A new way of production
We are running out of oil and the climate is changing drastically due to the emission of greenhouse gasses such as carbon-dioxide. This is the world as we know it, but it is not the way it has to be. Imagine a world where carbon-dioxide is a useful resource instead of a wasteful pollutant. A resource that could even replace oil. We believe that this world is much closer than you might think, when we start exploiting the full potential of cell-factories.
Bacteria as microscopic powerhouses
Many bacteria can be used to create so called cell-factories. The idea of a cell-factory is to exploit the native metabolism of a certain bacterium to let it produce a compound we need. This can be accomplished by creating a specific environment or by editing the genome of said bacterium. Organisms that are used most for this purpose are E.coli and yeast. These organisms do not use oil to produce valuable compounds, which is a large leap towards a bio-based economy!
Picky eaters
However, while chemoheterotrophic bacteria such as E.coli and yeast do not need oil to make products, they do need to eat. The food they need is glucose, or a similar complex carbon compound. Glucose is expensive, and the production of glucose is slow and takes up a lot of arable land. Furthermore, there are steps in the production of glucose which lead to additional efficiency loss, such as photosynthesis in plants and the creation of glucose from plant biomass. An ideal cell-factory would be able to skip all these steps and directly convert carbon-dioxide into a useful product, as shown in the picture below.
Mean green CO2-eating machine
Bacteria that can live off carbon-dioxide have been around on our planet for a very long time, they are called cyanobacteria and are the guys that were the first to pump oxygen into the atmosphere billions of years ago! These bacteria are still around, and while we are less apt in editing their genome than we are with E.coli and yeast, we have come a long way. Using these bacteria we can indeed circumvent the efficiency loss and cost increase involved in glucose production, and directly convert carbon-dioxide into a useful product.
Continuous cultivation
Cyanobacteria only need carbon-dioxide and some salts to grow, which makes cultivation over longer periods of time an affordable and attractive option. When performing continuous cultivation you do not need to keep killing and re-inoculating batch cultures, and you will have continuous product formation.
Genetic instability
However, a big problem that comes into play with continuous cultivation is that of genetic instability. When an organism is genetically modified to create a product, resources need to be allocated towards product formation. If one of the modified organisms now obtains a mutation somewhere in the production pathway, these resources can be allocated towards growth. This means that the mutated - non-producing - organism will grow faster and take over the population.
Growth-coupling
This problem of genetic instability has been tackled before by team Amsterdam in 2015 and it entails coupling production to growth. This essentially means that an increase in growth inevitably leads to an increase in production. Because evolution selects heavily on growth rate, this is a parameter that is naturally optimised. By coupling production to growth we have a way of naturally selecting for production rate!
Producing fumarate
We have found a way to produce growth coupled fumarate in Synechocystis, a well-studied species of cyanobacteria. This is not just exciting because it is the first reported case of an autotrophic organism (organisms that do not need organic compounds to grow) being able to produce extracellular fumarate, but also because fumarate is a highly valued industrial commodity that is currently made from petroleum-based chemicals. We have achieved this by looking at team Amsterdam 2015’s growth coupling of acetate production, which inspired us to couple production of fumarate to growth in a similar manner. We will not stop here, but add to it by taking several approaches to incorporate a light dependent glyoxylate shunt within the TCA cycle to increase nighttime production of fumarate. This will lead to a unique combination of growth coupled production during the day and inducible production during the night.
Finishing touch
In order to be able to easily test how much fumarate our strains are producing, we will develop a fumarate biosensor for determining extracellular concentrations. And last but not least, in an attempt to face any future limitations on high fumarate production head-on, we are going to improve fumarate transport out of Synechocystis by integrating heterologous fumarate transport systems.
