Optimization of methane monooxygenase to convert methane to methanol on

an industrial scale
Lani Kim
Stephen Mayo and Alex Nisthal
Abstract
Methane (CH4) is the second most prevalent greenhouse gas emitted in the United States,
accounting for 10% of all greenhouse gas emissions. By converting methane to methanol, the
methane gas, instead of going to waste, can be used to create biofuels and other higher value
chemicals. Currently, the industrial methane-to-methanol conversion process is extremely
energy-intensive and environmentally unfriendly. Instead, the biological oxidation of methane to
methanol using the methane monooxygenase enzyme (MMO) is a more desirable conversion
process. By optimizing spmoB, an MMO variant, highly active methane-oxidizing enzymes can
be created for industrial use. In order to produce large amounts of spmoB, the variant is
transformed into a Pichia yeast secretion system. The effectiveness of the Pichia secretion system
is then measured through absorbance and sGFP fluorescence levels.
I. Introduction
Methane gas is a byproduct of natural gas
production. It can be used as a source of fuel
itself, since methane is used in many
chemical processes. However, there are
currently a few challenges concerning the
use of methane. First of all, methane exists
as a gas at ambient temperatures, therefore
making it difficult to transport. In addition,
methane is an extremely potent greenhouse
gas. While methanes lifespan in the Earths
atmosphere is shorter than that of carbon
dioxide (CO ), it is twenty times more
efficient at trapping heat, making its impact
on climate change much higher. Therefore,
the danger of transporting it over long
distances and risking leakage is very high.
One way to resolve these issues is by
converting methane to methanol for easier
transportation. Currently, the industrial
methane-to-methanol conversion process is
extremely energy-intensive, requiring high
temperature and pressure conditions, and
environmentally
unfriendly,
having
synthetic gas byproducts. On the other hand,
the biological conversion of methane to
methanol using the methane monooxygenase
2

(MMO) enzyme is a much simpler process,

involving a highly selective reaction that
requires only mild conditions. There are two
types of methane monooxygenase, soluble
(sMMO) and particulate (pMMO) enzyme.
The pMMO enzyme is more favorable to
use, as the membrane delivers methane at
increased solubility levels in the lipid
bilayer.
So far, the Mayo Lab has engineered
multiple variants of the pMMO catalytic
domain (named spmoB) that express solubly
in Escherichia coli. The variants seem to
retain MMO activity similar to that of the
original spmoB, which is very promising, as
previous attempts to solubly express an
MMO in E. coli have been unsuccessful.
Now, the lab group is focusing on
optimizing spmoB in order to create highly
active methane oxidizing enzymes for
industrial use. In order to develop a better
spmoB protein, variants that can be pushed
through the Pichia secretion system are
selected. Pichia is a type of methylotrophic
yeast that is well-suited for protein
expression, as it has a high growth rate and
grows on simple media. By transforming the

variants into Pichia strains, we can produce

large amounts of protein.
II. Materials and Methods
Around-the-horn mutagenesis
A vector is a DNA molecule that is used
to artificially transport genetic material into
a cell for replication or expression. The type
of vector used in this experiment is a
plasmid vector. When a plasmid is placed in
bacteria, it is carried through generations of
bacterial growth. Due to its circular, doublestranded structure, a plasmid will
automatically replicate when placed in a
host cell. The plasmid also features a
multiple cloning site (MCS), where there are
a number of restriction enzyme cleavage
sites. In order to create millions of copies of
the plasmid, bacteria is placed on an agar
plate and an antibiotic, like ampicillin, is
injected to select for the desired plasmid.
After this process is complete, the amplified
vectors can then be extracted from the
bacteria for further use.
In this experiment, we amplified genomic
DNA from Escherichia coli to create our
sGFP tag insert. Since the DNA strand was
very long, oligos were used as base pair
markers. A polymerase chain reaction (PCR)
was then performed in order to copy the
section marked by the oligos. After the PCR,
a single band of DNA was extracted, which
was then used as the insert.
For vector amplification, a 5 uM primer
mix was made by mixing 10 uL of each
primer with 180 uL of water. 5 uL of this
primer mix, 1 uL of the desired pBGP3
plasmid, 25 uL of the 2x Q5 Mastermix, and
19 uL of water were placed in a 0.5 mL
Eppendorf micro-centrifuge tube. Three
tubes were prepared: spmoB/pBGP3,
NeMCO/pBGP3, and Cel6a/pBGP3. The
tubes were then placed in the Eppendorf
Mastercycler to anneal through a preset
cycler program. After vector amplification,

10 uL of the vector was set aside for agarose

gel analysis, and the remaining 40 uL was
used for the template digest.
The template digest was run to prepare
the vector for further analysis. In this
process, the DNA was cut at specific sites,
in order to create fragments of the same size.
1 uL of Dpn1 enzyme and 4 uL of 10x
CutSmart buffer were mixed with 45 uL of
the amplified vector. Dpn1 was used for this
process since it only cuts methylated DNA.
This mixture was then left to incubate at
37C for 30 minutes in the Eppendorf
Mastercycler.
The agarose gel analysis was run on the
amplified vector in order to check its size
against the original DNA ladder. To prepare
the gel box, 0.5 g agarose and 50 mL TAE
buffer were swirled together in an
Erlenmeyer flask. The flask was then
microwaved for 1 minute, swirled,
microwaved for another 30 seconds, and
then swirled again. After placing the plate in
the gel box, the side reservoirs were filled
with TAE, taking particular caution with
wetting the rubber sides of the plate. Since
the flask is now cooled, 50 uL of GelRed
stain was pipetted into the mixture. (GelRed
stain is preferred over ethidium bromide, as
it is less toxic and more sensitive.). The
mixture was then poured onto the plate, and
a ten-toothed DNA comb was placed into
the plate, with the wider spacing facedown
to allow for more room.
After the gel has fully firmed, samples
were loaded onto the gel plate. A piece of
Parafilm was used for mixing. 1 uL of the
DNA ladder (control) and 5 uL of each
sample were pipetted onto the Parafilm. 2
uL of GelBlue dye was placed underneath
the ladder and each sample. To mix the dye
with the ladder/sample, 10 uL of TAE buffer
from the side reservoir was drawn up in a
micropipette, and then the buffer was
pipetted into the middle space. 10 uL of the
final mixture was pipetted into a gel plate

well. After the box cover was put on, the

plugs were connected to the electrodes and
the reaction was run at 125 V for 45
minutes.
Next, a PCR cleanup was run, which uses
spin columns and filters to bind the desired
DNA while getting rid of unwanted small
DNA fragments, proteins, and enzymes in
the process. The Zymo Research DNA
Clean & Concentrator-5 Kit was used for
this process. After following the instructions
from the kit (eluted with 15 uL water), the
resulting product was a single band of
extracted genomic DNA from Escherichia
coli. The total amount extracted was then
measured using the Thermo Scientific
NanoDrop, which shines light through the
nucleotides at 260 nanometers and then
measures the absorbance of the sample.
To phosphorylate the double-stranded
DNA band, 8 uL of the elution was
incubated at 70C for 10 minutes in the
Eppendorf Mastercycler in order to melt the
ends of the band. After the initial incubation,
1 uL of T4 ligase buffer and 1 uL of
polynucleotide kinase (PNK) were added to
the elution. This mixture was then left to
incubate at 37C for 45 minutes. Next,
ligation was performed in order to connect
the sGFP tag insert DNA into the vector
backbone. 10 uL of the phosphorylated
DNA and 1 uL of T4 ligase are combined
and left at room temperature on the bench
for 2 hours.
The final step of the mutagenesis was the
transformation. 50 uL of Top10 Zcomp cells
were placed on top of the entire 11 uL of
ligated mixture while on ice. After 10
minutes, the entire mixture was plated onto
LB-amp plates, along with an additional 50
uL of lysogeny broth (LB). These plates
were then incubated overnight at 37C.

Pichia transformation
Colonies on each plate were selected and
miniprepped using the Omega Bio-Tek
Plasmid Mini Kit. After following the
instructions from the kit, the samples were
sent in for DNA sequencing by Laragen.
In order to transform the plasmid DNA
into Pichia, electroporation was used. In this
process, an electrical field was applied to the
Pichia cells in order to increase the
permeability of the cell membrane, thus
allowing the plasmid DNA to be inserted
into the cell. 1 uL of uncut plasmid DNA
was mixed with 50 uL of cells in a cuvette,
then incubated on ice for 2 minutes. In this
experiment, the X33 and SM5 strains of
Pichia cells were used. This cuvette was
then placed in the Bio-Rad Gene Pulser II
with a cuvette gap of 1 mm, charging
voltage of 750 V, resistance of 200 ohms,
and a capacitance of 25 uF. Immediately
after the electroporation was performed, the
samples were re-suspended with 1 mL of a
50/50 mixture of 1 M sorbitol and YPD
medium. The samples were then incubated
in the Innova shaker at 30C for 1 hour,
shaking at 100 rpm. After incubation, 100
uL of each sample was plated onto an
YPD/G418 300 ug/mL plate. These plates
were left to incubate overnight at 30C.
Assay for sGFP signal
Before the Pichia secretion systems could
be tested, multiple reagents had to be
prepared. In addition to the 1 M potassium
phosphate buffer and the GFP1-10 reagent,
both of the minimal media BMG and
BMGY were also prepared, in order to
compare their effects to the YPD medium.
First, 1 colony from each YPD/G418
plate was inoculated into 3 mL of medium in
a 14 mL Falcon tube. These tubes were then
incubated at 30C for 2 days in the Innova
shaker, shaking at 250 rpm.

After the 2 days of growth,

measurements of OD600 (absorbance at 600
nm) and sGFP fluorescence were taken for
each sample. 1 mL from each culture was
pipetted into a cuvette and OD600 was
measured using the Bio-Rad SmartSpec
Spectrophotometer. Next, the volume was
recovered from the cuvette and placed back
into the Falcon tube. The tube was spun
down at 5000xg for 10 minutes. The
supernatant was used as the sample for
sGFP fluorescence. 20 uL of the supernatant
was mixted with 180 uL of the GFP1-10
reagent in a Nunc 96-well clear microtiter
plate. In addition to all the samples, a
negative control (replaced the sample with
20 uL of TNG buffer) and a positive control
(replaced the sample with 5 uL spmoB7
protein and 15 uL water) were included in
the plate. The plate, covered with a lid, was
left on the Heidolph Titramax platform
shaker at 900 rpm for 4 hours. Afterwards,
sGFP fluorescence was measured using the
Tecan Safire microplate reader (excitation of
488 nm, emission of 530 nm).
BCA Copper Assay
Before metal loading assays with proteins
could be performed, a standard curve had to
be generated for the bicinchoninic acid
(BCA) assay. (While we tried a total of three
different methodologies to create a standard
curve, only one was successful. Therefore, I
have detailed the methods for the successful
one below, and will discuss the other two in
the Results section.) For this assay, three
buffers were prepared. Buffer A was
prepared with 75 g of trichloroacetic (TCA)
acid in 25 mL of water. Buffer B was
prepared with 8.95 g of ascorbic acid in 25
mL of water (this buffer must be prepared
fresh for every assay). Buffer C was
prepared with 1.8 g of sodium hydroxide,
7.8 g of HEPES, and 3 mL of 1% BCA
reagent dissolved in water to result in a final
buffer volume of 45 mL. To make the 155.7

uM copper standard solution, a 1:1000

dilution of the AAS Copper Standard was
prepared.
750 uL of the copper standard solution
and 250 uL of Buffer A were combined in a
1.5 mL Eppendorf micro-centrifuge tube.
The tube was then centrifuged at maximum
speed for 5 minutes in the Eppendorf
Microcentrifuge. Next, 500 uL of this
solution was combined with 100 uL of
Buffer B, then centrifuged for 5 minutes at
maximum speed. Finally, 400 uL of Buffer
C was added to the solution, followed by
another 5 minutes of centrifuging at
maximum speed. The tubes were left at
room temperature for 5 minutes. Afterwards,
the solutions in the tubes were pipetted into
a microtiter plate, and absorbance was
measured at 360 nm and 562 nm.
For the BCA copper assay, fresh samples
of purified protein were needed to serve as
controls. Cultures of spmoB7 and NeMCO
in BL21 DE3 cells were inoculated in 3 mL
of LB/amp100 overnight at 37C, shaking at
250 rpm. 500 uL of these cultures were used
to inoculate 100 mL of auto-induction LB in
Erlenmeyer flasks. These new cultures were
then grown for 24 hours at 25C, shaking at
250 rpm. Afterwards, the cultures were
centrifuged down in 50 mL Falcon tubes at
4000xg for 10 minutes. The supernatants
were then dumped, and the remaining pellets
were frozen at -20C. For purification, three
buffers needed to be prepared: lysis, wash,
and elution. After thawing the pellets, they
were each re-suspended in 5 mL of lysis
buffer and then shaken at 250 rpm for 5
minutes at 25C. The samples were then
incubated at 4C for 10 minutes. Lysate
clarification was then performed at 4000xg
for 10 minutes. After loading the gravity
columns with 300 uL of streptactin resin,
they were equilibrated with 3 mL of wash
buffer, then filled with the clarified lysate.
The columns were then washed with 10 mL

of wash buffer. To elute the protein, 2 mL of

elution buffer was applied.
To load the protein with metal, 750 uL of
the protein sample was used, replacing the
750 uL of copper standard solution. After
the metal loading was performed, the
amount of copper loaded onto the protein
was calculated.
III. Results
After our first attempt at the Pichia
transformation, the sequences given to us by
Laragen showed that only spmoB had
successfully transformed. For NeMCO, the
sequence was of poor quality. For cel6a, one
of the samples did not have the sGFP tag

insertion at all, and the other sample had a

poor quality sequence. The second attempt
yielded a successful NeMCO variant, but
cel6a still exhibited a poor quality sequence.
It took four around-the-horn reactions to
finally yield a correct insertion; the plate had
only three colonies, which was worrisome at
first. However, after miniprepping all three
colonies, the sequences all had the correct
insertion and an identical extra sequence
after the stop codon.
The fluorescence values obtained were
too low in comparison to the control values.
Since the sample values should be around
the level of the positive control value, and
they are much lower, this initial test was not
successful.

Table 1: OD600 and Fluorescence Values, Set 1

Table 2: OD600 and Fluorescence Values, Set 2

After two unsuccessful attempts at obtaining

a standard curve for the BCA assay, I finally
obtained one from the 360 nm data, which
had an absorbance range of ~0.3-2.0. The

right hand side of the curve became slightly

distorted, which might be due to the high
sensitivity of the BCA reagent.

Table 3: BCA Copper Assay Results

Graph 1: Standard Curve at 360 nm

After the metal loading was performed
for both the spmoB and NeMCO proteins, I
calculated how much copper was actually
loaded onto the protein. Ideally, all four
loading docks in each protein should be
loaded with copper. For spmoB, I measured
~3.4 copper ions/molecule. For NeMCO, the
number was ~4+. While I initially thought

there was a glitch in my calculations, a

measurement greater than 4 was actually
possible; performing multiple metal loading
assays and taking the average of them would
account for these abnormal values. Overall,
the first round of metal loading assays went
pretty well, as the copper loaded
successfully onto both proteins.

IV. Acknowledgments
I am grateful for all of the guidance I
received from Dr. Alex Nisthal this summer.
Under his mentorship, I was able to delve
into a field completely unbeknownst to me,
and learn a great amount in the brief ten
weeks of SURF. I would also like to thank
Monica in the Mayo Lab for helping me
day-to-day with even the smallest of tasks.
Finally, thank you to the Caltech SFP
Office, who made this entire experience
possible.