Team:NCKU Tainan/Analysis

CO2 utilization result analysis

Amount of CO2 uptake

At first, we set up a closed system to model CO2 uptake by E. coli. After a period of time, concentration of CO2 uptake reached a balance. The result can tell that more than 50% air CO2 diffuse into E. coli in a closed system.

Fig 1. CO2 uptake under closed system

However, we cannot set a CO2 utilization system in a closed system. No matter our application is continuous culture or fed-batch culture, air CO2 concentration will be constant, which means a boundary condition. Therefore, we model again under open system. Under constant air CO2 condition, CO2 diffusion speed changed with time and we categorized them into three phases, which are steady climb phase, transition phase, and saturated phase. The time segments of these three phases depend on CA activity. We assume A, B, C as time intervals of three phases. The cover area in the result shows that different time interval refers to different amount of CO2 uptake by E. coli. Its CO2 uptake rate change with time as well. As a result, we collect the CO2 uptake rate and then calculate with the xylose consumed rate and pyruvate produced rate. What’s more, the main reaction of CO2 in engineered E. coli happened between RuBP to 3PGA, which is another part we are going to discuss.

  1. Engineered E. coli with CA

    2. Time A was about 10 min (or 700 s) with [CO2 uptake] reach 1 mM, causing 25% CO2 uptake in total.

    Time B was about 1 hour (or 3500 s) with [CO2 uptake] equals to [air CO2], causing 75% CO2 uptake in an hour.

    Time C was about 1.5 hour (or 8000 s) for [CO2 uptake] reach balance with the highest CO2 uptake percentage, 90%.

    Time interval Xylose supplied Concentration CO2 uptake (mM) Xylose (mM/s) Pyruvate (mM/s) Total CO2 uptake percentage
    A 0.2% 1 0.008 0.016 25%
    B 0.2% 2.045 0.00264 0.00528 75%
    C 0.2% 2.15 0.001131 0.002263 90%

    Table 1

    xylose consumed rate and pyruvate produced rate under different CO2 uptake time interval with CA

  2. Engineered E. coli without CA

    3. Time A was about 10 min (or 700 s) with [CO2 uptake] reach 1 mM, causing 25% CO2 uptake in total.

    Time B was about 1 hour (or 3500 s) with [CO2 uptake] equals to [air CO2], causing 75% CO2 uptake in an hour.

    Time C was about 1.5 hour (or 8000 s) for [CO2 uptake] reach balance with the highest CO2 uptake percentage, 88%.

    Time interval Xylose supplied Concentration CO2 uptake (mM) Xylose (mM/s) Pyruvate (mM/s) Total CO2 uptake percentage
    A 0.2% 1 0.0036345 0.007504 28%
    B 0.2% 2.045 0.0017486 0.003888 72%
    C 0.2% 2.15 0.001631 0.00388 88%

    Table 2

    xylose consumed rate and pyruvate produced rate under different CO2 uptake time interval without CA

    From table 1 and table 2, we knew that the highest pyruvate produced rate happened under the lowest CO2 uptake percentage. Besides, pyruvate produced rate of engineered E. coli with CA is higher than that of engineered E. coli without CA. Although our working space was open system that we cannot sense precise data of the change of CO2 concentration. Through pyruvate produced rate, we can easily recognize which phase of CO2 uptake and then figure out the percentage of total CO2 uptake. It is the method we calculate how much CO2 uptake by our engineered E. coli.

    A

    B

    C

    Fig 2. result of xylose and pyruvate under A, B, C, time interval

    Actually, xylose consumed rate is slightly related to has little relationship with the CO2 uptake rate, since that xylose metabolism wasn’t just a single pathway. What we can analysis is that the pyruvate produced rate being correlation with CO2 uptake rate, which help us to define the question that how much CO2 uptake by engineered E. coli. It can also fit with experimental data easily. Next, we discuss about the true CO2 reaction in E. coli CO2 utilization bypass pathway. Every single mole of CO2 uptake will react with one mole of RuBP and then produce 2 mole of 3PGA.

    Fig 3. result of RuBP and 3PGA during CO2 uptake

    Since that RuBP and 3PGA are just intermediate products in metabolism, their concentration is quite low. Besides, results of three CO2 uptake time interval showed similar. We still can see that 3PGA produced is 2 times larger than RuBP produced. We then calculate their produced rate in three CO2 uptake time interval.

    Time interval RuBp produced rate (mM/s) 3PGA produced rate (mM/s)
    A 4.25E-09 6.35E-10
    B 3.89E-09 8.42E-09
    C 1.76E-09 3.71E-09

Carbon metabolism flux

The main metabolic pathway of xylose in E. coli is PP pathway and glycolysis. As for recombinant E. coli, it has multiple xylose metabolic pathways, and we can simplify them into original pathway and CO2 Bypass pathway. Therefore, we need to define the percentage of xylose, which is consumed by engineered E. coli, entering CO2 bypass pathway and utilize CO2.

It takes a lot of time to get absolute metabolic flux of CO2 in engineered E. coli and require feed of 13CO2 during cultivation. Since the metabolic flux of the original metabolic pathway is quite stable, the relative metabolic flux of CO2-utilization over that of the original metabolic pathway could show a quantitative understanding on the CO2 utilization efficiency. This relative value was MFICO2[1], a term as metabolic flux index of CO2-utilization pathway in heterotrophic engineered E. coli. This is the percentage of xylose that we mentioned above.

Fig 4. carbon flux in engineered E. coli

X：Actual 3PGA detected from the original pathway = 3PGA0

Y：Actual 3PAG detected from CO2 bypass pathway = 3PGA’

a：3PGA generated from the central pathway

b：CO2 fixed by the CO2 bypass pathway

c：mol of 3PGA0 into downstream

d : mol of 3PGA’ into downstream

To define the MFICO2, we use CO2 fixed by the CO2 bypass pathway, noted as b, divided by the 3PGA generated from the central pathway, noted as a. We also assume c is mol of 3PGA¬0 and d is mol of 3PGA’ that channels into downsteam metabolism. After metabolism, (a+b) mol of 3PGA0 and b mol of 3PGA’ are generated.

Besides, X and Y represent the actual 3PGA detected from the original pathway and CO2 bypass pathway, which show in 3PGA0 and 3PGA’ in the Fig 1., respectively. In the experiment, we use 13C-labeled CO2 and unlabeled sugar to get the amount of 3PGA0 and 3PGA’. However, it was reported that 3.45% of unlabeled 3PGA, which is noted as 3PGA’, will convert to its isotopic during the culturing E. coli strains in medium. Eventually, we concluded these situation into two equations.

$${y = b + 3.45\% \times (a+b) - d ......(1)}$$

$${x = (1-3.45\%) \times (a+b) - c ....(2)}$$

Since d/c = y/x under a metabolic steady-state, we derive equation (1) and (2) into a final relationship between a, b, x, and y.

$${MFI(Metabolic \ flux \ index) = {b \over a} = {{0.97y-0.03x} \over {1.03x-0.97y}}}$$

As a result, we only need the amount of 3PGA0 and 3PGA’ to calculate MFICO2. Through modelling, we supply 0.4% xylose and 5% CO2 to get the data of 3PGA0 and 3PGA’, which helps us to adjust the rate between xylose and CO2 sources.

Fig 6. The result of 3PGA produced form PP pathway (original metabolism) and from CO2 bypass pathway.

Time MFICO2
10min 0.35256
1 hr 0.35247
2.5 hr 0.3524

Table 3 MFICO2 at different time

Fitting Experimental data

The purpose of modelling is to predict the result before doing experimental data. Our model focus on the metabolism pathway in engineered E. coli, trying to understand how E. coli utilize CO2. The result can fit with our experiment result to see if our model method was correct. However, most of our experiments is about cell growth under different condition. There are too many factors affect biomass and we cannot list all of them into model. Therefore, pyruvate, was chosen to represent the trend of biomass since that the metabolism of pyruvate downstream process is quite clear with abundant research have been done.

Fig 7. pyruvate produced under different CO2 uptake condition (model result)

Fig 8. cell growth under different CO2 conditions (experimental data)

The final goal of our project is to prove that our engineered E. coli could successfully consumed CO2 into its metabolism. CO2 convert into pyruvate through E. coli and then express on its cell growth. Therefore, we can conclude that pyruvate production will have correlation with biomass, which confirms that our model is reasonable to show the result with pyruvate production.

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