Improved Lipid Accumulation Through Metabolic Engineering of Diatoms

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1 Improved Lipid Accumulation Through Metabolic Engineering of Diatoms Aubrey Davis Hildebrand Lab Scripps Institution of Oceanography, University of California, San Diego

2 Falkowski and Oliver 2007 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 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 ( Thalassiosira pseudonana Cyclotella cryptica

3 Fundamental Analysis of Short-term Lipid Accumulation Resulting from Silicon Limitation in T. pseudonana Fatty Acids Photosynthesis Sarah Smith

4 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)

5 The Key Issue in Producing Useful Biomolecules: Carbon Flux Regulating Carbon Partitioning is Required to Increase Production of Desired Biomolecules

6 Elucidation of Carbon Flux Processes: Transient Induction of Gluconeogensis Genes Storage Carbohydrate Genes Key Finding: The initial response to Si starvation is induction of gluconeogenesis and carbohydrate storage, followed later by TAG accumulation. Sarah Smith

7 General Carbon Flux Response During Si Limitation TAG

8 Knockdown of a Chrysolaminarin Synthase Gene Improves TAG Accumulation Three genes encoding steps in chrysolaminarin synthesis are transiently upregulated during silicon starvation Several knockdown strains exhibit improved TAG accumulation The best knockdowns resulted in fold improvement in TAG levels over wild-type. We are characterizing knockdowns in the two other genes and generating simultaneous knockdowns in multiple genes. Most strains grow as well as wild-type Key Finding: Inhibition of carbon flux to a competing pathway (carbohydrate storage) improves TAG accumulation, in some cases without detriment to growth. Kalpana Manandhar-Shrestha

9 Overexpression of a DGAT Improves TAG Accumulation Overexpression of DGAT resulted in 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

10 Characterization of CGI-58 Lipase Homologs Emily Trentacoste, Jennifer Hull, Bill Gerwick Relative change in mrna 1 Thaps3_ TAG Time (hours) Relative change in TAG TAG Catabolism CGI-58 knockdowns in Arabidopsis and mutations in humans resulted in high TAG accumulation

11 Characterization of CGI-58 Lipase Homolog Knockdowns Emily Trentacoste, Jennifer Hull, Bill Gerwick Growth Curves BODIPY Total Lipid TAG Polar Lipid 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 fold

12 A Variety of Manipulations Lead to Increased TAG Redirecting carbon flux Increasing TAG synthesis Inhibiting lipid catabolism fold improvement TAG fold improvement TAG Catabolism fold improvement TAG Flexibility in choosing gene targets could minimize detrimental effects and maintain fast growth and high biomass accumulation

13 There are More Gene Targets to Consider Mitochondrion Cytoplasm Chloroplast Not only specific metabolic steps, but compartmentation needs to be considered Sarah Smith

14 Diatoms as a System to Investigate Genetic Manipulation Approaches To Improve Productivity Controlled Expression Overexpression Knockdowns Fluorescent Tagging 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

15 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 Silimitation conditions. Increased viability plus increased fluorescence (BODIPY and Chlorophyll) are selectable phenotypes. Emily Trentacoste

16 Native Sequence, Non-GMO Selection Approach promoter nat1 antisense fragment terminator promoter antisense fragment terminator promoter antisense fragment terminator 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. Transform and Select 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.

17 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.

18 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