• Development of Algal Genetic Tools


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Development of Algal Genetic Tools
Tools
Mark Hildebrand
Marine Biology Research Division,
Scripps Institution of Oceanography, University of California, San Diego
Growth Conditions Affect Carbon Partitioning In Some Algae
Algae
CO2 CO2
Photosynthesis Photosynthesis
C C
Partitioning Partitioning
Regulators Regulators
Lipid Carbohydrate Lipid Carbohydrate
Synthesis Synthesis Synthesis Synthesis
Nutrient Replete Nutrient Deficient
(Abundant Growth) (Poor Growth)
Identification of pathway-level regulation may be especially useful in metabolic

engineering to increase lipid yields under non-limiting growth conditions.
The Value of Genetic Approaches to Microalgal Biofuels Production
n
• Genetic approaches can aid in understanding the regulation of algal lipid
metabolism and carbon partitioning under different growth conditions.
• Genetic approaches can be used to metabolically engineer or select for
abundant lipid production coupled with high biomass accumulation.
• Genetic approaches can be used to facilitate large scale processing of
microalgae.
Genetic Approaches to be Considered
Considered
• Genomics
Pathway-level metabolic regulation can be investigated by genome-wide surveys of
gene content and mRNA responses
• Genetic engineering
Alteration of gene sequence or expression should enable metabolic engineering
• Directed Evolution / Selection
Foreknowledge of genes involved is not necessarily required, and this approach
could facilitate identification of genes and regulatory steps
• Sexual crossing
It works for crop plants, why not algae?
Growth Conditions Affect Carbon Partitioning In Some Algae
Algae
CO2 CO2
Photosynthesis Photosynthesis
C C
Partitioning Partitioning
Regulators Regulators
Lipid Carbohydrate Lipid Carbohydrate
Synthesis Synthesis Synthesis Synthesis
Nutrient Replete Nutrient Deficient
(Abundant Growth) (Poor Growth)
Recombinant DNA Technology II,
1994, 721: 250-256.
Attempts to Genetically Manipulate Diatom Lipid Metabolism
Metabolism
in the ASP
ASP
• Introduction of multiple ACCase copies into Cyclotella cryptica resulted in increased
mRNA and ACCase activity, but not increased lipid synthesis.
• Perhaps feedback regulation of lipid synthesis pathways occurred, or carbon was not sufficiently
repartitioned into lipid synthesis.
• After transformation of N. saprophila with the C. cryptica ACCase, mRNA was detected but not protein,
suggesting a level of translational control.
• Ribozymes designed to cleave UGP/PMGase mRNA were introduced into C. cryptica,
but were unsuccessful.
Better understanding of regulatory processes and better genetic manipulation tools
may enable these approaches
Genomics Approaches Can Facilitate Understanding of thethe
Regulation of Lipid Synthesis at Both the Individual Gene
Gene
and Pathway Levels
Levels
• Full Genome Sequence
• ESTs
• Microarrays
• SAGE
Diatom Genomes Sequenced or In Progress
Progress
• Thalassiosira pseudonana model centric species, small genome
• Phaeodactylum tricornutum model pennate species, small genome
• Thalassiosira oceanica centric species, open ocean, small genome
• Fragilariopsis cylindrus pennate species, cold water environments
• Pseudonitzschia multiseries pennate species, domoic acid producer
1 µm 2 µm
Species-specific Effects of Si Depletion
on Diatom Lipid Accumulation
Cylindrotheca fusiformis a cryptica Thalassiosira pseudonana
40
Cyclotell
25
25
30
20
Lipid Content
20
(% AFDM)
15
15
20
10
10
10
5
5
0 24 48 72
0 24 48 72
0 24 48 72
Hours After Si Depletion
Hours After Si Depletion
Hours After Si Depletion
Cyclotella cryptica accumulated more lipid, and more rapidly after Si depletion
Species-specific effects are likely due to differential regulation

Comparative analysis may shed light on regulatory differences
EST / Partial Genome Sequence of C. cryptica
cryptica
AFOSR Project Collaboration with Andy Allen at JCVI
Cyclotella cryptica
40
30
Lipid Content
(% AFDM)
20
10
0 24 48 72
Hours After Si Depletion
• Genome sizing and cell cycle stage arrest will be determined soon
• EST libraries during early stage of Si depletion will be generated and sequenced
• Also determining Si depletion response and sequencing ESTs for T. oceanica
Comparative analysis of transcript responses in the different genomes
may shed light on regulation involved in different lipid accumulation responses
Genetic Manipulation / Gene Expression Control Approaches
Approaches
AFOSR Project
Project
1. Develop new selectable transformation markers
To enable sequential addition of genes for multi-component metabolic engineering.
2. Develop “universal” transformation vectors for use in a variety of species
To reduce the time required to construct a new vector for each species.
3. Develop new gene expression manipulation tools
To enable sophisticated control over expression levels and timing.
4. Develop new gene tagging approaches
To determine protein expression levels and intracellular localization.
Selectable Marker Genes For Diatom Transformation
Major Study Species
Thalassiosira pseudonana Phaeodactylum tricornutum Cylindrotheca fusiformis
Marker Gene Mechanism of Action Species Reference
Cyclotella cryptica, Navicula
neomycin phosphotransferase II Inactivates G418 by Dunahay et al. 1995,
saprophila, Phaeodactylum
(nptII) phosphorlyation Zaslavskaia et al. 2001
tricorntum
Falciatore et al. 1999,
Zeocin resistance by Phaeodactylum tricorntum,
sh ble Fischer et al. 1999,
stoichiometric binding Cylindrotheca fusiformis
Zaslavskaia et al. 2001
Nourseothricin
Phaeodactylum tricornutum, Zaslavskaia et al. 2001,
nat1, sat-1 resistance by enzymatic
Thalassiosira pseudonana Poulsen et al. 2006
acetylation
Dual selection achieved with
Phaeodactylum tricornutum Zaslavskaia et al. 2001
nptII or nat1 and sh ble
We are exploring conserved mutations to antibiotic resistance in ribosomal protein genes
Promoters to Drive Selectable Marker Gene Expression
Expression
For Diatom Transformation
Transformation
Promoter Species Reference
Cyclotella cryptica, Navicula
ACCase Dunahay et al. 1995
saprophila
Falciatore et al. 1999,
Phaeodactylum tricorntum,
Fischer et al. 1999,
fcp Cylindrotheca fusiformis,
Zaslavskaia et al. 2001,
Thalassiosira pseudonana
Poulsen et al. 2006
A “Universal” Transformation Vector Would be Very Useful
We Have Successful Transformation to nat Resistance With:
Promoter Species
fcp T. pseudonana, T. oceanica, T. weissflogii
ACCase: T. pseudonana & Nitzschia alba
rpl41: N. alba
SV40: T. pseudonana
Development of New Gene Manipulation Tools
Tools
• Inducible Promoters
• Homologous Gene Replacement
• Knockout or Knockdown Approaches
• Protein Tagging
Development of New Gene Manipulation Tools: Inducible Promoters
Promoters
The Nitrate Reductase (NR) Gene (Poulsen et al. 2005)
Prestarvation for N decreases
response time
Protein expression levels are
partly titratable
Translational Regulation
Nitrate Reductase is Light and Cell Cycle Regulated
Development of New Gene Manipulation Tools: Inducible Promoters
Promoters
Copper Induced (cue) Genes (Davis et al. 2005)
Cue Genes are Induced 1000 – fold after exposure to Cu
Cue Gene mRNA Levels are Titratable
Development of New Gene Manipulation Tools:
Tools:
Homologous Gene Replacement
Replacement
Homologous Recombination
Genomic DNA
Gene
Synthetic X
Construct
Genomic DNA
X
Homologous gene replacement can be used to insert a mutation into a gene
or knock it out, since the native gene is replaced, phenotypic interference is
eliminated.
Current Status
A restriction site and termination codon was inserted into a T. pseudonana fcp gene
Large flanking regions were used (3 kbp)
Initial results demonstrated integration but not homologous gene replacement
Next attempt is to use single-stranded DNA
Development of New Gene Manipulation Tools:
Tools:
RNA Interference (RNAi)
)
RNAi is working in P. tricornutum (Chris Bowler pers. commun.), we are developing for T. pseudonana
Development of RNAi in Chlamydomonas
Chlamydomonas
Rohr et al. 2004
Marker Inverted Repeat
This construct was inefficient at RNAi
Marker & Inverted Repeat
F
Maa7 encodes β-tryptophan synthase which converts
5-fluoroindole to 5-flourotryptophan, a cytotoxin
Additional Gene to
Be Silenced All 5-FI resistant transformants had efficient
RNAi against both the Maa7 and additional genes
We have constructed a Maa7 vector under control of the nitrate reductase promoter
and will introduce it into T. pseudonana
The Importance of Protein Tagging in Metabolic Engineering
Engineering
Allows monitoring of protein expression
Localization and translational regulation
Simply turning a gene “on” or “off” at the mRNA level is no guarantee

of completely controlling it’s expression
Development of New Gene Tagging Approaches:
Approaches:
What’s Wrong with GFP?
GFP?
ER Mitochondria Plastid
Poulsen and Kröger, 2005 Apt et al., 2002
Problems - lack of fluorescence, finicky growth conditions, and localization artifacts
Development of New Gene Tagging Approaches:
Approaches:
The TC tag
tag
ESSGSFLNCCPGCCMEPGGR
Griffin et al. 1998 Giepmans et al. 2006
Advantages – small tag, no protein folding required, combined fluorescence and TEM imaging
Disadvantages - cells need to be fixed, only two colors, expensive
Development of New Gene Tagging Approaches:
Approaches:
The SNAP Tag - Covalys
Covalys
Protein of interest
20 Kd enzymatic activity
Fluorescent para-substituted benzyl guanine
Advantages – multiple colors
Disadvantages – no real-time imaging, relatively large tag, requires activity
Development of “New” Gene Tagging Approaches:
Approaches:
Antibody Epitope Tags
Tags
FLAG DYKDDDDK

c-MYC EQKLISEEDL
HA YPYDVPDYA
VSV-G YTDIEMNRLGK
HSV QPELAPEDPED
V5 GKPIPNPLLGLDST
Advantages – well established technology, multiple colors
Disadvantages - cells need to be fixed
Directed Evolution / Selection Approaches
Foreknowledge of genes involved is not necessarily required
Example: increased lipid production
Selected Selected Selected
Wild-type Wild-type
Cell Cell Cell
Cell Cell
Round 1 Round 2 Round 3
Nile Red Selection Selection Selection
Staining
Nile Red Staining of Lipids
Desirable Traits
• Abundant lipid and biomass accumulation
• Growth under extreme conditions (temperature, salinity, pH)
• Efficient light utilization
Selection Approaches can be Coupled with Mutagenesis
• Chemical or UV mutagenesis
• Tagging-based mutagenesis – enables identification of genes
• Genetic engineering
Sexual Crossing Approaches – Algal Breeding
Breeding
Induction of gametogenesis
in Thalassiosira weissflogii
Armbrust 1999
Conclusions
Conclusions
Genetic approaches are essential to understand metabolic regulatory processes
and to manipulate cellular metabolism to facilitate high lipid yields
Broad-based genetic manipulation and selection approaches should be developed
for diverse algal species
Genetic approaches should not only be considered important in the initial
stages of development of algal biofuels technology, but also in its continuing
refinement and optimization in concert with engineering and processing needs.
Personnel Involved
Involved
Luciano Frigeri
Aubrey Davis
Jeff Carlson
Mark Hildebrand
Thanks to AFOSR / Walt Kozumbo for Funding


Use: 0.6205