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Cited by 176Algal biology and biofuel productionMicrobial Metabolic Engineering and BioproductionLipid metabolism and biosynthesis

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Oil accumulation mechanisms of the oleaginous microalga Chlorella protothecoides revealed through its genome, transcriptomes, and proteomes

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Gao, Chunfang, Wang, Yun, Shen, Yue, Yan, Dong, He, Xi, Dai, Junbiao, Wu, Qingyu·2014·BMC Genomics
DOI 10.1186/1471-2164-15-582PMC PMC4111847
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Abstract

BACKGROUND: Microalgae-derived biodiesel is a promising substitute for conventional fossil fuels. In particular, the green alga Chlorella protothecoides sp. 0710 is regarded as one of the best candidates for commercial manufacture of microalgae-derived biofuel. This is due not only to its ability to live autotrophically through photosynthesis, but also to its capacity to produce a large amount of biomass and lipid through fermentation of glucose. However, until the present study, neither its genome sequence nor the platform required for molecular manipulations were available. RESULTS: We generated a draft genome for C. protothecoides, and compared its genome size and gene content with that of Chlorella variabilis NC64A and Coccomyxa subellipsoidea C-169. This comparison revealed that C. protothecoides has a reduced genome size of 22.9 Mbp, about half that of its close relatives. The C. protothecoides genome encodes a smaller number of genes, fewer multi-copy genes, fewer unique genes, and fewer genome rearrangements compared with its close relatives. In addition, three Chlorella-specific hexose-proton symporter (HUP)-like genes were identified that enable the consumption of glucose and, consequently, heterotrophic growth. Furthermore, through comparative transcriptomic and proteomic studies, we generated a global perspective regarding the changes in metabolic pathways under autotrophic and heterotrophic growth conditions. Under heterotrophic conditions, enzymes involved in photosynthesis and CO2 fixation were almost completely degraded, either as mRNAs or as proteins. Meanwhile, the cells were not only capable of quickly assimilating glucose but also showed accelerated glucose catabolism through the upregulation of glycolysis and the tricarboxylic acid (TCA) cycle. Moreover, the rapid synthesis of pyruvate, upregulation of most enzymes involved in fatty acid synthesis, and downregulation of enzymes involved in fatty acid degradation favor the synthesis of fatty acids within the cell. CONCLUSIONS: Despite similarities to other Chlorella, C. protothecoides has a smaller genome than its close relatives. Genes involved in glucose utilization were identified, and these genes explained its ability to grow heterotrophically. Transcriptomic and proteomic results provided insight into its extraordinary ability to accumulate large amounts of lipid. The C. protothecoides draft genome will promote the use of this species as a research model.

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Figure 1 Chlorella protothecoides sp. 0710 is closely related to Chlorella variabilis NC64A. A). Genes in Cp0710, C. variabilis NC64A, and C. subellipsoidea C-169 were annotated by GO. Each bar represents the number of genes, and the different species are color-coded. B) The proteins in Cp0710 were searched by BLASTP against the TrEMBL protein database. The number of best-hit proteins in each organism is presented. C) A maximum likelihood phylogenetic tree was constructed using 300 single-copy orthologous genes shared in the seven species. D) The gene families in the three sequenced Trebouxiophyceae strains were compared. The Venn diagram shows the shared and unique gene families among the species. The number of gene families is indicated in black and the total number of genes is in yellow. CPRO: C. protothecoides, CVAR: C. variabilis NC64A; CSUB: C. subellipsoidea C-169.
Figure 1 Chlorella protothecoides sp. 0710 is closely related to Chlorella variabilis NC64A. A). Genes in Cp0710, C. variabilis NC64A, and C. subellipsoidea C-169 were annotated by GO. Each bar represents the number of genes, and the different species are color-coded. B) The proteins in Cp0710 were searched by BLASTP against the TrEMBL protein database. The number of best-hit proteins in each organism is presented. C) A maximum likelihood phylogenetic tree was constructed using 300 single-copy orthologous genes shared in the seven species. D) The gene families in the three sequenced Trebouxiophyceae strains were compared. The Venn diagram shows the shared and unique gene families among the species. The number of gene families is indicated in black and the total number of genes is in yellow. CPRO: C. protothecoides, CVAR: C. variabilis NC64A; CSUB: C. subellipsoidea C-169.CC BY · PubMed Central
Figure 2 Cp0710 has a compact genome. (A) Comparison of genes in three sequenced Trebouxiophyceae. Genes are classified as core orthologues, pairwise orthologues, or homologous and unique genes, and were marked with different colors. The height of the bar represents the number of genes in each category. (B) Synteny comparison between Cp0710 and C. variabilis NC64A. The conserved blocks of synteny are indicated by a different color. Scaffold ID of Cp0710 and C. variabilis NC64A are indicated in green and red, respectively. Only scaffolds with a length greater than 1 Mbp and 400 Kbp in Cp0710 and C. variabilis NC64A, respectively, and the lengths of syntenic regions greater than 50 Kbp are shown.
Figure 2 Cp0710 has a compact genome. (A) Comparison of genes in three sequenced Trebouxiophyceae. Genes are classified as core orthologues, pairwise orthologues, or homologous and unique genes, and were marked with different colors. The height of the bar represents the number of genes in each category. (B) Synteny comparison between Cp0710 and C. variabilis NC64A. The conserved blocks of synteny are indicated by a different color. Scaffold ID of Cp0710 and C. variabilis NC64A are indicated in green and red, respectively. Only scaffolds with a length greater than 1 Mbp and 400 Kbp in Cp0710 and C. variabilis NC64A, respectively, and the lengths of syntenic regions greater than 50 Kbp are shown.CC BY · PubMed Central
Figure 3 Multiple metabolic pathways are differentially regulated under heterotrophic conditions. (A) The whole KEGG pathway map after integrating the RNA-seq data. The gray lines indicate pathways not present in Cp0710 and the blue lines represent pathways that showed no significant changes in transcription. Pathways enriched in genes that are upregulated or downregulated are labeled in red and green, respectively. Significance is defined as log2 (fold-change) >1 and P < 0.01. Transcripts for enzymes with multiple subunits or isoforms were summed. (B) Selected pathways that are uniformly changed in heterotrophic cells. Only carbohydrate metabolism, energy metabolism, and lipid metabolism are listed here. See Additional file 1 Figure S9 for a complete list of affected pathways.
Figure 3 Multiple metabolic pathways are differentially regulated under heterotrophic conditions. (A) The whole KEGG pathway map after integrating the RNA-seq data. The gray lines indicate pathways not present in Cp0710 and the blue lines represent pathways that showed no significant changes in transcription. Pathways enriched in genes that are upregulated or downregulated are labeled in red and green, respectively. Significance is defined as log2 (fold-change) >1 and P < 0.01. Transcripts for enzymes with multiple subunits or isoforms were summed. (B) Selected pathways that are uniformly changed in heterotrophic cells. Only carbohydrate metabolism, energy metabolism, and lipid metabolism are listed here. See Additional file 1 Figure S9 for a complete list of affected pathways.CC BY · PubMed Central
Figure 4 Glycolysis and the triacylglycerol biosynthetic pathway in Cp0710. (A) Change of enzyme expression in glycolysis under heterotrophic growth conditions. The name of each enzyme is in red and the corresponding protein ID in Cp0710 is shown in blue. Values in brackets indicate changes in RNA (left) and protein (right) levels, respectively. U: only identified in heterotrophic approach (upregulation). D: only identified in autotrophic approach (downregulation). N: not detected. (B) Change in enzyme expression in the triacylglycerol biosynthetic pathway under heterotrophic growth conditions. The color code is the same as in (A). Abbreviations of enzymes are as follows: HT, H+/hexose co-transporter; HK, hexokinase; GPI, glucose-6-phosphate isomerase; PFK, 6-phosphofructokinase; ALDO, fructose-bisphosphate aldolase; TPI, triose phosphate isomerase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGAM, phosphoglycerate mutase; ENO, enolase; PK, pyruvate kinase; Accase, acetyl-CoA carboxylase; MCAT, acyl-carrier protein (ACP) S-malonyltransferase; KAS, 3-oxoacyl-ACP synthase II; KAR, 3-oxoacyl-ACP reductase; HD, 3-hydroxyacyl-ACP dehydratase; ENR, enoyl-ACP reductase I; FATA, fatty acyl-ACP thioesterase A; ACS, long-chain acyl-CoA synthetase; G3PDH, glycerol-3-phosphate dehydrogenase; GPAT, glycerol-3-phosphate O-acyltransferase 3/4; LPAAT, lysophosphatidate acyltransferase; PAP, phosphatidate phosphatase; DGAT, diacylglycerol O-acyltransferase 1; MLDP, major lipid droplet protein.
Figure 4 Glycolysis and the triacylglycerol biosynthetic pathway in Cp0710. (A) Change of enzyme expression in glycolysis under heterotrophic growth conditions. The name of each enzyme is in red and the corresponding protein ID in Cp0710 is shown in blue. Values in brackets indicate changes in RNA (left) and protein (right) levels, respectively. U: only identified in heterotrophic approach (upregulation). D: only identified in autotrophic approach (downregulation). N: not detected. (B) Change in enzyme expression in the triacylglycerol biosynthetic pathway under heterotrophic growth conditions. The color code is the same as in (A). Abbreviations of enzymes are as follows: HT, H+/hexose co-transporter; HK, hexokinase; GPI, glucose-6-phosphate isomerase; PFK, 6-phosphofructokinase; ALDO, fructose-bisphosphate aldolase; TPI, triose phosphate isomerase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGAM, phosphoglycerate mutase; ENO, enolase; PK, pyruvate kinase; Accase, acetyl-CoA carboxylase; MCAT, acyl-carrier protein (ACP) S-malonyltransferase; KAS, 3-oxoacyl-ACP synthase II; KAR, 3-oxoacyl-ACP reductase; HD, 3-hydroxyacyl-ACP dehydratase; ENR, enoyl-ACP reductase I; FATA, fatty acyl-ACP thioesterase A; ACS, long-chain acyl-CoA synthetase; G3PDH, glycerol-3-phosphate dehydrogenase; GPAT, glycerol-3-phosphate O-acyltransferase 3/4; LPAAT, lysophosphatidate acyltransferase; PAP, phosphatidate phosphatase; DGAT, diacylglycerol O-acyltransferase 1; MLDP, major lipid droplet protein.CC BY · PubMed Central

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Metadata

Authors
Gao, C., Wang, Y., Shen, Y., Yan, D., He, X., Dai, J., & Wu, Q.
Affiliations
  • China People's Public Security University (CN)
  • BGI Group (China) (CN)
  • Tsinghua University (CN)
Corresponding author
  • Dai, Junbiao
  • Wu, Qingyu
ORCID
Venue
BMC Genomics
Publisher
Springer Science and Business Media LLC
Published
2014-07-10
Volume
15
Issue
1
Pages
582-582
ISSN
1471-2164 (ISSN-L)
DOI
10.1186/1471-2164-15-582
PMC
PMC4111847
OpenAlex
W1984532384
Cited by
175
References
65
Citations by year
2026: 5 · 2025: 9 · 2024: 15 · 2023: 10 · 2022: 14 · 2021: 17 · 2020: 16 · 2019: 25 · 2018: 18 · 2017: 16 · 2016: 18 · 2015: 7 · 2014: 5
FWCI
5.06
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • BMC Genomics (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Natural Science Foundation of China (41030210, 31370282)
  • Tsinghua University (2012Z08128)
Topics
  • Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Algal biology and biofuel production
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Lipid metabolism and biosynthesis
Keywords
Chlorella protothecoides, lipid accumulation, draft genome sequence, genome size reduction, comparative genomics, heterotrophic growth, autotrophic growth, glucose catabolism, glycolysis, TCA cycle, fatty acid synthesis, Chlorella variabilis
MeSH terms
Carbon, Chlorella, Citric Acid Cycle, Energy Metabolism, Glycolysis, Plant Oils, Genome, Plant, Proteome, Metabolic Networks and Pathways, Comparative Genomic Hybridization, Biofuels, Transcriptome

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