to note that some of the values represent actual production yields from large-scale cultivation (Soy, Palm)2831, whereas other values are extrapolated from small scale cultures with 24 h light (Synechococcus elongatus Isobutyraldehyde and Isobutanol). The N. gaditana lipid production yields have been derived from small scale cultures with 12 h light/12 h dark cycles and therefore provide a more realistic estimation relative to S. elongatus. Robust lipid yields from Nannochloropsis scale from 25 ml cultures to 8 l cultures under laboratory conditions, to 10-hectare outdoor ponds where it is grown on a commercial scale (Hairong Electric Company and Seambiotic). The high lipid content of N. gaditana cells is apparent on fluorescent labelling of algal triglycerides with the lipophilic dye, BODIPY. Actively growing cells have a constitutive lipid droplet that expands within cells in stationary phase or during nitrogen deprivation (Supplementary Fig. S1a,b respectively). The large majority of lipids in N. gaditana are composed of palmitic and palmitoleic acid with a minor content of myristic and oleic acid (Supplementary Fig. S1c), resulting in a relatively simple fatty acid profile, and these fatty acids can be used for the production of biodiesel or biopetrol.


Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana.

Radakovits R, Jinkerson RE, Fuerstenberg SI, Tae H, Settlage RE, Boore JL, Posewitz MC - Nat Commun (2012)

Biomass production by N. gaditana.(a) N. gaditana production of biomass, lipids, protein and sugars quantified during continuous growth over a period of 3 months in 50% salinity seawater medium supplemented with nitrate, phosphate and CO2 with continuous 1,000 μE light. Every week, half of the culture was collected and replaced with fresh medium. Inset values show the yield in mg l−1 per day. Values are from 12 measurements and error bars show the standard deviation. (b) Chart illustrating collected biomass compositions, the majority of which consists of lipids even under nutrient replete conditions. Inset values show percentage of total biomass. (c) Comparison of N. gaditana lipid production rates with other algae examined in this work. Values are from at least three separate experiments and error bars show the standard deviation. (d) Comparison of N. gaditana large-scale production rates with other biofuel production platforms. Bars in green indicate our estimations; bars in grey indicate estimations by Atsumi et al. 30 The values for N. gaditana have been extrapolated from 1 l cultures and adjusted for our observed productivity in 12 h/12 h light/dark cycles. The S. elongatus production values are for 24 h light and would presumably be lower in 12 h/12 h light/dark cycles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: Biomass production by N. gaditana.(a) N. gaditana production of biomass, lipids, protein and sugars quantified during continuous growth over a period of 3 months in 50% salinity seawater medium supplemented with nitrate, phosphate and CO2 with continuous 1,000 μE light. Every week, half of the culture was collected and replaced with fresh medium. Inset values show the yield in mg l−1 per day. Values are from 12 measurements and error bars show the standard deviation. (b) Chart illustrating collected biomass compositions, the majority of which consists of lipids even under nutrient replete conditions. Inset values show percentage of total biomass. (c) Comparison of N. gaditana lipid production rates with other algae examined in this work. Values are from at least three separate experiments and error bars show the standard deviation. (d) Comparison of N. gaditana large-scale production rates with other biofuel production platforms. Bars in green indicate our estimations; bars in grey indicate estimations by Atsumi et al. 30 The values for N. gaditana have been extrapolated from 1 l cultures and adjusted for our observed productivity in 12 h/12 h light/dark cycles. The S. elongatus production values are for 24 h light and would presumably be lower in 12 h/12 h light/dark cycles.
Mentions: N. gaditana is a robust producer of both biomass and lipids under a wide array of culture conditions, including minimal f/2 seawater medium and artificial seawater (10–120% seawater salinity, pH 7–10) supplemented with nitrate, phosphate and CO2. The yields from N. gaditana cultures grown in f/2 medium at 50% seawater salinity are shown in Figure 1a,b. Yields of 0.65 g l−1 d−1 biomass and 0.31 g l−1 d−1 total lipids were achieved over a period of 3 months in 1 l Roux Flasks sparged with air/2% CO2, when half the cultures were exchanged for fresh medium every week. Lipid body accumulation can be triggered/enhanced in most algae by nitrogen deprivation or other stress conditions27, and the high lipid content (47.5%) in actively growing cultures of N. gaditana is likely facilitated by the rapid depletion of nitrate in dense cultures (3–8 g l−1) during growth. Optimal lipid yields were obtained with a starting culture density of ~3.6 g l−1. It is likely that lack of light penetration due to self-shading is the main limiting factor for cultures at higher starting densities. Low-density cultures (<0.5 g l−1) can be growth inhibited by high light (>200 μE) but the higher density cultures have good production between 1,000 μE and 2,000 μE. For medium to high-density cultures (3–10 g l−1), no substantial increase in productivity is observed on increasing the light from 1,000 μE to 2,000 μE, supporting the hypothesis that self shading becomes the limiting factor at these densities. The laboratory productivity numbers have been extrapolated to calculate potential lipid yields in comparison with other algae (Fig. 1c) and to other biofuel production platforms (Fig. 1d). In Figure 1d, the green bars indicate our extrapolations based on data from Chisti et al.28 and Chen et al.29, whereas grey bars indicate estimations originally provided by Atsumi et al.30 It is important to note that some of the values represent actual production yields from large-scale cultivation (Soy, Palm)2831, whereas other values are extrapolated from small scale cultures with 24 h light (Synechococcus elongatus Isobutyraldehyde and Isobutanol). The N. gaditana lipid production yields have been derived from small scale cultures with 12 h light/12 h dark cycles and therefore provide a more realistic estimation relative to S. elongatus. Robust lipid yields from Nannochloropsis scale from 25 ml cultures to 8 l cultures under laboratory conditions, to 10-hectare outdoor ponds where it is grown on a commercial scale (Hairong Electric Company and Seambiotic). The high lipid content of N. gaditana cells is apparent on fluorescent labelling of algal triglycerides with the lipophilic dye, BODIPY. Actively growing cells have a constitutive lipid droplet that expands within cells in stationary phase or during nitrogen deprivation (Supplementary Fig. S1a,b respectively). The large majority of lipids in N. gaditana are composed of palmitic and palmitoleic acid with a minor content of myristic and oleic acid (Supplementary Fig. S1c), resulting in a relatively simple fatty acid profile, and these fatty acids can be used for the production of biodiesel or biopetrol.

Bottom Line: The potential use of algae in biofuels applications is receiving significant attention.We define the genes required for glycerolipid biogenesis and detail the differential regulation of genes during nitrogen-limited lipid biosynthesis.Phylogenomic analysis identifies genetic attributes of this organism, including unique stramenopile photosynthesis genes and gene expansions that may explain the distinguishing photoautotrophic phenotypes observed.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, Colorado 80401, USA.

ABSTRACT
The potential use of algae in biofuels applications is receiving significant attention. However, none of the current algal model species are competitive production strains. Here we present a draft genome sequence and a genetic transformation method for the marine microalga Nannochloropsis gaditana CCMP526. We show that N. gaditana has highly favourable lipid yields, and is a promising production organism. The genome assembly includes nuclear (~29 Mb) and organellar genomes, and contains 9,052 gene models. We define the genes required for glycerolipid biogenesis and detail the differential regulation of genes during nitrogen-limited lipid biosynthesis. Phylogenomic analysis identifies genetic attributes of this organism, including unique stramenopile photosynthesis genes and gene expansions that may explain the distinguishing photoautotrophic phenotypes observed. The availability of a genome sequence and transformation methods will facilitate investigations into N. gaditana lipid biosynthesis and permit genetic engineering strategies to further improve this naturally productive alga.

Show MeSH
Related in: MedlinePlus