Presentation on theme: "HHMI/Johnson Summer Internship Davis Weymann Mentors: Dr. Christine Kelly Dr. Curtis Lajoie Summer 2011 Identifying and Cloning Xylose Isomerase Gene for."— Presentation transcript:
HHMI/Johnson Summer Internship Davis Weymann Mentors: Dr. Christine Kelly Dr. Curtis Lajoie Summer 2011 Identifying and Cloning Xylose Isomerase Gene for Biofuel Production
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
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
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
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.
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)
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.
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
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:
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?
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
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
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/18.104.22.168ach.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
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