Davis Weymann - Oregon State University
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Transcript Davis Weymann - Oregon State University
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.