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 Cheap
Energy 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

 Biofuels produced by algae
Biodiesel, triglycerides and fatty acids
Lipids, long chain hydrocarbons - botryococcene

Carbohydates: sugars and starches

Ethanol or other alcohols
Cellulose or other biomass
 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

Need an accelerated time frame for
“domestication” of algae

Corn Domesticated 4000 B.C.

Steel plow, large scale agriculture 1837
Corn “varieties” 1863
Green Revolution 1944
Genetically modified corn 2000

• Need the same for algae only quicker

 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
 What do we have so far?

• We have many species identified with limited

characterization, 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
 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
 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, cytoplasm or export
• Gene silencing is presently a limiting factor
 1 Initial Transformation
Day 1

2 Selection of Primary Transformants 3 Homoplasmic Lines with High

Day 10 Expression Levels
Day 14

4 Scale-up to Multi-liter Volumes

4 weeks

5 Scale-up to 64,000 Liters

6 weeks

6 Grow out to 1.2 Million L/acre

12 weeks
 Chloroplast Genome

 Complete set of genetic material

 Simple prokaryotic promoters

C. reinhardtii
Chloroplast genome
203,395 bp
 Stable uncapped non-polyA mRNAs

 Bacteria-like ribosomes

 Easily transformed
Expression of recombinant protein
in C. reinhardtii chloroplasts

atpA promoter
5’ UTR rbcL 3’ UTR

Codon optimized gene

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
atpA 3’ UTR
rbcL promoter
5’ UTR rbcL 3’ UTR

psbA promoter
+ + =
psbA 3’ UTR
5’ UTR
psbD promoter gfpCt tRNA 3’ UTR
5’ UTR
Accumulation of chimeric gfp mRNAs
in C. reinhardtii chloroplast
atpA rbcL psbA

16S-T7/rbcL
rbcL/tRNA
psbA/psbA

psbD/psbA
atpA/psbA

rbcL/psbA

psbA/rbcL
atpA/atpA
atpA/rbcL

rbcL/rbcL

16S/rbcL
gfp probe

psbA probe

RNA stain

Northern blot
19
25
36
61
80
19
25
36
61
80
wt
atpA/atpA
atpA/rbcL
atpA

atpA/psbA
rbcL/rbcL
rbcL/psbA
rbcL/tRNA
psbA/psbA
rbcL psbA

psbA/rbcL

psbD/psbA
16S/rbcL
16S-T7/rbcL
rbcL/rbcL
1/2 psbD/pabA
1/4 psbD/psbA
1/8 psbD/psbA
stain
GFP accumulation in transgenic lines

Coomassie
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 D1 3’ genomic flanking

psbA coding
Expression of SAA from psbA KO vector

SAA-22 mem
total Sol Mem

Saa-22 Sol
SAA-22

SAA-22

SAA-22

Wt total
SAA-1

Wt Sol
Wt

Wt

Wt

Coomassie stain gel Western anti-SAA3

 10
19
39
60
115
mw
Wt

SAA22

SAA22-psbD/psbA

SAA22-psbA/psbA

Stain gel
1/2 SAA22-psbD/psbA

1/4 SAA22-psbD/psbA

Wt

SAA22

SAA22-psbD/psbA
in a photosynthetic strain

SAA22-psbA/psbA

1/2 SAA22-psbD/psbA

1/4 SAA22-psbD/psbA
Western anti-SAA
Robust expression of recombinant proteins
 Light activation requires PSI and is
proportional to light flux through the
photosystem complex

Constant light increasing time Constant time increasing light

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 -ve

Chlamydomonas Ble::GFP

E. Coli GFP
Secretion of proteins from C. 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
Fatty Acids to Biodiesel
Expression of a bacterial PPTase
in C. reinhardtii chloroplasts

Michael Burkart UCSD

 Hydrocarbon Biosynthesis
Isopentyl-PP
Monoterpenes
Geranyl-PP synthase

Geranyl-PP
Geranylgeranyl-PP synthase

Geranylgeranyl-PP
Phytoene synthase

Phytoene
Diterpenes
Carotenoid isomerase

Lycopene

Xanthophyll Beta-Carotene
Cycle
 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
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, 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
 Cellulose Biomass Degrading Enzymes

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

Endoxylanase
BD-11 XYN I Acting on the hemicellulose crosslinking .
4 BD-12 XYN II
 Exo-β-glucanase (BD-01)
Exoglucanase gene-specific amplicon Wild-type psbA-specific amlicon
1 2 3 7 8 9 10 1 2 3 7 8 9 10

11 11

4 4

5 6 12 13 14 5 6 12 13 14

Western Blot analysis of clones identified by PCR

Wt 5 8 10 11 12 13

Activity determined by filter paper assay

Sample Abs540nm
Purification of enzyme from clone 10 blank 0
in
o te Exoglucanase clone 10 0
e tei
n Pr in
t d te
y sa Pro o un Pro Endoglucanase clone 6 0.20
L
de uble i n -b ed
u l s lut Exoglucanase clone 10 and 0.45
Cr So Re E Endoglucanase clone 6
Quantitation of cellulase expression in chloroplast
BD-5-26
24h light 48h light FLAG control protein
5ug 10ug 20ug 5ug 10ug 20ug 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
 The Scripps Research Institute

• Andrea Manuell • Veronica Beligni

• Julia Marin
• Mike Mendez • Dwight Barnes
• Brian O’Neill • Kenichi Yamaguchi
• Emma Brown
• Kari Mickelson • Scott Franklin
• Miller Tran • Aravind Somanchi
• Susana Prieto
• Phil Lee • David Siefker
• Par Petterssen • Jason Schultz
• Ryan Henry
• Machi Muta • Anna Coragliotti
• Karen Espina
• Rick Bruick
• Chris Yohn

DOE, NIH, Skaggs