Draft genome sequence and detailed characterization of biofuel production by oleaginous microalga Scenedesmus quadricauda LWG002611

Abstract

Background: Due to scarcity of fossil fuel, the importance of alternative energy sources is ever increasing. The oleaginous microalgae have demonstrated their potential as an alternative source of energy, but have not achieved commercialization owing to some biological and technical inefficiency. Modern methods of recombinant strain development for improved efficacy are suffering due to inadequate knowledge of genome and limited molecular tools available for their manipulation.

Results: In the present study, microalga Scenedesmus quadricauda LWG002611 was selected as the preferred organism for lipid production as it contained high biomass (0.37 g L^-1 day^-1) and lipid (102 mg L^-1 day^-1), compared to other oleaginous algae examined in the present study as well as earlier reports. It possessed suitable biodiesel properties as per the range defined by the European biodiesel standard EN14214 and petro-diesel standard EN590:2013. To investigate the potential of S. quadricauda LWG002611 in details, the genome of the organism was assembled and annotated. This was the first genome sequencing and assembly of S. quadricauda, which predicted a genome size of 65.35 Mb with 13,514 genes identified by de novo and 16,739 genes identified by reference guided annotation. Comparative genomics revealed that it belongs to class Chlorophyceae and order Sphaeropleales. Further, small subunit ribosomal RNA gene (18S rRNA) sequencing was carried out to confirm its molecular identification. S. quadricauda LWG002611 exhibited higher number of genes related to major activities compared to other potential algae reported earlier with a total of 283 genes identified in lipid metabolism. Metabolic pathways were reconstructed and multiple gene homologs responsible for carbon fixation and triacylglycerol (TAG) biosynthesis pathway were identified to further improve this potential algal strain for biofuel production by metabolic engineering approaches.

Conclusion: Here we present the first draft genome sequence, genetic characterization and comparative evaluation of S. quadricauda LWG002611 which exhibit high biomass as well as high lipid productivity. The knowledge of genome sequence, reconstructed metabolic pathways and identification of rate-limiting steps in TAG biosynthesis pathway will strengthen the development of molecular tools towards further improving this potentially one of the major algal strains for biofuel production.

Keywords: Biofuel; Draft genome sequence; Lipid metabolism; Metabolic pathways; Oleaginous microalgae; Phylogenetic analysis; Scenedesmus quadricauda.

Fig. 1

Comparison of a biomass productivity (g L⁻¹ day⁻¹) and b lipid productivity (mg L⁻¹ day⁻¹) of S. quadricauda LWG002611 with other eight isolated oleaginous microalgae grown in autotrophic cultivation condition using BBM and mixotrophic cultivation condition using TAP media where acetate is supplemented as external carbon source in uniform cultivation condition at a temperature of 27 °C ± 0.5 °C, a photoperiod of 14:10 h light/dark cycle and fluorescent illumination of 3000 lux. Productivity was estimated in logarithmic phase of growth. c Comparison of biomass productivity (g L⁻¹ day⁻¹) and lipid productivity (mg L⁻¹ day⁻¹) of S. quadricauda LWG002611 with earlier reports of microalgae grown in different carbon sources i.e. mixotrophic (mixo) growth in acetate and autotrophic (auto) growth in CO₂ and air. Bar (i) indicate our estimation, bar (ii, vi, vii, ix) indicate the estimation by Rodolfi et al. [9], bar (iii, iv) by Mandal and Mallick [26], bar (v) by Yoo et al. [27], bar (viii) by Chiu et al. [28] and bar (x, xi) by Liang et al. [29]. d Comparison of S. quadricauda LWG002611 large-scale production rates (TOE ha⁻¹ year⁻¹) with other promising oleaginous microalgae as well as with other biofuel feedstocks. Bar (i) indicate the average global productivity of microalgal lipid [11], bar (ii) indicate our estimations for S. quadricauda LWG002611, extrapolated from 1 L cultures cultivated in TAP medium at a temperature of 27 °C ± 0.5 °C, a photoperiod of 14:10 h light/dark cycle and fluorescent illumination of 3000 lux; bar (iii) indicate extrapolated estimations of lipid productivity of Nannochloropsis gaditana in nitrogen deficient condition [18], bar (iv) indicate the lipid productivity of Chlorella sp. [18], bar (v), (vi), (vii) and (viii) represent the oil productivity of Jatropha [18], Palm [1], Sunflower [1] and Rapeseed [1] respectively. e The extracted lipid (mg) from biomass (g) of S. quadricauda LWG002611 was refluxed for 5 h at 50 °C in the presence of methanol and 2% sulphuric acid for transesterification. After removal of impurities the FAME mix (mg mg⁻¹ of lipid) was dissolved in hexane and estimated for the percentage of Fatty acid methyl ester (FAME) (weight/weight) by Gas-Chromatography (Thermo Fisher Scientific) and quantified against a standard FAME mix (Supelco, USA) (values are from three separate experiments and error bars show the standard deviation)

Fig. 2

a Phylogenetic tree constructed from de novo genome sequence annotation using Uniprot hits from Cellular component of the algae families and species. b Photomicrograph of S. quadricauda LWG002611 under light microscope (×63). c Phylogenetic relationship of S. quadricauda LWG002611 with other algae based on 18S rDNA sequences using NR database of NCBI blast and figure was generated by Mega 5 software

Fig. 3

The above figures represent the gene ontology (GOs) of S. quadricauda LWG002611 in Panther gene ontology. a The number of genes involved in cellular component. b The number of genes involved in biological process. c The number of genes involved in molecular function. d The number of genes involved in lipid metabolism. e Comparative gene numbers related to membrane, catalytic activity, binding, electron carrier activity, metabolic process, lipid metabolic process, lipid biosynthetic process of S. quadricauda LWG002611, N. gaditana, M. neglectum and C. reinhardtii (the gene ontology of S. quadricauda LWG002611 is given in Additional file 5) [18, 21, 22]

Fig. 4

a Reconstructed metabolic pathways (KEGG) of S. quadricauda LWG002611. The pathways encoded by it are represented by green colour and not encoded are light grey in colour. b Reconstructed carbon fixation pathway showing the number of homologous genes found for the respective enzyme (number of the homologous gene given in the first bracket) in genome sequence of S. quadricauda LWG002611: PEP, phosphoenolpyruvate; OAA, oxaloacetate; MA, malic acid; Pyr, pyruvate; 3PGA, 3-phosphoglycerate; 1,3BPG, 1,3-bisphosphoglycerate; G3P, glyceraldehyde 3-phosphate; FBP, fructose 1,6-bisphosphatase; F6P, Fructose 6-phosphate; ERU4P, Erythrose 4-phosphate; SDP, Sedoheptulose-1,7-bisphosphatase; S7P, sedoheptulose 7-phosphate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; RuBP, ribulose-1,5-bisphosphate; enzymes (in red): PEPC, phosphoenolpyruvate carboxylase [EC:4.1.1.31]; MDH, malate dehydrogenase [EC:1.1.1.82]; PEPCK, Phosphoenolpyruvate carboxykinase [EC:4.1.1.49]; PPDK, pyruvate, orthophosphate dikinase [EC:2.7.9.1]; AST, aspartate aminotransferase [EC:2.6.1.1]; ALT, alanine transaminase [EC:2.6.1.2]; PGK, phosphoglycerate kinase [EC:2.7.2.3]; GAPDH, glyceraldehyde 3-phosphate dehydrogenase [EC:1.2.1.12]; ALDO, fructose-bisphosphate aldolase, class I [EC:4.1.2.13]; FBP, fructose-1,6-bisphosphatase I [EC:3.1.3.11]; TKT, transketolase [EC:2.2.1.1]; SEBP, sedoheptulose-1,7-bisphosphatase [EC: 3.1.3.37], RpiA, ribose 5-phosphate isomerase A [EC:5.3.1.6]; PRK, phosphoribulokinase [EC:2.7.1.19]; TPI, triosephosphate isomerase [EC:5.3.1.1]; Xfp, xylulose-5-phosphate [EC:4.1.2.9]; RPE, ribulose-phosphate 3-epimerase [EC:5.1.3.1]; CA, carbonic anhydrase [EC 4.2.1.1]; dn, de novo; ref, reference guided. c Reconstructed fatty acids and triacylglycerols (TAG) biosynthesis pathway showing the homologous gene found for the respective enzyme (number of the homologous gene given in the first bracket) in genome sequence of S. quadricauda LWG002611: FFA, free fatty acid; LPA, lysophosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerol; TAG, triacylglycerol; ACP, acyl carrier protein; CoA, coenzyme A; enzymes (in red): PDH, pyruvate dehydrogenase complex [EC 1.2.4.1]; ACC, acetyl-CoA carboxylase [EC 6.4.1.2]; MAT, malonyl-CoA:ACP transacylase [EC 2.3.1.39]; KAS, β-ketoacyl-ACP synthase [EC 2.3.1.41]; KAR, β-ketoacyl-ACP reductase [EC 1.1.1.100]; HD, 3-hydroxyacyl-ACP dehydratase [EC 4.2.1.59]; ENR, enoyl-ACP reductase [EC 1.3.1.9]; FAT, fatty acyl-ACP thioesterase [EC 3.1.2.14]; G3PDH, gycerol-3-phosphate dehydrogenase [EC 1.1.1.8]; GPAT, glycerol-3-phosphate acyltransferase [EC 2.3.1.15]; LPAAT, lyso-phosphatidic acid acyltransferase [EC 2.3.1.51]; DAGK Diacylglycerol kinase [2.7.1.107]; PAP, Phosphatidic acid phosphatase [EC 3.1.3.4]; PDAT, phospholipid:diacylglycerol acyltransferase [EC 2.3.1.158]; DGAT, diacylglycerol acyltransferase [EC 2.3.1.20]; dn, de novo; ref, reference guided