Hi there, my name is Cordelia. Myself and my colleague, Nico, will be introducing our synthetic biology project, which is to engineer a bioplastic-producing E. coli cell factory. And the problem with most plastic production currently is that plastic is derived from crude oil, which is then refined into petroleum. And when you’re obtaining this crude oil from the ground, you often end up with horrible environmental consequences.
Such as this oil spill here. Another problem is, because plastic is so cheap, people often throw it away without consequence and so you end up with these enormous landfill sites which are just full of this plastic, which won’t break down for up to a thousand years. In addition, sea birds and animals will also pick up this plastic and ingest it, and it then sits in their gut where it can’t break down, and often this leads to death.
As you can see with this unfortunate bird. Another problem is that, plastic actually breaks down over time into these microplastics, which end up accumulating in the sea.
And you can see here the currents have actually caused these great big garbage patches in the Pacific Ocean. So, a solution we have is to produce this bioplastic out of biomass. So, biomass is any kind of plant material really in this situation, particularly lignocellulose, which is a very hard to break down product.
And in this case, you can break it down into sugars, which is then a carbon source for our engineered bacteria. And these engineered bacteria can then produce a biodegradable bioplastic.
And the good thing about this is that it’s actually cyclical, so it’s a renewable cycle where environmental bacteria can then break down this biodegradable plastic, release carbon dioxide, which is then sequestered by the plant, and you can feed the whole process again. Now it’s time for the practical part of our project.
How are we going to implement bioplastic production in a bacterium? We chose to work with PHB, which stands for poly(3-hydroxybutyrate), as you can see it right here. PHB is actually a natural storage polymer in many microorganisms.
However, we would like to work with E. coli and E. coli does naturally not produce PHB. But E. coli is a really nice microorganism to work with in the lab, as it’s very fast growing, and a lot of molecular biology and synthetic biology tools are available for E.
Coli. E. coli’s natural central metabolism can convert glucose up here into the common precursor, acetyl CoA. And acetyl CoA can actually be a precursor for PHB production. So we can add a pathway that converts acetyl CoA to PHB.
You can see a pathway here. We decided to work with the pathway of Cupriavidus necator, which is a natural organism making PHB. The pathway consists of three enzymes doing the whole conversion. In addition to engineering this pathway, we like to improve the PHB production further by testing some E. coli knockout mutants.
The first knockout mutant which we are testing is this knockout here, of this enzyme which is one of the first enzymes in glycolysis. So basically blocking all the flux into glycolysis and forcing it in other ways. In addition, we’re testing a single knockout mutant in the pentose phosphate pathway, which basically means no flux is going into this pathway. A third very interesting mutant we think also based on metabolic modeling would be knocking out the Pta, Pta is the first gene to what’s acetate fermentation, converting acetyl CoA in the end into acetate. And we think that this fermentation pathway takes a lot of the acetyl CoA, which we would actually like to force into the PHB production.
So, we’d like to test all those three and adding the pathway. So it’s time to get into the lab and get some bacteria going and isolate some DNA. But wait! Before we continue, we have to know how are we going to introduce these three genes, which encode those three enzymes for PHB production into E. coli.
There are actually multiple approaches possible, and we chose to investigate two approaches. The first approach we’re looking into is introducing the three genes on a plasmid. A plasmid is a short circular DNA that replicates itself in a cell like E. coli. And there will be multiple copies of this plasmid, so you get a relatively high dosage of the gene, which might be interesting for getting a lot of production.
On the other hand, it might be a high metabolic stress for the cell. In addition, plasmids have a small drawback in that they’re not always stable in a cell. And one of the ways to keep them in is actually having to add antibiotic all the time, as there’s an antibiotic resistance marker on the plasmid. As an alternative approach, we tried to introduce the three genes for the bioplastic production and an antibiotic marker onto the chromosome of E. coli, which is potentially way more stable.
Therefore, we use a tool called transposons. Transposons are natural genetic elements and they can also be used as a synthetic biology tool to introduce stuff on a chromosome.
They have the property that they introduce genes on a random place in the chromosome, which might be interesting because you might get many different expression levels dependent on the place of the chromosome where it gets integrated. And in addition, it seems to be really stable. So, we’re also going to try that.
So, we managed in the mean time to get the plasmids into our E. coli strains by transformation. We didn’t manage completely yet to get the transposons into the chromosomes. So, more about that later. But we already have some data on the plasmids expressions of the genes.
If we express those genes from the plasmid we seem to get PHB and we found that out by using an indicator molecule, which is called Nile red. As you can see here if we add Nile red to our cell cultures, they get very red if we have the PHB producing strains.
And this looks nice, but of course we’d like to quantify how the different mutants and the wild type produce PHB. So that’s why we went to measure in a 96-well plate the fluorescence of this red compound. After putting this plate in a plate reader, we get the data on the PHB production.
And as you can see, this is quite interesting. All the strains produce some PHB, but the Pta knockout, if you remember that was the one going into the acetate fermentation, seems to produce far more PHB than all of the other knockouts. So this is a really interesting strain to continue with. In addition of course we’d like to see what transposons on the chromosome would do.
We didn’t manage to do it ourselves completely yet, it’s not practical, but there’s already some data available from literature on this.
Last year, it was published that production from the chromosome in the same strain actually we are using gives a bit lower production than optimal production with a knockout, the one we used actually, gives a bit lower production than that one, about 78%. That’s probably because there is lower gene dosage, having one integration into the chromosome instead of all the plasmids in your cell. However, in this paper they also showed that the cell with the chromosome knockin has a way better growth than the other cell.
So, it might still be an advantage to use transposons. Of course it’s nice to see red colors in cells, but in the end, we would like to see real PHB.
So, we had to do some more real work. We had to use our strain, the one with the plasmid to purify the PHB using extraction. And we succeeded. And here we have our overnight culture of bacteria in an Erlenmeyer flask, and we then freeze dried this in a lyophilizer and our next step was to do a solvent extraction, and this was a two phase process using acetone and chloroform, and we then were able to precipitate out our plastic, our final product, poly(3-hydroxybutyrate).
In conclusion, we have found that PHB production in E.
coli is feasible, however, it’s a very expensive process. And in our pilot study, we found that we only got 1% yield from our biomass. And so this obviously isn’t particularly viable as a commercial enterprise. So what we actually plan to do is to optimize the strains and perhaps look at different host organisms, maybe to use yeast instead. The high cost we found was mostly associated with the acetone and the chloroform extraction method we used, so we could look at using alternatives.
The problem with these solvents is that they’re very difficult to get rid of. In the future, perhaps we could use synthetic biological approaches to develop new bioplastics and perhaps even replace many of the conventional plastics that we use.