HHMI /Johnson Summer Internship

1. Identifying and Cloning Xylose Isomerase Gene for Biofuel Production Davis Weymann Mentors: Dr. Christine Kelly Dr. Curtis Lajoie Summer 2011 HHMI/Johnson Summer Internship

2. Background • There is growing interest in alternatives to petroleum fuels • Biofuels are promising, but require economical mass production methods before expanding • Lignocellulosic biomass is cheap and widely available resource that does not share role as a food source • Saccharomyces cerevisiaecannot break down all of the sugars in lignocellulosicbiomass Fermentation Cellulose Ethanol (and CO2)

3. Xylose • Lignocellulose = Cellulose + hemicelulose + lignin • 20-40% of lignocellulosic biomass is composed of hemicellulose • Hemicellulose is easy to hydrolyze, but it yields mostly xylose. • Xylose cannot be metabolized by S. cerevisiae • Xylose isomerase (XI) converts xylose into xylulose, which then can be utilized by S. cerevisiae • Attempts to engineer S. cerevisiaeto produce XI have failed • Common XI is active at high temperatures and pH’s. Not compatible with S. cerevisiae Xylose isomerase Xylulose Xylose

4. Goal • A certain yeast (Y1) is thought to produce a xylose isomerase (XIy) that is compatible with ideal fermentation conditions of S. cerevisiae • S. cerevisiae fermentation: • XI from Y1 (XIy): • pH ~5 35°C • pH 4.5 37°C • Ultimate goal: genetically engineer an organism to mass-produce XIy, which will then be used in fermentation with S. cerevisiae • Challenge: The location of the XIy gene on Y1’s genome is unknown • Project goal: Identify and isolate the XIy gene

5. Fermentation Diagram Current method Goal Ethanol to distillation Yeast, enzymes, pre-treated biomass Yeast, enzymes, pre-treated biomass Isomerization reactor Contains immobilized XI pH 7.5, 55°C Xylose fermenter pH 5, 30°C ↑ ← xylulose ← Biomass hydrolysis and glucose fermentation Biomass hydrolysis and glucose & xylose fermentation (contains immobilized compatible XI) pH 5, 30°C Heat exchange xylose → ↑ NH4OH injector to raise pH Ion exchange Xylose & EtOH Ethanol to distillation Solid/liquid separation Solid/liquid separation → → Solids to energy recovery Solids to energy recovery Technology by Trillium FiberFuels, Inc.

6. PCR (Initial Method) • P.C.R.= Polymerase Chain Reaction • Used to duplicate and isolate a specific genetic sequence • Two other eukaryotic XIs are known, and XIywas suspected to be similar to them • Search Y1’s genome for sequences similar to known XI genes • Degenerate primers attach to sites that match target with discrepancies • Degenerate primers matching known XI genes will target sequences that are similar (don’t need to be identical)

7. PCR results • We know the expected size of the copied sequences because they matched known XI genes • None of the copied gene sequences were of the expected length • The degenerate primers copied other sequences, but not the ones expected • No obvious XI gene matches were copied by the degenerate primers The blue arrow represents the size that was hypothesized. There are no bands at that location.

8. Genomic sequence • Not any great matches in entire genome • pXI1: Codes for a protein (probably an endonuclease) with similar 3D structure as XI • pXI2: Codes for a phosphorylated sugar isomerase with the expected molecular weight • Still is the question of if Y1 actually produces XI • The strain used in original study was lost • It has been difficult to detect XI activity from Y1

9. Functional Search with vectors • Testing function of a gene by isolating then inserting it into an organism and observing if it makes the desired protein • Insert the putative XI genes into E. coli and mutant Hansenula p. using a vector plasmid • If the lysate from the transformed E. coli has XI activity (plasmid coded for inter-cellular proteins), it might have accepted the XIy gene. Bacteria can’t always make eukaryotic proteins, however. • If the transformed mutant Hansenula p. can grow on xylose, either the XIy gene or xylitol gene was probably accepted Hansenula p. plates: Some colonies: XIy gene might be present No colonies: no XIy gene Control: No colonies

10. Plasmid Vectors Culture the E. coli w/ plasmids Target Sequence Genome Extract the plasmids Plasmid Insert plasmids into Hansenula p.Can it ferment xylsose? Cut plasmid and genome w/ enzymes Mutant Hansenula p. can’t grow on xylose Insert the sequence into plasmid Heat-shock plasmid into E. coli

11. Plasmid insertion results • E. coli cells showed significantly increased activity • Activity was over an unreasonably long time, however • First attempt to insert into yeast yielded no activity

12. Conclusion • The gene for XIy has not yet been identified • The results of the ongoing tests will determine if the line of research is continued • Does out strain of Y1 indeed contain the gene for the supposed XIy? Maybe

13. Bibliography • Sources: • Christine, K. Development of a Fermentation Compatible Xylose Isomerase Enzyme. 2010. Trillium FiberFuels, Inc. • Christine, K. Sungrant Proposal. 2010. • Trillium FiberFuels, Inc. • Wikipedia (general reference only) • Image Credits (In order of appearance) • http://www.uq.edu.au/_School_Science_Lessons/16.3.1.6ach.GIF • http://upload.wikimedia.org/wikipedia/commons/thumb/e/e8/Ethanol-structure.svg/529px-Ethanol-structure.svg.png • http://upload.wikimedia.org/wikipedia/commons/6/6a/Xylose.png • http://upload.wikimedia.org/wikipedia/commons/archive/b/b9/20100510164614!Xylulose.png • http://www.alvinziegler.com/gridlock/wp-content/uploads/2009/12/Genome-white.jpg • http://schoolworkhelper.net/wp-content/uploads/2011/06/PCR1.jpg • http://blog-images.microscopesblog.com/uploaded_images/pipet-701236.jpg • http://www.usascientific.com/productimages/16155500_300.jpg • Christine, K. Development of a Fermentation Compatible Xylose Isomerase Enzyme. 2010. Trillium FiberFuels, Inc. • Weymann, Davis. July 2011.

14. Acknowledgements Dr. Christine Kelly Dr. Curtis Lajoie Pete and Rosaline Johnson Dr. “Skip” Rochefort Howard Hughes Medical Institute (HHMI) Dr. Kevin Ahern Trillium FiberFuels, Inc.