Hi, my name is Jay Keasling.
I'm from the Joint BioEnergy Institute,
the University of California at Berkeley,
and Lawrence Berkeley National Laboratory.
And today I'm going to be talking about engineering
microbes to solve global challenges.
It's probably no surprise to anyone that we derive
most of our fuels, chemicals, and even drugs from
petroleum. We pump it up from underground, we refine it
in a refinery, and then from that refinery, we produce all of
the fuels, chemicals, and drugs that we use on a day to day
basis. Now, if you just look at a typical barrel of oil.
Roughly, 80-85% of that barrel of oil goes to producing
all of the fuel that we use. In the U.S. that's on the order
of 225 billion gallons of transportation fuels every year.
The other 15-20% goes into producing all of the other
products, from carpet fiber to paint to plastic bottles, to even
some of the drugs that we ingest into our body.
Now, we'd like to have an economy that's not just
based on petroleum, but that's also based on renewable
feedstocks. Things like sugarcane, corn, and even cellulose
biomass in the form of grasses. Now if you were a farmer,
and you were to grow those various crops depending on where
you are in the world, you'd send them to a bio-refinery. And that
bio-refinery, just like a petroleum refinery, would split
those molecules apart, those sugar containing compounds apart,
and transform them into fuels, into chemicals, and even
into drugs. If you burn those fuels in your car, you'd still
be producing CO2 like you do when you produce
and burn petroleum based fuels, but that carbon dioxide
would be taken back up by the plant and incorporated
into the plant containing sugars, that then would be
refined into fuel. And in this sense, you'd have a carbon
neutral cycle of producing and consuming carbon dioxide.
Now if we just look at the volume and the value
of these molecules, at the very top of the volume and the bottom
of the value are the fuels. They have the lowest
value of many of the molecules that we use on a regular
basis. And yet, they have the highest volume. Remember I
told you that's about 225 billion gallons of transportation
fuel in the U.S. alone on an annual basis.
At the top of the values, and at the bottom of the
volume are the drugs we use. Extremely high value
molecules produced in small volumes. And then
in the middle are all of the other molecules and products
that we use on a day-to-day basis, like carpet on the
floor, the plastic bottles, the paints on the wall, mouthwash,
perfume, etc ... Now when we think about flexibility for
substitution, we know that with our drugs we don't want
anything that's just kind of like the drug we use but
exactly like the drug we know will cure a particular
disease. But we often think that there's a great deal more
substitution we can have for things like carpet fiber,
for mouthwash, and even more for fuels. However, that's
not really the case. Our fuels are regulated, what we put
in our tank is highly regulated, because that's what gets
exhausted into the atmosphere. And that ensures that our
planes stay up at the altitude. Same case for plastic bottles,
and carpeting, and paint. We need to know that those
products will work the same as they've always worked
regardless of whether they're produced from petroleum
or from carbon that comes from plants. Now today, I'm going
to be talking about engineering microbes, namely yeast
and E. coli, to produce a variety of these products.
And to produce products that behave or are identical
to the products that we would normally get from petroleum.
And I'm going to start off with a story about drugs.
And in this case, the story about artemisinin, a drug that
cures malaria. This guy, his name is George, I met him when
I traveled to Kisumu, Kenya. Kisumu is on Lake Victoria
and it's about sea level. And it has a very high incidence rate
of malaria. In fact, 80% of the population has malaria.
When I visited Kisumu that day, it was very warm,
but yet George came into this clinic wearing a long
coat and pants, and he had a runny nose. And it was pretty
clear that he wasn't feeling well. This is the clinic that he
came into, it's a government run clinic, and although it's
difficult to see, there are children that are lined up outside
the clinic waiting to get in. This at the top is a picture of
the typical home, a home that George might live in. It has
mud walls, a steel roof, but you could tell that the steel isn't
in very good shape. The nurse saw George and she knew that
he probably had malaria. So she pulled out a brand new
test kit that she just had access to. She pricked George's
finger, put a drop of blood at the bottom, and then
followed that with a couple of drops of buffer. A few seconds
later, the blood ran up the gradient. The two lines that
appeared, the top line is the control line to say that the kit
worked, and the bottom line by the T is the test line.
And that indicated that George had malaria.
The nurse then took George and weighed him, and
from his weight, got a package of Coartem
which contains artemisinin, an antimalarial drug.
She gave him the first tablet and then she instructed
his brother to give him a tablet 12 hours later, the third
tablet 12 hours after that, the fourth tablet 12 hours
after that, and so on. She repeated the instructions and
sent them on their way. And then she turned to me and
she said, "Jay, after the third tablet, George is going to be
feeling great. And his mother's going to take the rest of
those tablets and tuck them away in a drawer, because
the next time he gets malaria, she may not have access
to this free Coartem that she was able to get."
And because they're living on less than $1 a day,
there's no way she could afford the Coartem or any other
anti-malarial drug that would be sold in the private pharmacies.
A little bit about malaria. Roughly 3 billion people
on the planet at any one time, are at risk of getting malaria.
Roughly 250 million people are infected with the malaria
plasmod, the causative agent, called plasmodium.
And at any one time, roughly 500,000 to 1 million
people die of malaria, 2/3 of them are children under the age
of 5. There's a treatment, a cure for malaria, it's called
artemisinin. And it comes from this plant Artemisia annua.
And it has a great history, it goes back to 168 B.C.
where it was first described in the writings for being used
for treating hemorrhoids. And then a few hundred years later,
there are writings of it being used to treat fevers.
Presumably fevers due to malaria. It was largely
forgotten, but in the '60s and '70s, China was fighting
in Vietnam and malaria was rampant in Vietnam.
And the drugs they currently had available, based on quinone
were no longer effective. So Mao sent his medical core out
to find a cure for malaria. This woman here, Youyou Tu,
was one of the people who went out and led the medical
core. She pored through the ancient literature and finally
she found a description, shown here, for using
sweet wormwood, making a tea of the wormwood,
extracting the drug, and patients drinking it.
They purified the active ingredient, found out how
best to extract it and then by 1972, had the active
ingredient isolated and described. In 2004, the World Health
Organization recommended artemisinin-based combination
therapy as the drug of choice for treating malaria.
So, what's the challenge? We've got this great cure for
malaria. It's an ancient Chinese therapy now been westernized.
What's the problem? Well, I alluded to a few of these
challenges earlier. The first one is availability. Remember
George's mother would give him 3 tablets, he'd be feeling
great, and then she'd tuck the rest away because it
wouldn't be available to them. And, if it were available to them
the cost would be too high. There are also challenges around
quality of the drugs. There are makers of artemisinin
therapies that don't put as much artemisinin in them as
they should or any at all, and try to sell them as artemisinin
drugs. And finally, there's the challenge due to resistance.
In many -- in a few parts of the world, where they've been
using artemisinin the most, resistance has -- plasmodium
has become resistant to artemisinin. And this has become
a big challenge. This is mainly occurring in regions around
Vietnam, where artemisinin was first used.
Now, this is the process by which we get artemisinin.
We grow plants on a plantation, we purify the artemisinin
from those plants, and then convert it chemically into the
derivatives that are used in artemisinin combination therapies.
I visited one of these plantations and actually saw
this in action. Now, the artemisinin is produced in
small trichomes, where it's held, that are on the leaf
of the plant. These trichomes store the artemisinin and when
you rub the plant, you can get the artemisinin off.
When you make a tea of the plant, these trichomes
burst and the artemisinin comes out. Now the way we
get artemisinin is that the seeds are planted in
small greenhouses, then once the plants grow up
to be about six inches to a foot tall, they're taken out
into the fields, they're planted by hand, where they grow
up over a growing season, and they grow to be about
8 feet tall. After they've grown up, they are then harvested,
the plants are chopped, generally by hand, and then laid out
on tarps to dry in the sun. The artemisinin is contained in
the leaves of the plants. So then they beat the plants
to get the leaves off, they collect all of those leaves
that are on the tarps, run them through sieves to get only
the smallest leaves, and then they take it into the factory
and extract it. Now, I mentioned that price and availability
were a challenge. In 2004, artemisinin was chosen as the drug
of choice for treating malaria. And by 2005, the price had shot
way up to over $1100 a kilogram. Farmers saw that they
could make money from producing artemisinin, so they
started producing lots of it. And shown in the blue bars
are the amount of artemisinin. And by 2007, they had
overproduced it so much so that the price fell to around
$200/kilogram. Farmers then stopped planting artemisinin,
there was a shortage by 2009, and the price started shooting
back up. So you see these swings in price and availability.
The other challenge with artemisinin is the long time
it takes to produce it. You can see that by the difference
in the peaks of the price and the availability. It's two years.
It takes anywhere from 18 months to 2 years for pharmaceutical
companies to decide how much artemisinin they need,
to then have contracts with farms to grow it, to get the proper
seeds that produce the most, for it to go through a growing
season, for the farmers to harvest it and get it back
into the factory where it's extracted, and then turned
into the pharmaceutical companies, where they then
make the derivatives that are produced and used in
artemisinin combination therapies. So that time window
of 18-24 months is also a challenge.
Now, what else could farmers plant if they didn't plant
artemisinin? Well, they could plant many food crops
and shown here are the prices of artemisinin compared
with food crops in China and Vietnam. Early in
the use of artemisinin in 2004, many farmers in Vietnam
started planting artemisinin. But those farmers went out
of business, since large plantations in China began
to sprout up. Now most of the artemisinin is produced
from plants grown in China. I mentioned that resistance
is rising to artemisinin. You can see here that several
countries allowed sale and production of mono-therapies.
Mono-therapies, because there's only one drug in them,
actually encourage resistance. If you have more than one
drug that fights the plasmodium with multiple modes
of action, then you have a chance of containing that
resistance. And as I mentioned earlier, there's been
resistance documented on the Cambodia-Thailand
border near Vietnam. So, several years ago, our
goal was to engineer a microbe to produce artemisinin
from an inexpensive renewable resource that would allow
us to stabilize and reduce the price. Stabilize and increase
supply. Reduce production time. And control resistance
by controlling the supply. This was our process.
We were going to replace the plant with microbes, either
E. coli or yeast. Rather than produce artemisinin, we
proposed producing artemisinic acid. Artemisinic acid is
less toxic than artemisinin to yeast and E. coli.
But there was a readily known conversion process that
would allow us to convert artemisinic acid into
the same derivatives that are currently produce from
artemisinin. So then it could be substituted directly
in the drug pipeline. This is the strategy we would use.
We would engineer a microbe to produce artemisinic acid,
we would then hope to get that artemisinic acid
out of the microbe, some way through a purification
process. And then chemically convert it into the derivatives.
We started with E. coli as our first chassis.
E. coli is an organism that we know a lot about, we have
a lot of genetic tools for engineering it. And so that
became our first chassis. E. coli produces farnesyl
pyrophosphate, a precursor to artemisinin.
And so that gave us the first step in that biosynthetic
pathway. Unfortunately, E. coli produces very little
farnesyl pyrophosphate, so we brought in heterologous
pathway from another organism, yeast. In yeast this
pathway produces a relative of cholesterol called
ergosterol, that's needed for the growth of the yeast.
We used this pathway to produce farnesyl pyrophosphate.
It tapped into acetyl-CoA, which E. coli has plentiful amounts
of. The next step in the process was to find the
enzyme that converts farnesyl pyrophosphate into
amorphadiene, the first committed precursor in the
artemisinin biosynthetic pathway. Luckily for us, that gene
had been cloned and all we thought we needed to do
was get access to the gene. Unfortunately, the person who'd
cloned that gene wouldn't give us access, so we had to
synthesize the gene directly. We also developed a number
of synthetic biology tools that would allow us to increase
the flux through this biosynthetic pathway. We developed
scaffolds that would allow us to attach the enzymes
in the biosynthetic pathway to a synthetic protein scaffold,
and thereby collect all the enzymes together so that they
could increase the flux through the pathway. We developed
regulators that could sense intermediates in the pathway,
like farnesyl pyrophosphate, which are toxic to organisms
like E. coli if they accumulate at too high a level.
And then those regulators would regulate the biosynthetic
pathway. We also developed a type of debugging tool
that would allow us to debug the inside of a cell and
determine which genes were turned on, which genes were
turned off, and what might be the cause of any toxicity
or any lack of production that we witnessed.
Through the use of those tools, through synthesis
of genes, we were able to increase the production
to about 10 million fold from where we started. And this
was work that was done both in my laboratory at UC
Berkeley, as well as in Amyris, a company that was started
out of my laboratory. Now, the next steps in the biosynthetic
pathway involve cytochrome P450s that would put an oxygen
on that amorphadiene. Unfortunately for us at the time,
the genes hadn't been cloned and hadn't been
described. But we know that it was going to involve
multiple steps and potentially multiple cytochrome P450s.
We were worried. E. coli is not a great host for expressing
these enzymes. So, the first thing we did was went about,
we went about building a yeast that would be a reagent
for us. An organism where we would engineer the production
of amorphadiene, and then use it to find the P450s that
might be involved in the biosynthetic pathways to convert
amorphadiene into artemisinic acid. We increased
the expression of the enzymes in the mevalonate pathway,
that same pathway that we imported into E. coli
to produce farnesyl pyrophosphate, and by upregulating
the genes in that biosynthetic pathway, we were able
to produce sufficient farnesyl pyrophosphate and then
amorphadiene, when we introduced amorphadiene synthase.
We could use that organism as a reagent to find the enzymes.
But we still had to go into artemisia and find the genes
in that last part of that pathway. Well, as I mentioned earlier,
artemisinin is produced in trichomes on the leaves.
And there are basal cells that produce that artemisinin.
They must be enriched in the enzymes that produce the
artemisinin, if that's where it's produced, and therefore
they'd be enriched in the transcripts that encode those
enzymes. So we went in looking for those transcripts.
We developed a method to purify the trichomes off the
leaf, which was a very difficult process. And then we made
cDNA libraries of those trichomes and looked for RNAs
that might be important for that biosynthetic pathway.
But it'd be like looking for a needle in a haystack.
So, a very creative postdoc in my laboratory said,
well, the artemisinin biosynthetic pathway is it looks
a lot like biosynthetic pathways that are in lettuce
and sunflower. If you look, the molecules are very
similar, and because the molecules look similar,
that must mean the enzymes are similar.
So he then sorted through a cDNA library that we had
available to us, of sunflower and lettuce. Looked for
P450s that were unusual, and compared those to the
P450s that we got in the cDNA library that we got from
Artemisia annua. And about that time, a bunch of
miracles started happening. The first miracle was that
the first enzyme we tried was the right enzyme.
The second miracle was that rather than just
catalyzing the first step in the biosynthetic pathway,
it catalyzed all three steps and the yeast produced
artemisinic acid. The third miracle was really where
the artemisinic acid ended up. We couldn't find it
inside the cells, we couldn't find it in the broth,
in the shake flasks. It turns out that artemisinic acid
is not as toxic as artemisinin to yeast, but toxic
enough that the yeast excreted it out of the cell,
turned on pumps to pump it out selectively,
so that it wouldn't be inside the cell and toxic
to the yeast. And because the pH of the fermentation
broth was relatively low, the artemisinic acid
precipitated on the yeast membrane. As we
increased the production of artemisinic acid,
it actually comes out of solution in the fermentation
broth. This is a picture contributed by Amyris
when they took their highly engineered yeast,
it actually precipitates, secretes the artemisinic
acid outside the cell, and it precipitates in the
fermentation broth. And this gives you the perfect
purification. Now, the yeast that we delivered
to Amyris was not ideal. It produced artemisinic
acid, but not at a high level, and it grew slowly
because of the toxicity. And through engineering
the P450, as well as the CPR, and controlling
the electron transfer between the CPR and the
P450, Amyris was able to increase the production
and give us a host that's highly productive.
That host is now being used by Sanofi, producing
artemisinic acid in large scale in Eastern Europe.
That artemisinic acid is then converted into
artemisinin, using a light catalyzed process.
Much like we think occurs in the plant, where
light catalyzes the conversion of artemisinic acid
to artemisinin. And this is the full-scale facility
that Sanofi built in Garessio, Italy, where they're
producing the artemisinin. That artemisinin,
shown here pallets of it, is then shipped to
Morocco, where it's tableted and there is the final
product, right down there. 3 million treatments
had been delivered to Africa as of about January,
by May of this year, 16 million treatments had been
shipped to Africa. And Sanofi will have the capacity to
produce between 100-150 million treatments on an
annual basis. That's roughly one half of the world's
needs. Now, what I've told you is a story about
producing artemisinin in yeast and E. coli.
There are many other products that we might want
to produce. Things like fuels. Now, as I mentioned
earlier, if you produce fuels using engineered organisms
and those fuels are derived from renewable resources,
like sugars from biomass, the carbon that you put into
the atmosphere that the plants took up in producing that
biomass. So you have this possibility of producing carbon
neutral fuels. Well, we had an organism engineered
that could produce hydrocarbons. Now ethanol is one
of the fuels that's most widely used, and it's commercially
produced in the U.S. and supplemented into gasoline.
But it can't substitute for diesel and jet fuel.
We need a fuel that behaves exactly like the fuels
we get from petroleum. Well, we had in our hands
a host that would produce hydrocarbons, and we thought
we could just convert that into a fuel producing host.
Here are some of the fuels that could possibly be
produced using an engineered organism and they
include short-chained hydrocarbons that you'd find
in gasolines, long-chained hydrocarbons that you'd find
in diesels and jet fuels, and even aromatics that you find
in all fuels. So we went about engineering that yeast
and E. coli that we had first engineered for producing
artemisinin, to now produce hydrocarbons that would have
similar properties to the hydrocarbons that we get from
petroleum. And that engineering involved replacing the
genes that were specific for producing artemisinin
with genes that were specific for producing the hydrocarbons
that we'd use as fuels. Now, one great thing about these
fuels, which is very similar to the story I told you
about artemisinin, is that they are pumped
or leak out of the cells. And shown here is
a tube that had E. coli growing in it and you can see
at the top, there is an oil film at the top of that tube,
and then shown in this picture are E. coli swimming
in their broth. And you can see the blebs of fuel
that are being excreted and are collecting. Now this
makes the purification process for these fuels
really simple. You grow the cells up on the sugar,
they produce the fuel, excrete it, and it floats to the top.
It's a lot like making a vinaigrette, the oil floats to the top,
you skim it off the top, and if you've engineered the biology
right, you can put that into your tank and drive away.
Now I've talked to you about fuels and drugs, I want
to just mention the possibility of engineering
microbes for producing all of the other chemicals
that we now get from petroleum. Things like mouthwash,
perfume, carpet, and paint. As I mentioned earlier,
about 15% of a barrel of oil goes into producing
all of those products. And yet that 15% of the barrel of the oil
generates as much profit for companies as the other
85%, because of the higher value of those commodity
and specialty chemicals. But again, we have to produce
products that behave exactly like the products that we get
from petroleum, not things that are kinda like it.
But, shown here are just a variety of the products
that we currently get from petroleum. And this is
a pared down list. The list is vast and extensive.
But we can use a lot of the same synthetic biology
that I talked about earlier. Engineering the microbe
to produce identical precursors to the ones you get
from petroleum that could then be incorporated into all
of these products. And there we have a renewable
economy. Now, one of the challenges that we have in
engineering biology to produce all the products we want
to is that it's expensive and time-consuming. That
project I described about engineering yeast to produce
artemisinic acid, that took $25 million just for the research
and development alone. And about 150 person years
of work to get it accomplished. We need to be able to do
these projects with a tenth or a hundredth of that if it's
ever to compete with petroleum processes.
Why did it take so long? Well, just to engineer
that yeast to produce artemisinic acid, we needed
over 40 components, the genes, all the regulatory
switches. We had no standards for those connections,
no accurate models, and no details could be ignored.
And what's more, there was no BioShack to go out to,
to buy any of those components. Biological engineering
is slow because we design something, we then go
into the laboratory, we build it, and then we test it.
And hopefully, we learn from our mistakes.
But one turn of this cycle can take weeks, if not
months. And many years ago, it took a year
or more to go around this cycle. We need to turn
the crank faster and in higher throughput, so that
eventually, we can design once and have it be correct
after the first design. What's more, companies don't share
when they've gotten good at this process. Amyris became
good at engineering artemisinin, Dupont became very good
at engineering 1,3-Propanediol, but when they become
good at that, that's a trade secret. And they keep it
in house and don't share it. So the timelines don't
get shorter and shorter as we hope they could.
We really need the biotech industry, the next
generation of biotech industry, to look more like
the microelectronics industry, so that we can have
really powerful things like we now have computers
and cell phones that won't cost as much to develop
as they currently do in biology. And so, I'm advocating
for a next generation of biological foundry, just like we
have silicon foundries, we need to have biological
foundries that will allow any user to come in, design
on the computer what they want, send it off
to a foundry to be build, and come back to them
and work as planned. This is my goal and something
that I think the future holds. I think there's a lot of
promise in this. And it's something we have a head start
on. I'd like to acknowledge all the funding and the work.
There was a lot of work from people from my laboratory,
Amyris, and Sanofi for the development of the semisynthetic
artemisinin. The work at jbei and synberc for the fuels
and chemicals. And I'd like to thank all the funding from
the Bill & Melinda Gates Foundation, the Department of
Energy, and the National Science Foundation for all the
work this enabled. And I'd like to thank you for listening
to this video.
