Solving the puzzle of different product profiles between race A and race B strains of Botryococcus braunii

Transcription

1 Solving the puzzle of different product profiles between race A and race B strains of Botryococcus braunii What can genome-scale metabolic modelling tell us about this unique microalgal species? Carolyn M.C. Lam, SPLASH consortium workpackage 2 (U. Cambridge, U. Bielefeld, U. Münster, DLO-PRI, WUR-BPE), Vitor A.P. Martins dos Santos LifeGlimmer GmbH, Berlin

2 a harmful microalga useful for industry Blooms of the colonial green algae Botryococcus braunii Kützing associated with massive fish mortality in Nozha Lake, Alexandria, Egypt. Fish-killing mechanism unknown... Speculations: - DO - viscosity - reactive oxygen compounds - free fatty acids Fig. 1: Golden-yellow colonies of B. braunii (top) associated with fish mortality (bottom). Labib et al The 15th International Conference on Harmful Algae,Korea, 2012.

3 unique hydrocarbons (HC) & exopolysaccharides (EPS) vs. vegetable oil triglyceride Triterpenes: e.g. squalene e.g. botryococcene e.g. alkadienes/alkatrienes Ref. Race Metabolites A Alkadienes, alkatrienes etc. e.g. C23-C33, C23-C31 Molnar et al., 2012 Li et al., 2013 B Botryococcenes, triterpenoids etc. e.g. C30-C37, C31-C36 Molnar et al., 2012 Li et al., 2013 L Tetraterpenoids (C40) Li et al., 2013

4 unique hydrocarbons (HC) & exopolysaccharides (EPS) Main backbone structure of common mannan isolated from green seaweeds. Plastics from plant(algae)-based sugars: Wang et al Marine Drugs 12(9): furandicarboxylic acid (FDCA), the main new building block for polyethylenefuranoate (PEF) FDCA from plant-based industrial sugars FDCA BASF and Avantium intend to establish Joint Venture 15 Mar , Press releases Production and marketing of furandicarboxylic acid (FDCA) based on renewable resources, the main new building block for polyethylenefuranoate (PEF) Further development and licensing of Avantium s production processes for FDCA and PEF at industrial scale Intention to build a reference plant for FDCA with an annual capacity of up to 50,000 tons at BASF s Verbund site in Antwerp, Belgium PEF bottles, films, fibres etc.

5 taxonomic relation w.r.t. other microalgae (Micro)algae are more genetically diverse than plants or animals (Gimpel et al., 2013 Curr Opin Chem Biol 17: )

6 taxonomic relation w.r.t. other microalgae Eukaryota Chlorophyta Chlorophyceae Prasinophytes Chlamydomonadales/Volvocales Trebouxiophyceae Trebouxiophyceae incertae sedis Mamiellophyceae Chlamydomonadaceae Dunaliellaceae Chlamydomonadales incertae sedis Mamiellales Chlorellales Coccomyxaceae Botryococcaceae Fig. 2: Taxonomic relationship among several sequenced microalgae. Dotted line refers to intermediate levels being omitted. (Source: NCBI) Chlorellaceae

7 Race B race B vs. race A strains Race A Botryococcus braunii strains (courtesy Thomas F. Bastet, 2013, Bioprocess Engineering Group. [BPE], Wageningen University, Wageningen, the Netherlands)

8 Race B race B vs. race A strains Race A Botryococcus braunii strains (courtesy Thomas F. Bastet, 2013, Bioprocess Engineering Group. [BPE], Wageningen University, Wageningen, the Netherlands)

9 a systems approach to understand its capacity Integrating omics data and literature/database information for B. braunii genome-scale metabolic modelling for understanding this unique microalga. Genomics Proteomics Literature Transcriptomics ideally Optimized selection of pathways for further engineering Metabolomics Databases Design of experiments Genome-scale Botryococcus metabolic model

10 a systems approach to understand its capacity Integrating omics data and literature/database information for B. braunii genome-scale metabolic modelling for understanding this unique microalga. Literature Metabolomics Transcriptomics Databases Proteomics Genomics for Botryococcus Optimized selection of pathways for further engineering Design of experiments Genome-scale Botryococcus metabolic model

11 a systems approach to understand its capacity Literature B. braunii specific pathways Isolated/Characterized enzymes, metabolites Biomass composition glyoxisome Established algal GSMM mitochondrion Chlamydomonas Reaction reversibility BRENDA Molecular formula MetaCyc MetaCyc ChEBI KEGG Enzyme localization UniProt Literature cytoplasm nucleus Pathway details MetaCyc KEGG chloroplast Golgi thylakoid lumen

12 examples of characteristic products Photosynthesis (biomass) Steroids biosynthesis Sugars/(Exo)polysaccharides biosynthesis Long-chain alkadienes/ alkatrienes biosynthesis Mevalonate pathway squalene Methylerythritol 4-phosphate (MEP) pathway presqualene diphosphate botryococcenes

13 examples of characteristic pathways Botryococcenes vs. (normal) squalene pathways Squalene pathway (essential; both races) Botryococcenes biosynthesis (race B only) Why?? (from central metabolism) H+ removed from figures for clarity

14 examples of characteristic pathways 'Race A' vs. race B specific HCs Race A (long-chain alkadienes/alkatrienes) H+ removed from figures for clarity Race B (botryococcenes)

15 examples of characteristic pathways (CO2, light) D- fructose-6phosphate D- fructose-6-phosphate D-mannose L-galactose 6-methyl-galactose D-glucose D-xylose Qualitative relative abundance arabinose galactose 3-methyl-arabinose 6-methyl-galactose glucose mannose L-arabinose (Weiss et al., 2012 Eukaryotic Cell 11, ) 3-methyl-arabinose Metabolic connectivity of major sugars in polysaccharides of extracellular matrix in B. braunii (race B) from photosynthetic pathway.

16 light a systems approach to understand CO2 its capacity thylakoid lumen chloroplast Autotrophic growth Race A: exopolyscchrides mitochondrion Golgi Race B: cytoplasm hydrocarbons products glyoxisome nucleus

17 a systems approach to understand its capacity thylakoid lumen chloroplast Different utilization of metabolic pathways - presqualene diphosphate / squalene biosynthesis Golgi mitochondrion cytoplasm - sugars/(exo)polysaccharides biosynthesis Race A > B Race B > A glyoxisome nucleus

18 Next/Future steps Race A (EPS) vs. race B (HC) Intracellular flux distribution/variability under various growth conditions Regulations leading to differences in race A vs. race B Which genes to overexpress / knockdown? Isolatable pathways? etc.

19 Why not seen in other microalgae? To summarize Triterpenes B A Alkadienes/Alkatrienes Genomics Transcriptomics Metabolomics Proteomics Literature Databases EPS Understanding other B. braunii races!!

20 Acknowledgement U. Muenster (proteomics preliminary results): Eugen Urzica Johannes Leufken Michael Hippler DLO-PRI (genomics): Sander Peters WUR-BPE (fermentation): Douwe van der Veen Joao Gouveia Rene Wijffel U. Cambridge (transformation): Mark Scaife Alison Smith U. Bielefeld (transcriptomics, metabolomics): Swapnil Sudhakar Chaudhari Olga Blifernez Anja Doebbe Olaf Kruse LifeGlimmer (metabolic modelling): Carolyn Ming Chi Lam Vitor Martins dos Santos