Achievement

In order to achieve these two goals, we try to figure out three main questions：

1. Amount of CO₂ uptake by E. coli
2. CO₂ flux in E. coli metabolism
3. Amount of Carbon fixed in biomass

Purpose

We aim to develop the biological model to make our experimental data more useful, even more effective to achieve the optimization parameters in the model. Therefore, we can save time to try and error on doing experiment. After that, we analyze the rate of production and consumption. In this way, we can calculate the amount of carbon dioxide uptake into the Escherichia coli (E. coli) and calculate how much CO₂ will be used in our system. In addition, we also want to realize how much carbon fix in our system. We have to understand the process in E. coli after uptaking CO₂. Since we integrated non-native carbon fixation pathway into E. coli to let E. coli utilize CO₂, we can simplify the CO₂ utilization pathway in engineered E. coli into two parts, CO₂ uptake and CO₂ metabolism.

Method of model

Follow our model progress diagram with a better sense of how we achieve our goal.

CO₂ uptake

CO₂ exists in the phase of gas. Therefore, E. coli uptake CO₂ by diffusion. However, the native diffusion rate is really low, not to mention the dissolved rate. We clone CA (carbonic anhydrase) gene to speed up the diffusion rate. The higher diffusion rate, the higher CO₂ concentration maintained in engineered E. coli. Higher CO₂ concentration in engineered E. coli provides more steady CO₂ condition for downstream pathway.

Table 1. parameters of CO₂ uptake pathway

CO2 uptake				Kf (mM)		Kr (mM)
R1	$${[CO_2]_{air} \rightarrow [CO_2]_{uptake}}$$			9.23E-04		4.78E-04
R2	$${[CO_2]_{uptake} + [H_2O] \rightarrow [H_2CO_3]}$$			0.062		23.7
R3	$${[H_2CO_3] \rightarrow [H^+] + [HCO_3^-]}$$			8000000		4.7E10
R4	$${[HCO_3^-] \rightarrow [H^+] + [CO_3^{2-}]}$$			3		5E10
R5	$${[H^+] + [HCO_3^-] \longleftrightarrow [H_2O] + [CO_2]_{uptake}}$$			Vmax		Km
				0.47333		0.0062

Result

Enzyme, CA, strongly catalyzes the hydration of CO₂ and its interaction appears to follow a Michaelis-Menten mechanism with a Hill constant of 0.0062 in our research. The result of Engineered E. coli cloned with or without CA gene showed below. A quite different diffusion time explains the function of CA.

Fig 1. CO₂ uptake amount change with time in engineered E. coli without CA gene.

Fig 2. CO₂ uptake amount change with time in engineered E. coli with CA gene.

CO₂ Metabolism

Xylose is the main source of our engineered E. coli to uptake CO₂. However, there is another pathway for xylose metabolism, which means that there is only a part of xylose that E. coli consumed will react with CO₂. We build a model to predict the different carbon flux between original xylose metabolism, PP pathway (Pentose Phosphate pathway), and our CO₂ bypass pathway.

Both of two pathways will end up at pyruvate, which is a key intersection in the network of metabolic pathways. We built a model of TCA cycle to explain how pyruvate play an energy precursor role and then is converted into biomass of E. coli.

Pathway

Fig 3. Metabolic pathway of CO₂-utilization E. coli.

The introduced CO₂-utilization bypass pathway composed of PRK and Rubisco is drawn in green line and noted by ” A” reaction and the double line was the pathway that composed by genetically modified, while the central carbon metabolic pathway including PP pathway and TCA cycle is drawn in blue line and yellow line and noted by “B” reaction and “C” reaction, respectively. See more detail in Kinetic law.

Xylose v.s. CO₂ analysis in CO₂ utilization efficiency

Two main sources of CO₂ metabolism in engineered E. coli are xylose and CO₂. CO₂ utilization rate varies under different condition. We use 5% CO₂ (about 2.6 mM) and 0.4% xylose (about 26 mM) in experiment as an optimization CO₂ utilization rate. Constant CO₂ condition and limited CO₂ condition also effects metabolism performance. In other words, open system and close system showed different results. Therefore, adjust initial concentration of CO₂ and Xylose and choose open or close system to see the change of pyruvate production.

Concentration of xylose 0.4 % 1 % 2 %

Concentration of CO₂ 5 % 10 % 15 %

Close system / Open system Close Open