Hildebrand Lab

Scripps Institution of Oceanography, University of California, San Diego

Improved Lipid Accumulation Through Metabolic

Engineering of Diatoms

Aubrey Davis

Diatoms and Lipid Accumulation

• Amongst unicellular algae, diatoms are one of the most productive classes, they

naturally grow under bloom conditions (maximum rate and biomass

accumulation in a short time period) and generally out-compete other

algae for growth (Brzezinski et al. 2001. Deep Sea Res. 48:3997; Carter et al., 2005, J. Exp. Mar. Biol. Ecol. 326:1).

• Diatoms are capable of abundant accumulation of neutral lipids (30-45%).

• Diatoms were a model organism in the Aquatic Species Program, out of a list of

50 strains selected from a pool of over 3,000 candidates that held the most promise

as biofuel production strains, 60% were diatoms (www.nrel.gov/docs/legosti/fy98/24190.pdf).

Thalassiosira Cyclotella pseudonana cryptica

Falkowski and Oliver 2007

Fundamental Analysis of Short-term Lipid Accumulation Resulting from Silicon Limitation in T. pseudonana

Sarah Smith

Fatty Acids

Photosynthesis

Whole-Genome mRNA Expression Using Microarrays

Lipid Induced

• RNA isolated from 6 timepoints (t=0h, t=4h, t=8h, t=12h, t=18h, t=24h)

• Affymetrix whole-genome tiling

microarray in duplicate (Andy Allen, JCVI)

• Bioinformatics (11,782 genes)

(Trina Norden-Krichmar, JCVI & Steve Federowicz, UCSD)

The Key Issue in Producing Useful Biomolecules: Carbon Flux

Regulating Carbon Partitioning is Required to Increase Production of Desired Biomolecules

Elucidation of Carbon Flux Processes:

Transient Induction of Gluconeogensis Genes

Sarah Smith

Storage Carbohydrate Genes

Key Finding:

The initial response to Si starvation is induction of

gluconeogenesis and carbohydrate storage,

followed later by TAG accumulation.

General Carbon Flux Response During Si Limitation

TAG

Knockdown of a Chrysolaminarin Synthase Gene

Improves TAG Accumulation

Three genes encoding steps in

chrysolaminarin synthesis are transiently

upregulated during silicon starvation

Several 12695 knockdown strains exhibit

improved TAG accumulation

The best knockdowns resulted in 1.5-3 fold

improvement in TAG levels over wild-type.

We are characterizing knockdowns in the two other

genes and generating simultaneous knockdowns in

multiple genes.

Key Finding:

Inhibition of carbon flux to a competing pathway

(carbohydrate storage) improves TAG

accumulation, in some cases without detriment to

growth.

Most strains grow as well as wild-type

Kalpana Manandhar-Shrestha

Overexpression of a DGAT Improves TAG Accumulation

Overexpression of DGAT resulted in 1.2-1.5 fold improvement in TAG levels over wild-type.

We are characterizing other enzymes involved in TAG synthesis and have generated overexpression strains.

Kalpana Manandhar-Shrestha

Characterization of CGI-58 Lipase Homologs Emily Trentacoste, Jennifer Hull, Bill Gerwick

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TAG

Catabolism

CGI-58 knockdowns in Arabidopsis and mutations in humans resulted in high TAG accumulation

Characterization of CGI-58 Lipase Homolog Knockdowns Emily Trentacoste, Jennifer Hull, Bill Gerwick

Total Lipid TAG Polar Lipid

Growth Curves BODIPY

Key Finding: CGI-58 homolog knockdowns improve both TAG and polar lipid

content with (in antisense cases) no detrimental affect on growth.

TAG was increased 1.9 - 4 fold

TAG TAG TAG

Catabolism

A Variety of Manipulations Lead to Increased TAG

Flexibility in choosing gene targets could minimize detrimental effects

and maintain fast growth and high biomass accumulation

Redirecting

carbon flux

Increasing

TAG synthesis Inhibiting lipid

catabolism

1.5-3 fold

improvement

1.2-1.5 fold

improvement

1.9-4 fold

improvement

Mitochondrion Cytoplasm Chloroplast

There are More Gene Targets to Consider

Not only specific metabolic steps, but compartmentation needs to be considered

Sarah Smith

Controlled Expression Overexpression Knockdowns Fluorescent Tagging

Diatoms as a System to Investigate Genetic Manipulation

Approaches To Improve Productivity

1. A versatile set of genetic manipulation tools has been developed, bringing the manipulation technology

for diatoms on a par with other model organisms.

2. Gene silencing does not occur.

3. G+C content is near 50% and codon optimization is not necessary (but it helps).

4. Tools developed in one species thus far have worked in other species.

Roshan Shrestha, Aubrey Davis, Luciano Frigeri

Characterization of CGI-58 Lipase Homologs:

Improved Lipid Content Plus a Non GMO Selection Approach Emily Trentacoste, Jennifer Hull, Bill Gerwick

Knockdowns exhibited a phenotype of increased TAG plus increased cell viability under Si-

limitation conditions.

Increased viability plus increased fluorescence (BODIPY and Chlorophyll) are selectable

phenotypes. Emily Trentacoste

Native Sequence, Non-GMO Selection Approach

Organisms are classified as GMOs only if DNA from another genus is introduced. Theoretically,

through recombination and cross-over such an arrangement could be generated by the native

host.

Transformants generated this way will not be classified as GMOs, can be selected, and may have

improved viability under stress than wild-type.

Additional genes can be manipulated by co-transformation.

Key Finding: Application of genetic manipulation approaches has led to the possibility

of native sequence-based metabolic engineering for field applications with no regulatory

issues.

promoter nat1 antisense fragment

terminator promoter antisense fragment

terminator

promoter antisense fragment

terminator

Transform and Select

Summary

Transcript-level changes are generally accurate indicators of metabolic changes.

Gene targets for metabolic engineering can be identified by transcriptomics analysis.

Manipulations affecting a variety of metabolic processes improved TAG accumulation

with no detriment to growth.

Improvements were substantial.

Genetic manipulation approaches led to the possibility of manipulations with non-

GMO classification.

Raffaela Abbriano

Orna Cook

Aubrey Davis

Mark Hildebrand

Jennifer Hull

Kalpana Manandhar-

Shrestha

Eva Sanchez

Roshan Shrestha

Sarah Smith

Benoit Tesson

Jesse Traller

Emily Trentacoste

Andrew Allen

Steve Federowicz

Trina M. Norden-Krichmar

Matteo Pellegrini

Sabeeha Merchant

Richard Sayre