Davis Weymann - Oregon State University

Report
Identifying and Cloning
Xylose Isomerase Gene
for Biofuel Production
Davis Weymann
Mentors: Dr. Christine Kelly
Dr. Curtis Lajoie
Summer 2011
HHMI/Johnson
Summer Internship
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 cerevisiae cannot break down all of the
sugars in lignocellulosic biomass
Fermentation
Cellulose
Ethanol (and CO2)
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. cerevisiae to produce XI have failed
 Common XI is active at high temperatures and pH’s. Not
compatible with S. cerevisiae
Xylose isomerase
Xylose
Xylulose
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: pH ~5 35°C
 XI from Y1 (XIy):
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
FERMENTATION DIAGRAM
Current method
Goal
Heat
exchange
xylose →
Xylose
& EtOH
Biomass
hydrolysis and
glucose
fermentation
→
↑
Ion
exchange
Solid/liquid
separation
Solids to
energy recovery
↑
NH4OH injector
to raise pH
Yeast, enzymes,
pre-treated
biomass
pH 7.5, 55°C
Xylose
fermenter
pH 5, 30°C
Contains immobilized XI
← xylulose ←
Isomerization reactor
Ethanol to distillation
Yeast, enzymes,
pre-treated
biomass
Biomass
hydrolysis and
glucose &
xylose
fermentation
(contains
immobilized
compatible XI)
pH 5, 30°C
Ethanol to
distillation
→
Solid/liquid
separation
Solids to
energy recovery
Technology by Trillium FiberFuels, Inc.
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 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 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.
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
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:
Control: No
colonies
No colonies:
no XIy gene
Some
colonies: XIy
gene might
be present
PLASMID VECTORS
Target
Sequence
Genome
Culture the E. coli
w/ plasmids
Plasmid
Insert
plasmids into
Hansenula p.
Can it ferment
xylsose?
Cut plasmid
and genome
w/ enzymes
Insert the
sequence
into plasmid
Extract the
plasmids
Heat-shock
plasmid into E. coli
Mutant
Hansenula p.
can’t grow
on xylose
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
XI activity of putative genes in E. coli
Xylulose Produced (g/L)
2.50
2.00
1.50
Control
Put1
Put2.1
Put2.2
1.00
0.50
0.00
0
20
40
60
Time (hours)
80
100
120
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
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/b9/201005101646
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.
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.

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