Torres Romero, Ismael

Universitat Autònoma de Barcelona. Facultat de Biociències

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Metabolic engineering in microalgae: Challenges in Dunaliella tertiolectaIsmael Torres RomeroB.Sc. in Biotechnology, Universitat Autònoma de BarcelonaIndustrial BiotechnologyProduct improvements: - ↑TAG (ACCase overexpression, polyamine activation of  biosynthetic genes, etc.)- Lipid composition: better properties for biodiesel, shortening lipid chains.ConclusionsGenetic tools for D. tertiolecta are still scarce to engineer its metabolism. It is essential to sequence its genome for directed genetic engineering and explore its potential.Process improvements:- Higher iron uptake, ferritin concentration enables magnetic-driven extraction (↓ extraction cost).- Co-products accumulation such as β-carotene (nutraceutical, food additives).Assets of  D. tertiolecta- High growth rate in hypersaline media (↓ contaminations).- Produces up to 67% lipids and 27% starch (dry weight).- No rigid wall or theca: easier product extraction and genetic manipulation.- Accumulates β-carotene: photoprotection and nutraceutical co-product.“omics” TechnologyReferences1. Image extracted from: http://eol.org/data_objects/2088133 Authors: David Patterson and Bob Andersen.2. Gomes de Oliveira Dal’Molin et al. AlgaGEM – a genome-scale metabolic reconstruction of  algae based on the Chlamydomonas reinhardtii genome. BMC Genomics (2011) 12(Suppl 4):S5.3. Transcriptome sequencing and annotation of  the microalgae Dunaliella tertiolecta: Pathway description and gene discovery for production of  next-generation biofuels. BMC Genomics (2011) 12:148.4. Chang RL et al. Metabolic network reconstruction of  Chlamydomonas offers insight into light-driven algal metabolism. Molecular Systems Biology (2011) 7:518.Improve-import-replace metabolic pathways forGenetic transformation methods:- Particle bombardment (DNA attached togold shot by gene gun): the best for marinealgae and to reach the chloroplast (plastidsof D. tertiolecta have been successfullytransformed).- Glass beads (good for D. tertiolecta thin walls)- Others: electroporation (also reported in D.tertiolecta), microinjection, artificialtransposon, recombinant eukaryotic algalviruses, Agrobacterium tumefaciens-mediated.Plastid transformation through homologousrecombination.Vector construction:- Insertion of  introns from native genes in heterologous sequences and 3′ - 5′ UTRs increases protein yield.- Promoters: DCY1 for stable nuclear transformation in D. salina, CaMV35S as universal for marine algal, D. tertiolectainduced high heterologous expression with psbD promoter and psbA terminator, also cab4 light-regulated promoter. - Codon usage optimization is essential.- Reporter genes: GFP interfere with algal pigments. X-Gluc. - Marker genes: chloramphenicol, erythromicin (ereB), bleomycin (ble) inhibited D. tertiolecta.- Gene copy number and homology-dependant gene silencing- RNA interference technology, detected in D. salina.Dunaliella tertiolecta.Differential interference microscopy (1).Eukaryotic microalgae industrial potentialTo yield biofuels.- Oils, e.g. tryacylglycerides (TAG), stored in the cytoplasm/interthylakoid space. Transesterification →biodiesel; distillation+cracking → gasoline/jet fuel.For molecular farming, heterologous expression of  proteins and production of  secondary metabolites.- High growth rates, short generation times.- The most efficient at converting solar energy.- Cheap and versatile cultures.- Good protein post-transcriptional/traductional profiles.Metabolic EngineeringSystems BiologySynthetic BiologyFull genome-scale metabolicnetwork reconstruction ofChlamydomonas reinhardtii withcompartments depicted, Chang et al2011 (4).Iterative cycle to achieve industrial goals in microbial engineering, where analysis provides new biological knowlege.The process for genome-scale model reconstruction, Gomes de Oliveira Dal’Molin et al 2011(2). A holistic perspective approach is needed to predictchanges in cell biology, e.g. if wanting to know how alack of some substance or the accumulation of certainby-product would affect cell growth or product yield.A proper model of ‘what would happen if ’ must besought to achieve bioengineering goals with lessiterations.By now, AlgaGEM, a genome-scale meta-bolic reconstruction based on C. reinhardtii, distinctsalgal behaviours such as catabolism or secretion(currently covering only primary metabolism).In silico modeling of metabolic or cellsignaling networks predicts fluxvariations.Genomics:D. Tertiolecta is not sequenced; but thesimilar D. salina is (NCBI).ObjectiveReview the state-of-the-art knowledge to engineer D. tertiolecta metabolism in order to design target-directed strategies.Transcriptomics:Functional genomics. D. tertiolectatranscriptome was sequenced underOne of  the schematic pathways reconstructed; fatty acid biosynthesis based on the de novo assembly and annotation of  D. tertiolecta transcriptome, Rismani-Yazdi H et al 2011(3). various growth conditions to stimulate reportedproduction and accumulation of lipids or starch:nitrogen limited or sufficient cultures, seawater or saltstressed, and harvesting during log or stationaryphases. BLASTx. Functional annotation and pathwayidentification (gene ontology and KEGG orthology).

Metabolic engineering in microalgae : challenges in Dunaliella tertiolecta

http://ddd.uab.cat/pub/tfg/2013/114621/TFG_ismaeltorresromero.pdf.pdf

Metabolic engineering in microalgae : challenges in Dunaliella tertiolecta

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