Genetic Engineering of Algae for Biofuel Production

Stephen Mayfield
Department of Cell Biology and The Skaggs Institute for Chemical Biology The Scripps Research Institute

Alga: Latin “Seaweed”
• Aquatic eukaryotic organisms that contain chlorophyll and other pigments and can carry on photosynthesis
– Range from microscopic single cells to very large multicellular structures resembling stems and leaves – Further categorized as brown algae, red algae, green algae, also organisms such as dinoflagellates and diatoms – Some prokaryotes are incorrectly called blue-green algae

Why algae as a biofuel platform

Cost Scalability Sustainability

A few numbers to consider for biofuel production from algae
• 140 billion gallons/year of liquid fuel consumed in US • Algae can produce 50 tons biomass or 1,600 gallon/acre-year • 90 million acres needed to fill liquid fuel requirements
• could be reduce 4 fold at maximum photosynthetic yields • 90 million in corn and 67 million in soybeans in 2008

• Cost of production estimated between $3 and $60 /gallon

Production of biofuels from terrestrial crops Perpetual motion Cheap Energy Cheap Food Biofuel .

What should an algal biofuel solution look like? • • • • • Sunlight energy converted directly to fuel No use of agricultural land or products Highly scalable process to meet demand Commodity energy prices Carbon neutral process .

Sunlight is the original source of all liquid fuels .

Challenges of producing fuels from algae Harvesting Recovery of fuel Improved strains .

botryococcene Carbohydates: sugars and starches Ethanol or other alcohols Cellulose or other biomass .Biofuels produced by algae Biodiesel. triglycerides and fatty acids Lipids. long chain hydrocarbons .

Guesses* about how to realize biofuel production from algae • Identify strains with desired traits – one strain unlikely to have everything we want • Need to modify those strains – to produce high levels of desired molecule – to fit harvest and fuel recovery requirements – Probably not naturally occurring traits – Will require genetic modification on an accelerated time frame *To predict without sufficient information .

large scale agriculture 1837 Corn “varieties” 1863 Green Revolution 1944 Genetically modified corn 2000 • Need the same for algae only quicker .C.Need an accelerated time frame for “domestication” of algae Corn Domesticated 4000 B. Steel plow.

What do we need to achieve rapid domestication of algae for biofuels? • We need a much bigger and better knowledge base on algae • We need to identify and characterize a large number of diverse algal species – Genomic. proteomic and metabolic profiles • We need to develop genetic tools for breeding • We need to develop molecular tools for engineering • We need to develop agricultural practices for algal growth. harvesting and processing .

but showing the potential • We know how to grow algae at a modest scale • We have a few algal genomes sequenced and annotated • We have nuclear and chloroplast transformation for a handful of species .What do we have so far? • We have many species identified with limited characterization.

Developing the tool for algal engineering Biofuels are all made in the chloroplast from photosynthesis .most of enzymes responsible are nuclear encoded Nucleus Chloroplast .

Genetic transformation of algae is relatively easy Although you need selectable markers for each species nucleus Chloroplast .

Chloroplast transformation proceeds by homologous recombination psbA promoter 5’ UTR rbcL 3’ UTR Codon optimized coding region • need promoter and UTRs flanking region of homology • recombinant proteins can accumulate to very high levels • chloroplast can express complex proteins • less sophisticated gene regulation in plastids .

cytoplasm or export • Gene silencing is presently a limiting factor .Nuclear transformation proceeds by random integration Promoters larger More complex 5’ UTR 3’ UTR Codon optimized coding region • Need more transformation events to get good expression • Gene expression more complex. regulation potential greater • Can target proteins to plastids.

1 2 Initial Transformation Day 1 Selection of Primary Transformants Day 10 3 Homoplasmic Lines with High Expression Levels Day 14 4 Scale-up to Multi-liter Volumes 4 weeks 5 Scale-up to 64.2 Million L/acre 12 weeks .000 Liters 6 weeks 6 Grow out to 1.

395 bp  Simple prokaryotic promoters  Stable uncapped non-polyA mRNAs  Bacteria-like ribosomes  Easily transformed .Chloroplast Genome  Complete set of genetic material C. reinhardtii Chloroplast genome 203.

reinhardtii chloroplasts atpA promoter 5’ UTR rbcL 3’ UTR Codon optimized gene .Expression of recombinant protein in C.

Synthetic codon optimize chloroplast GFP gene .

Analysis of codon optimized gfp expression in transgenic chloroplast .

Promoter and UTR combinations for increased chloroplast expression atpA promoter 5’ UTR rbcL promoter 5’ UTR psbA promoter 5’ UTR psbD promoter 5’ UTR atpA 3’ UTR + gfpCt + rbcL 3’ UTR psbA 3’ UTR tRNA 3’ UTR = .

Accumulation of chimeric gfp mRNAs in C. reinhardtii chloroplast atpA/atpA atpA/rbcL atpA/psbA rbcL/rbcL rbcL/psbA rbcL/tRNA psbA/psbA psbA/rbcL psbD/psbA 16S/rbcL 16S-T7/rbcL gfp probe psbA probe RNA stain atpA rbcL psbA Northern blot .

GFP accumulation in transgenic lines 80 61 36 25 19 80 61 36 25 19 atpA rbcL psbA psbD/psbA 16S/rbcL 16S-T7/rbcL rbcL/rbcL 1/2 psbD/pabA 1/4 psbD/psbA 1/8 psbD/psbA wt atpA/atpA atpA/rbcL atpA/psbA rbcL/rbcL rbcL/psbA rbcL/tRNA psbA/psbA psbA/rbcL Coomassie stain GFP Western .

Protein and mRNA levels correlate poorly for many transgenic mRNAs .

The 5’ UTR largely determines protein expression levels .

Gene replacement vector for improved Recombinant protein expression D1 promoter and UTR D1 3’UTR SAA gene D1 5’ genomic flanking psbA coding D1 3’ genomic flanking .

Expression of SAA from psbA KO vector Saa-22 Sol total SAA-22 SAA-1 Sol SAA-22 Mem SAA-22 Wt total SAA-22 mem Coomassie stain gel Western anti-SAA3 Wt Sol Wt Wt Wt .

115 60 39 Wt SAA22 SAA22-psbD/psbA SAA22-psbA/psbA 1/2 SAA22-psbD/psbA 1/4 SAA22-psbD/psbA mw 10 19 Robust expression of recombinant proteins in a photosynthetic strain Stain gel Wt SAA22 SAA22-psbD/psbA SAA22-psbA/psbA 1/2 SAA22-psbD/psbA 1/4 SAA22-psbD/psbA Western anti-SAA .

Light activation requires PSI and is proportional to light flux through the photosystem complex Constant light increasing time Constant time increasing light .

Coli GFP -ve .Expression of recombinant proteins from the nuclear genome of C. reinhardtii HSP70A promoter rbcS2 5’UTR ble gene rbcS2 3’UTR NcoI HSP70 x IE rbcS2 Ble GFP rbcS2 pBleEGFP4 XbaI NdeI BamHI KpnI rbcS2 intron 1 (x4) rbcS2 intron 1 Gene (GFP) pBleEGFP4 Chlamydomonas Ble::GFP E.

reinhardtii cells Signal peptide NcoI HSP70 x IE rbcS2 Ble GFP rbcS2 XbaI NdeI BamHI KpnI FMDV 2A peptide GFP fluorescence Chlamydomonas Ble::GFP Chlamydomonas 2AGFP .Secretion of proteins from C.

Fatty Acids to Biodiesel .

reinhardtii chloroplasts Michael Burkart UCSD .Expression of a bacterial PPTase in C.

Hydrocarbon Biosynthesis Isopentyl-PP Geranyl-PP synthase Monoterpenes Geranyl-PP Geranylgeranyl-PP synthase Geranylgeranyl-PP Phytoene synthase Phytoene Diterpenes Carotenoid isomerase Lycopene Xanthophyll Cycle Beta-Carotene .

A few numbers to consider for ethanol production form cellulose • 140 billion gallons/year of liquid fuel consumed in US/year • Requires 100 g cellulase/gallon EtOH • 10% of fuel as EtOH would require 1.4 B kg of enzyme • 1 B kg of enzyme requires ~100 B liters of fermentation broth .

as we know it now. will preclude it’s use at this scale and cost • Algae can be grown at agricultural scale at cost approaching agricultural commodities • Algae can be engineered to produce recombinant cellulases • The by products of algal production can be used for biodiesel and/or animal feed .Reasons for the Engineering Production of Cellulose Degrading Enzymes in Algae • Production of cellulase at agricultural cost and scale is the only way to achieve economic production at the levels required • CapEx of fermentation.

2 BD-04 BD-05 BD-06 BD-07 Acts on interior portion of cellulose chain. . 3 4 BD-08 BD-09 BD-10 Used to avoid reaction inhibitors by cellobiose.Cellulose Biomass Degrading Enzymes Sapphire ID Exo-β-glucanase CBH I CBH II CBH I Endo-β-glucanase EG I EG III EG V EGL A β-glucosidase BGL I BGL II BGL I Endoxylanase 1 BD-01 BD-02 BD-03 Acting from the cellulose chain end. BD-11 BD-12 XYN I XYN II Acting on the hemicellulose crosslinking .

20 0.Exo-β-glucanase (BD-01) Exoglucanase gene-specific amplicon 1 2 3 7 8 11 4 5 6 12 13 14 5 6 4 12 13 14 9 10 Wild-type psbA-specific amlicon 1 2 3 7 8 9 10 11 Western Blot analysis of clones identified by PCR Wt 5 8 10 11 12 13 Activity determined by filter paper assay Purification of enzyme from clone 10 n ei ot in Pr n te ote d tei r sa un ro Ly le P bo d P e b n.45 .e si lut ud olu S Cr Re E Sample blank Exoglucanase clone 10 Endoglucanase clone 6 Exoglucanase clone 10 and Endoglucanase clone 6 Abs540nm 0 0 0.

Quantitation of cellulase expression in chloroplast BD-5-26 24h light 5ug 10ug 20ug 48h light 5ug 10ug 20ug FLAG control protein 10ng 50ng 100ng 200ng Western anti-flag > 2% of soluble protein .

Biofuel production in algal photobioreactor protein biofuel sunlight cellulase Algae secreting cellulase Municipal wastewater Waste carbon dioxide .

Where do we go from here? • We need a national center for algal research .San Diego • Develop the knowledge base of algal • Develop the molecular tools to make algae a biotechnology platform • Develop strains of algae for economic biofuel production • Develop industrial practices for growth harvest and recovery of biofuel .

NIH.The Scripps Research Institute • • • • • • • • Andrea Manuell Mike Mendez Brian O’Neill Kari Mickelson Miller Tran Phil Lee Par Petterssen Machi Muta • • • • • • • • • • • • • • • Veronica Beligni Julia Marin Dwight Barnes Kenichi Yamaguchi Emma Brown Scott Franklin Aravind Somanchi Susana Prieto David Siefker Jason Schultz Ryan Henry Anna Coragliotti Karen Espina Rick Bruick Chris Yohn DOE. Skaggs .

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