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

2.  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 cerevisiae cannot break down all of the sugars in lignocellulosic biomass BACKGROUND CelluloseEthanol (and CO 2 ) Fermentation

3.  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. cerevisiae to produce XI have failed  Common XI is active at high temperatures and pH’s. Not compatible with S. cerevisiae XYLOSE Xylose isomerase Xylose Xylulose

4.  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): GOAL  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 pH ~5 35°C pH 4.5 37°C

5. FERMENTATION DIAGRAM Biomass hydrolysis and glucose fermentation → Solid/liquid separation ↑ Xylose fermenter pH 5, 30°C xylose → ← xylulose ← ↑ Heat exchange Ion exchange NH 4 OH injector to raise pH Xylose & EtOH Yeast, enzymes, pre-treated biomass Isomerization reactor Contains immobilized XI pH 7.5, 55°C Ethanol to distillation Solids to energy recovery Current method Goal Biomass hydrolysis and glucose & xylose fermentation (contains immobilized compatible XI) pH 5, 30°C → Solid/liquid separation Yeast, enzymes, pre-treated biomass Solids to energy recovery Ethanol to distillation Technology by Trillium FiberFuels, Inc.

6.  P.C.R.= Polymerase Chain Reaction  Used to duplicate and isolate a specific genetic sequence  Two other eukaryotic XIs are known, and XIy was 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) PCR (INITIAL METHOD)

7.  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 PCR RESULTS The blue arrow represents the size that was hypothesized. There are no bands at that location.

8.  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 GENOMIC SEQUENCE

9.  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 FUNCTIONAL SEARCH WITH VECTORS Control: No colonies Some colonies: XIy gene might be present No colonies: no XIy gene Hansenula p. plates:

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

11.  E. coli cells showed significantly increased activity  Activity was over an unreasonably long time, however  First attempt to insert into yeast yielded no activity PLASMID INSERTION RESULTS

12.  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? CONCLUSION Maybe

13.  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/b9/201005101646 14!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. BIBLIOGRAPHY

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