Team:SDU-CHINA/Demonstrate
Demonstration
This year we desired to create a light-induced metabolic flux redirection platform in engineered bacteria. To achieve this idea, we improved the light sensor CcaS-CcaR system. In addition, we measured the type-I-E CRSPRi system and combined it with advanced photoreceptor to control the metabolic flux dynamically.
Now we are proud to present our results:
(1) We successfully created three new types of CcaS-CcaR system: #3, #4, #10.
(2) We constructed a metabolic flux redirection platform dynamically controlled by light.
(3) We demonstrated our system through the production of Polyhydroxybutyrate(PHB).
(4) We manufactured illuminated hardware through reconstructing the shakers suitable for further fermentation.
We removed two PAS domains in CcaS and acquired #3、#4、#10 variants of CcaS. We tested these three variants with the wild type and measured their expressions of sfGFP in the dark state or under light (green or red) state respectively. The measurements showed that #3 and #10 have similar response to green/red light and are better than the original one.
To demonstrate our dynamic light-activated redirection system, we tried to change the induction conditions at different stages of growth. The samples were illuminated by red light at the beginning, then induced with green light at early-log phase (2h), mid-log phase(5h) and late-log phase(9h). As you can see in the figure that among all conditions, bacteria growth was repressed the most obviously when induced at mid-log phase (5h), whereas induction at late-log phase (9h) had little impact on bacteria growth. Through this dynamic platform, we can arbitrarily regulate the expression of targeted gene at different stages to control the metabolism in bacteria to optimize the production of biological products.
In addition, we analyzed the metabolic flux before and after the shift through the Flux Balance Analysis model, and the computational result also certifies that original metabolic flux was affected by targeting gltA gene as well as TCA Cycle.
To introduce this system into metabolic engineering and demonstrate that it would make sense in production, we took the fermentation of producing PHB as an example. The results show…
(waiting for the result. I still don’t have it…)
For the convenience of the experiments and further fermentation, we also reconstructed the shakers for erlenmeyers in which the volume ranging from 50ml to 1000ml. This equipment can change the light color, light intensity, even the irradiation frequency. Such a multi-functional ameliorated hardware provides a solid foundation for further study in light.
In a conclusion, we successfully constructed a dynamic photoactivated metabolic flux redirection platform. Our idea worked out for PHB production. (I hope, I hope, too…)
