Team:BIT-China/Engineering

Engineering

The metabolic pathway of eriodictyol is L- tyrosine as carbon source to L- tyrosine to produce p-coumaric acid under the action of TAL; p-coumaric acid is then mediated by 4CL to form its corresponding p-coumaroyl COA; and then synthesis of chalcone by p-coumaryl COA by CHS; chalcone is transformed into naringin; Finally, F3’H catalyzes the hydroxylation of naringenin, adding a hydroxyl group to the position of naringenin 3' to convert it into eriodictyol.

The above synthetic pathway is long and the metabolic pressure on the bacteria is high. We consider cutting the pathway and then dividing it into two bacteria. The heterologous biosynthesis pathway will cause metabolic pressure, which makes eukaryotes grow slowly and culture for a long time, and prokaryotic cells can not provide the membrane structure required for post-translational modification of P450 in turn, so we consider co-culture of E. coli with S.cerevisiae to produce eriodictyol. Co-culture has some advantages, including division of metabolic pathways, sharing the burden of gene expression, cross-feeding metabolites, and improving metabolic efficiency and robustness. However, there are some problems, such as: it is difficult to realize the mass transport of intermediate products, and it is difficult to maintain the stability of the proportion of each strain in the co-culture system.

In order to solve the problems raised at the beginning of the project, we proposed our design for the project.

PH regulation is very important in fermentation. Appropriate PH value can reduce byproducts and increase the growth rate of strains. The TAL enzyme only works in an alkaline environment. We pay a lot of attention to the PH regulation. We first consider inducing E.coli to express GadA to automatically adjust PH value. But in the modern fermentation workshop, we found the equipment to automatically detect and adjust the PH value, so we decided to delete the PH adjustment part in the E.coli. In addition, in industry, improving the robustness of strains is generally a more efficient approach.

E. coli is a prokaryote and yeast is a eukaryote. The growth rates of the two differ greatly. In order to solve the problem that the proportion of strains in the co-culture system cannot be maintained stable due to the large difference in the growth rate between E. coli and yeast, we developed a set of high strength and resource saving QS system for e. coli. In this system,We induced the lux promoter through Coumarin HSL and RPAR conjugates to express the downstream toxin protein and lactose metabolizing enzyme lines.The main process is Coumarin HSL and RPAR combine to induce the Lux promoter to express toxic protein CbtA, lactose metabolizing enzyme system and RPAI respectively. RPAI continues to act on p-coumaryl COA to produce Coumarin HSL, and the above process again forms a cycle.

However, when the toxic protein produced reaches a certain amount, it can stop the division of E. coli and keep the quantity of E. coli within a certain range. After a period of time, the toxic protein CbtA will degrade automatically and the E. coli will continue to divide and grow.

The lactose metabolome enzyme converts lactose to galactose. After entering yeast, galactose binds to the galactose-induced promoter and induces the expression of downstream metabolic pathways. This not only controls the time when the yeast starts metabolic production so that the yeast can grow fully before this, but also reduces the accumulation of intermediate products.

We chose toxic proteins to control the population of E. coli. We hoped that after the population density of E. coli reached a certain value, the population of E. coli would stop growing and form a shock effect. At first, we chose phage cracking protein φX174. Phage cracking protein φX174 is a kind of small molecule protein derived from the phage, in the inner and outer membrane fusion of gram-negative bacteria, and on the membrane formation is 40 ~ 200 nm hole size, material in the cytoplasm in the osmotic pressure difference inside and outside, under the action of discharge cells, and lead to cell death. but in the process of communication with cofco, we know that phage cracking protein system has a long delay, and if we're going to have the shock effect ,the amount of cleavage protein requires a very delicate balance point. So we select the CbtA system again. CbtA is a toxic protein that e. coli carries on its own and is easy to produce. The system of the toxic protein in block under the premise of preventing cell elongation and division, does not affect the normal physiological activities necessary to sustain life, reducing internal friction.

Under the synergistic action of toxic protein, lactose metabolizing enzyme system and QS system, we formed the characteristic model of our project -- double oscillation dynamic regulation co-culture. It is under the condition that the growth rate of E.coli population oscillates and the expression intensity of upstream and downstream metabolic pathways oscillates, the population density and upstream and downstream metabolic pathways of Escherichia coli and Saccharomyces cerevisiae were dynamically matched. Here we introduce the story of the race between the tortoise and the hare to explain, We compared faster-growing E. coli to rabbits, yeast compared to turtle. During the production and metabolism of E. coli, the accumulation of coumaric acid has not yet reached the concentration of the opening toxic protein and galactose. Yeast grows normally but does not produce, just as rabbits run faster than turtles. After the accumulation of p-coumaric acid to a certain extent, p-coumaric acid is generated into Coumarin HSL, and Coumarin HSL is combined with RPAR, which turns on quorum sensing to produce toxic proteins, and controls the stop growth of E. coli and the accumulation of coumaric acid. The lactose is converted into galactose, and the galactose is transferred to the yeast to open the downstream metabolic pathway, and the product is obtained. This part is like the rabbit sleeping, the tortoise catches up with the hare, the tortoise goes on but the hare is sleeping. When the large intestine is exhausted of coumaric acid and natural decomposition of Coumarin HSL, Lux promoter is shut down, toxic protein is broken down, and galactosidase is shut down. The large intestine resumes normal growth and production, while yeast does not normally grow and produce, opening a new cycle.

Finally, regarding the transport of intermediate products, in order to increase the transport of products, we chose p-coumaric acid as the intermediate transport product because of its easy transmembrane and non-toxic characteristics. And balanced the metabolic pressure between large intestine yeast.

That's all we've designed.