Synthetic Biology and Materials Science Part 2: Nature-Made Plastics

In the previous tutorial, we talked about the 
ways in which biotechnology is revolutionizing   the production of important materials like 
plastics. Instead of having to perform chemical   reactions in the traditional way, like we do 
in a laboratory, we can refer to a catalog of   enzymes, and select a series of enzymes that 
catalyze a sequence of chemical reactions   which will transform some readily available 
starting material, often plant-based,   into an industrial material of our choosing. 
We can then encode those enzymes in a plasmid,   insert the plasmid into an 
appropriate microorganism,   and allow it to express the genes that result in 
the enzymes of interest. With these enzymes now   present inside the microorganism, it will rapidly 
generate mass quantities of the desired product,   in a way that has more in common with brewing 
beer than with traditional chemical manufacturing.   As far as industrial processes go, it’s cheap, 
it’s environmentally friendly, it’s everything we   want in new technology.

So now let’s get a little 
bit more specific. Let’s examine the production   of a particular compound that is currently being 
optimized, as well as the challenges associated   with this process and its optimization.
As we know, petroleum, or crude oil,   is pumped from the ground and refined so as 
to isolate individual organic components.   Some of these components are used to 
synthesize materials like plastics.   One such component is methyl methacrylate, an 
alpha-beta unsaturated methyl ester, abbreviated   as MMA. This compound can undergo polymerization 
to generate polymethyl methacrylate, or PMMA,   which is a durable transparent plastic also 
known by brand names like Plexiglas, Acrylite,   and others, though we can generally refer to it as 
acrylic plastic. This is a useful material in that   it acts as a lightweight and shatter-resistant 
alternative to glass, as well as an additive in   acrylic paints.

However, this method of producing 
PMMA is non-sustainable. Extraction of petroleum   from the ground is harmful to the environment, 
as is indicated by the 1.8 billion metric tons   of carbon dioxide emission due to plastics 
production in 2015. Also, oil in the ground will   eventually run out, meaning this process can’t be 
carried out indefinitely. So what is the solution?  One solution is being developed by a 
protein engineering company named Arzeda,   and I reached out to project lead Aaron Korkegian, 
who was gracious enough to fill me in on some of   the details. As it happens, there is a compound 
named alpha-methylene gamma-butyrolactone,   or MBL, also known as Tulipalin A. It is named 
as such because it is produced in tulips.   This compound has properties that 
are extremely similar to those of   MMA. Like MMA, it can polymerize to form 
PMBL, which is also a transparent plastic.   But unlike PMMA, PMBL is more thermal and scratch 
resistant, which makes it more durable than PMMA,   so this is a more desirable process.

The only 
problem is that it is not a major product of   tulips. The amount of tulips that would have 
to be grown to then harvest and purify an   amount of Tulipalin A that would be industrially 
useful is not even remotely practical. However,   given our new technique utilizing enzymes, we 
should be able to find a biological approach to   synthesizing this compound ourselves. After all, 
tulips use enzymes to make it, so why can’t we?  Ok, so let’s get to work. As per the scheme 
we outlined in the previous tutorial,   we can start with some biologically-derived 
sugars that are found in plants. Then we select   a fermentation host, which is some microorganism 
like yeast or a bacterial species. Then we have to   engineer a metabolic pathway. We need some number 
of enzyme-catalyzed reactions which will transform   that sugar, or more likely some metabolite the 
organism naturally generates from the sugar,   into mass quantities of Tulipalin A, which we can 
then purify and polymerize to make our plastics.   As we mentioned, these enzymes do not need 
to be native to this microorganism.

They can   be any enzymes from any biological species, 
which can be referred to as source organisms.   In fact, they don’t even have to exist in nature 
at all, completely novel enzymes can be designed.   They just have to do the chemistry we are 
looking for, because we can insert the genes   that encode these enzymes into the host organism 
via a plasmid, and it will make those enzymes,   thereby promoting the relevant chemistry.
Now of course, this task should not be portrayed   as trivial.

In fact, it represents the bulk of 
the challenge. What is the sequence of enzymes   that will be successful? Finding the answer to 
this question would have been near-impossible just   a few decades ago. But thanks to advancements in 
computer science, many new avenues have opened up   for solving such a problem. Recent developments 
in computation have allowed us to take massive   enzyme databases and combine them with efficient 
algorithms which can search and identify potential   enzymatic pathways from any starting material to 
any potential product.

It should be made clear   that although enzymes tend to have a highly 
specific substrate within biological systems,   when removed from their typical role in 
biosynthesis, they can technically operate on a   variety of molecules. Anything that fits into the 
active site and promotes the achievement of the   transition state for the enzymatic reaction will 
be a suitable substrate. This means that slight   structural variants of the natural substrate may 
be totally viable. For example, say there is an   enzyme class that is known to operate on phenol. 
It may be the case that some enzymes within that   class work just as well on 2-methylphenol, as it 
is possible that when inserting into the active   site, this additional methyl group can point 
outwards and not interfere with the chemistry.   By testing a wide variety of enzymes within 
that class, we may find some that function   on 2-methylphenol, even though they are 
only documented as working on phenol.   Therefore, this adds an element of testing for the 
researcher, as if we are set on this compound as   an intermediate in our pathway, we may need to 
screen a multitude of enzymes that are known to   operate on phenol and see how they work with this 
slightly altered substrate.

For some of them the   methyl group will clash in the active site and 
it won’t work. For others there will be plenty   of space, and the reaction will proceed without a 
problem. Such an enzyme can be described as being   “promiscuous” between the substrates.
Now imagine the multitude of potential   intermediates, and the ways in which they could 
resemble a known enzymatic substrate or another.   The reality of the matter is that it will almost 
never be the case that each intermediate in some   desired pathway lines up flawlessly with the 
precise structures that are documented as   known substrates for enzymes in a catalog. 
We just need them to be close enough that   the chemistry may work the same way. So 
when building the pathway, for each step,   it may be necessary to select hundreds or even 
thousands of different enzymes that each carry   out the transformation you are looking for, but 
on a substrate that is similar to what you’ve got.   Again, say we have 2-methylphenol.

Enzymes that 
are documented to promote the desired reaction   on phenol could be tested, as could any number of 
other untested proteins with sequence identities   close to these known ones. Or perhaps activity on 
2-methylphenol is also documented for a particular   enzyme, but the activity is poor. Then perhaps 
the structure of the enzyme can be modified,   again driven by computational design. We can 
model the active site and predict where that   additional methyl group must be sitting, 
such that it interferes with the activity.   Perhaps we can see that there is a bit of 
space for the methyl group, but one amino   acid in particular seems to be getting in the 
way, perhaps due to a bulky side chain.

We can   swap that residue out for a different one with a 
smaller side chain, and this singular modification   may result in a hundred-fold better reactivity. 
In fact, such modifications may result in the   enzyme preferring the novel substrate over 
the native one. And it doesn’t have to be just   one residue. Our developing understanding of 
protein structure and function relationships   also allows for more aggressive designs that 
change twenty to thirty residues at once,   thereby significantly influencing enzyme 
activity, selectivity, and expression.  So as you can see, designing the pathway 
is a mountain of work.

The possibilities   are staggering, given how many enzymes there 
are, and the wide range of substrates they   could potentially act upon, particularly once 
enzyme modification is taken into account.   But one way or another, the pathway is 
planned with specific reactions in mind,   like for example, the reduction of an aldehyde to 
an alcohol. For each step, some number of enzymes   which perform that reaction on substrates similar 
to yours are screened meticulously, and options   that work for each step are either identified or 
designed. This process continues until the whole   pathway is covered. Pathways with fewer steps 
are preferred, and once a pathway is selected,   each enzyme is tested individually to ensure that 
it can perform the transformation intended for it.  To confirm their activity, the enzymes first 
have to be generated. This entails identifying   DNA sequences that encode each enzyme, and then 
optimizing them for expression within the host   organism. There are many ways we can do this, 
and one example involves codon-optimizing for   the intended host organism, like perhaps E.

Coli. 
What this means is that since multiple codons can   code for the same amino acid residue, as we recall 
from learning about transcription and translation,   there is significant variability that is possible 
in the DNA sequence without altering the resulting   protein. But certain sequences may be preferential 
over others in a particular host organism for   subtle reasons, such as the ratios of specific 
tRNA production in that host. Essentially,   in a particular organism, some codons are 
used frequently, and some very infrequently.   So if introducing a gene that contains many 
instances of the codons that are almost never   used, the necessary tRNA molecules may not be 
available and gene expression becomes difficult.   Once this is finalized, each enzyme can 
then be individually expressed and purified,   and the desired reaction is tested in vitro to 
confirm that the intended chemistry is occurring.   Thousands of different variants of a given 
enzyme or enzyme expression system can be   screened all at once in massive screening sets 
so that quantitative data on their ability to   promote the desired reaction can be gathered 
quickly and efficiently. Once enzymes have been   identified that can conduct the desired series 
of reactions, their properties can be further   optimized by computational design.

This entails 
using computers to get ideas about how we might be   able to modify an enzyme so as to improve reaction 
rates, stability at a certain temperature,   binding affinity, or selectivity. In 
other words, we can use our growing   knowledge of the relationship between enzyme 
structure and function to tweak the enzymes   so as to improve their activity on the desired 
substrate, and therefore their ability to generate   the desired product. These novel designs can again 
be converted into corresponding DNA sequences   for expression and testing, and this process can 
be repeated, with further refinement each time.  Once the activity of the complete enzymatic 
pathway is confirmed in vitro, their genes can be   assembled together on an operon, and transformed 
into the manufacturing host for further testing.   What this means is that because the genes 
are part of the same operon, all the enzymes   will be expressed by the microorganism at 
once, in a singular act of transcription.   After the initial test, further refinement and 
iteration will typically be required to tune   the expression of each enzyme in the context 
of the overall efficiency of the pathway.   This could involve modifying the individual 
ribosomal binding site sequences in front of each   enzyme sequence in order to adjust the translation 
initiation rate separately for each enzyme, so as   to modify the relative rates of enzyme production. 
Additional work may also be required to engineer   the host organism to better push carbon flow into 
the desired product and away from other existing   metabolic processes to increase the yield of the 
target.

Once the numbers look good, scale up can   occur sequentially with further refinement, 
until in the long run, the large-scale process   just looks pretty much like a vat of beer brewing, 
and is essentially just as simple to execute.  Though this technology is in its budding 
stages, it promises to completely change the way   entire industries operate. Whether aiming for a 
target molecule like Tulipalin A or any other,   the better these designer organisms become, 
the closer we can get to supplanting current   manufacturing techniques, thereby leading to 
more diverse products that can be produced   not only cheaply and efficiently, but from 
simple plant-based starting materials which   are grown from nothing but sunlight, water, and 
air.

Any reaction can be run in the same vat   with precisely the same materials, all that 
changes is the specific engineered microorganism   that is employed. This means we don’t need 
a variety of expensive industrial machinery,   there are no significant reagent expenses, nor any 
reagent waste. Scaling up in this case just means   buying a bigger vat. And again, the 
whole process is carbon-capturing,   or at the very least carbon-neutral, rather than 
carbon-releasing.

It’s as clean and sustainable as   manufacturing can get, and with companies like 
Arzeda leading the way, it’s only a matter of   time before we begin to see the societal 
ramifications of these daring innovations..

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