Team:Amsterdam/test/joeri/index

Production

A major requisite of cyano-cell factories, according to expert's opinion, is that they must be able to produce in a stable fashion under industrial conditions. A recent quantitative analysis of the various ways to convert the energy of photons to chemical bonds has revealed that the direct utilization of sunlight is the most efficient [1]. This however means that cells will be exposed to diurnal regimes in which they will inevitably be exposed to periods of darkness. Our goal here is to achieve the first photoautotrophic cell factories that are able to stably produce fumarate around the clock.


Produce

Overview

Synechocystis does not naturally produce fumarate. However, model guided engineering found that removing a single gene within Synechocystis leads to a stable cell factory that produces fumarate as it grows during the day. Nevertheless, at night our cells do not produce fumarate, since at night, they don't grow. To overcome this challenge, we have taken a systems biology approach which interweaves theory, modeling, and experimentation to implement stable nighttime production of fumarate. We theorized that we can redirect the nighttime flux towards fumarate production by removing a competing pathway via knockout of the zwf gene. Additionally, we also took inspiration from nature and speculated that the incorporation of the glyoxylate shunt would further increase our nighttime production of fumarate. Our models corroborate these predictions, however, they also suggest that the stability of the glyoxylate shunt is sensitive to the timing of when the shunt is turned on (i.e. expressed). We therefore took a robust approach to incorporate the glyoxylate shunt enzymes under ideal expression conditions.

Highlights

  • Engineered a ΔfumCΔzwf Synechocystis strain, that uses different fumarate production strategies during day and night.
  • Developed a method to make fully segregated libraries in polyploid organisms
  • Created the first fully segregated library representing the entire genome (99.9% confidence) of Synechocystis upstream of the glyoxylate shunt genes. This library is now ready to be tested to further increase nighttime fumarate production.
  • Stable production of fumarate directly from CO2 around the clock (Nighttime fumarate production rate of 2.96 mM grDW-1hour-1 Daytime fumarate production rate of 9.24 l mM grDW-1hour-1Titer of 48.48 mg L-1) [Disclaimer: our experimental design was aimed mostly at proof-of-principle. Much higher titers (>230 mg/L) are possible if economically more favorable due to downstream costs].

References

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