Team:Amsterdam/test/thijs/project

PHOTOSYNTHETIC CELL-FACTORY

Produce, export, and detect fumarate

The Project

Introduction

Irrespective of where you come from, we all share the global responsibility of ensuring that our societies are sustainable. We have been depleting the world’s resources and filling the atmosphere with abnormal levels of CO2 for too long. But CO2 can also be used as a resource, plants and certain bacteria have been doing so for billions of years. This is the foundation for the bio-based economy, reducing and eventually replacing the use of oil. Up to now the focus in bio-based production has been on producing biofuels with plants and producing sugars to use in bacterial fermentation processes. But there is a better way, using cyanobacteria to take up CO2 from the atmosphere and directly converting CO2 into useful products. Cyanobacteria do not compete for arable land, as plants do, and by taking up CO2 and directly converting it into product you eliminate the sugar producing step that is necessary for fermentation. What's more, we calculate that plants have a production rate of 0.0257 mmolcarbon gDW-1 hr-1, which is well below what has already been reported for the cyanobacterium Synechocystis sp. PCC6803 (hereafter Synechocystis) for a variety of chemicals.

Goal

We aim to create photosynthetic cell factories for fumarate using Synechocystis. Not only do we want our cell factories to produce fumarate, but we also want them to export it out of the cell, and when the fumarate has been produced and exported, we want to be able to detect this. In essence our goal is to stably produce, export and detect fumarate under conditions mimicking industrial settings.

Did you remember that....
Fumarate is used to make certain plastics, food additives, and medicine and is currently made from petroleum.

‘Mimicking industrial settings’ is an important note. This means we want to be able to perform continuous cultivation and for this purpose, the fumarate producing strains must be stable. A producing strain is stable when it is not beneficial, or simply not possible, for them to stop production. Moreover, in an industrial setting, the light our cell factories will receive comes from the sun, meaning they are cultivated in a day/night rhythm. We have divided the work on our project into three ‘modules’ which can be seen below.

MODULES

Stable Production

When an organism is genetically modified to create a product, it causes a decrease in growth rate. This means that a mutated - non-producing - organism will grow faster and take over the population. This problem is tackled by coupling fumarate production to growth. Given that evolution selects heavily on growth rate, we now have a way of naturally selecting for production rate [4]! Knocking out the fumarate degrading reaction in the TCA cycle causes fumarate to be produced in a growth coupled way.
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Transporter

In anticipation of high fumarate production, transport of fumarate out of the cell can be a limiting factor. This mechanism is largely unknown in Synechocystis and will therefore be -guided by bioinformatics- characterized by means of knock-out and over- expression experiments.
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Biosensor

A biosensor for fumarate is constructed in order to facilitate a high throughput screening of extracellular fumarate. This is essential for low measurement time and costs.
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