Introduction to the project

Nowadays, environmental pollution and energy depletion have become crucial problems. We need to find alternative energy to replace the running out fossil fuel. Due to the pollution issues, this alternative energy should be environmental friendly. Up until now, ethanol is the most common biomass fuel because the final product is harmless water. However, ethanol will corrode metallic surface of the engines lead to higher cost than fossil fuel usage. Unlike ethanol, isobutanol do not corrode metal and contain higher ratio of the heat of combustion than ethanol. Besides, isobutanol doesn’t produce pollutants such as sulfur dioxide, nitric oxide and nitric dioxide. Isobutanol has widely utilized in many applications as a organic solvent, and antifreeze. Just as what we wanted, in order to find clean energy, we chose isobutanol to be our project. We believe that isobutanol is a potential eco fuel in the future. However, currently isobutanol production wasn't very promising. According to the previous studies, the low yield of isobutanol was caused by the toxicity of isobutanol which kill E.coli hosts. In this study, we introduced two innovative and brilliant solutions to solve this serious problem. Now, let’s take a deeper look in our amazing ideas!

　Project details

　Enzyme for isobutanol

According to the previous study, we use four enzymes to catalyze pyruvate to produce isobutanol. The genes are cloned from different bacteria and encode four enzymes─ AlsS, ilvC, ilvD, KivD.Figure 1 shows the overall pathway. As glucose can be catalyzed into pyruvate by glycolysis, we chose glucose as the starting point of our biosynthetic pathway. Then, pyruvate will be converted into isobutanol by the enzymes shown in Figure 2.

Figure 1

Figure 2

　Temperature control system

To allow E.coli to produce isobutanol efficiently,we introduced the low temperature release system in to our circuit (BBa_K887002). The low temperature system could allow E.coli to produce the optimum production of isobutanol before being poisoned by isobutyaldehyde. The following picture is our system.

First, we incubated E.coli in 37°C environment. After accumulating enough 2-ketoisovalerate , we move E.coli into 30°C environment. The accumulated non-toxic intermediate would be converted into the final product , isobutanol. Therefore, producing an efficient method to obtain the excellent biofuel.

This is our biobrick. The most important gene of our biobrick is 37°C ribosome binding site gene. There are two circuits in our biobrick. The first circuit is the one encodes 37℃ ribosome binding site gene and the second circuit is the one that encodes kivD gene.

Now, let us introduce how our system works.

When being in 37°C environment, the first circuit will be translated and produce TetR protein to inhibit Ptet promoter. So, the second circuit will not be translated. Therefore, we can obtain the intermediate , 2-ketoisovalerate , at this step.

After having enough of 2-ketoisovalerate , we move E.coli into 30°C environment. This way, the ribosome will not bind the 37°C ribosome binding site and tetR genes will not be translated. Therefore, the second circuit will be translated successfully. In the end , we can get the isobutanol efficiently.

　Result

1. The preliminary experiments

Figure 1. Pre-culture of the E.coli through overnight incubation. Then, transfer it into the new medium with the microaerobic environment until the O.D. reach 0.2. Afterwards, measure the O.D. value every 4 hours.

We did an experiment to prove that isobutanol is really toxic to E.coli. The data shows that the higher the concentration of the isobutanol in the medium, the lower the O.D value obtained.

Figure 2. Culture of DH5a, DH10B, JM109, MG1655,EPI300 strains in the 3.6% glucose M9 medium and 37℃ environment until the O.D. value reach 0.2. Then, continue culture it for 24 hours and measure the production of isobutanol.

First, we tested how much isobutanol would be produced by the five different strains using our low temperature release system. According to the result, we knew that the DH5α is the strain which produced the most of isobutanol. Therefore, we chose the DH5α as the main strain to produce isobutanol in our project.

Figure 3. Incubation of the DH5α strain in the common medium, M9, and 2 other mediums, M9T(M9+ trace metal mix) and M9TY(M9+ trace metal mix+ yeast extract).

In addition, we also cultured the DH5α strain in the common medium, M9, and 2 other mediums to see which medium is the best to produce isobutanol in low temperature release system. In this data, we found that M9TY medium is the most appropriate medium for DH5α strain. As a result, we decide to use the M9TY medium.

Figure 4. Culture our E.coli in the 37゜C environment which is appropriate for E.coli growing at the beginning. Then, change the temperature after being 0 hour or 4 hours or other hours.

This is our main idea to design the experiment of the temperature control system. It is that we cultured our E.coli in the 37゜C environment which is appropriate for E.coli growing at the beginning. Then, we would change the temperature after being 0 hr or 4hrs or other times showed by the figure 4.

(1)GC analysis of the isobutanol production-turning from 37゜C environment into 32゜C environment sample

Figure 5. Culture our E.coli in the 37゜C environment at the beginning. Then, change the environment into 32゜C after being 0 hour, 12 hours , 16 hours, 20 hours and 24 hours.

The report indicates that changing from the 37゜C environment into the lower temperature environment did truly get more isobutanol. We could see that E.coli being in 37゜C environment for 20 hours and in 32゜C environment for 4 hours have the highest % concentration of isobutanol.

Figure 6. Mark the second circuit with the fluorescent protein to test the expression of kivD enzyme.

We also used the fluorescent protein to mark the second circuit of our biobrick. The data tells us that kivD enzyme being in the 37゜C environment had the lower expression than being in the 30゜C environment and the 25゜C environment.

All of the reports mean that our low temperature release system does truly work!

(2)GC analysis of the isobutanol production-turning from 37゜C environment into 42゜C environment sample

Figure 7. Culture our E.coli in the 37゜C environment at the beginning. Then, change the environment into 42゜C after being 0 hour, 12 hours , 16 hours, 20 hours and 24 hours.

According to the data, we discovered that E.coli being in 42゜C environment for 24 hours would have higher % concentration of isobutanol than being in 37゜C environment at the beginning. It had the high reproducibility and is really surprising to all of us because it is out of our expectation ! We pondered the possible reason might that the kivD enzyme being in the 37゜C environment had lower activity than being in the 42゜C environment. In addition, there would be lack of toxic byproducts because of low expression of kivD enzyme in the 42゜C environment.

Figure 8. Culture our DH5α strain in the medium of 3.6% glucose or 5% glycerol for three days. Test the sample by GC every 24 hours.

After learning that glycerol is the redundant product of the petroleum pyrolysis, we wanted to reduce the useless byproduct of petroleum Industry on earth. We did another experiment to see whether glycerol is possible to be our E.coli ‘s ingredient or not. We discovered that using glycerol as the ingredient could get the 1/3 of the yield of isobutanol produced by using glucose as the ingredient. We also observed that the % concentration of isobutanol being in the 42゜C environment is the most highest in the three kinds of temperature environments. We proved the reproducibility of it and considered that we might optimize our temperature control system according to these reports.

　Zinc finger

Zinc finger proteins contain a DNA binding domain and a functional domain. DNA binding domain could recognize specific DNA sequence, which named DNA program. Zinc fingers could tightly bind to specific DNA or RNA sequence. We replace the zinc fingers' functional domains with our enzymes to create fusion proteins. With the zinc finger's "hand", the enzyme could binds to the specific DNA program we made. By doing so, the enzymes would no longer disperse around the cell. Therefore the productivity of isobutanol will be higher.

With this feature, we expected to build a production line to help us make isobutanol. We put the enzymes in order. When the intermediates are produced, it could have the next reaction as quickly as possible. The final product, isobutyraldehyde will be converted into isobutanol by ADH in E.coli.

(Point mutation)

We found that there are only five nucleotides between the genes, HIVC and ilvD after ligating. (ATG are the first three nucleotides of the ilvD gene.) According to the triplet nature of gene expression by codons, it would cause a frameshift mutation, which cause the condons code for incorrect amino acid.

(1)So we inserted an A into the site after the HIVC gene. As a result, the sequence between the two genes accomplishes six nucleotides.

(2)However, we found a stop codon, TAG in every connection between the zinc fingers and the enzymes that surprised us.

(3)We made a point mutation again to change the fifth nucleotide from A to T. Therefore; the stop codon (TAG) is changed to TTG codes for leucine.

　Result

(developing)

　Instrument

(1)37°C

At the first step, we put the E.coli with the plasmid we designed in M9 medium ,containing 36 g/L glucose, 5 g/L yeast extract,100 μg/ml ampicillin, 30 μg/ml kanamycin, and 1,000th dilution of Trace Metal Mix A5 (2.86 g H3BO3, 1.81 g MnCl2 ⋅4H2O, 0.222 g ZnSO4 ⋅7H2O, 0.39 g Na2MoO4⋅2H2O, 0.079 g CuSO4⋅5H2O, 49.4 mg Co(NO3)2⋅6H2O per liter water) ,into the first tank. Then, we culture the E.coli in the 37°C environment for three hours which means that we put the tank in the warm bath to let E.coli produce the intermediate,2-ketoisovalerate.

(2)30°C

Afterward, we put our E.coli into 30°C environment maintained by warm bath for 3 days incubation. Our low temperature system would initiate expressing of kivd which would convert 2-ketoisovalerate to isobutyraldehyde. Then, isobutyraldehyde would be converted into isobutanol by E.coli's own ADH.

(3) preliminary distillation

After incubating the “E.coline” in the 30°C environment for three days, the concentration of isobutanol is high enough to be collected. We prepared two flasks which half-filled cold water and each of them is equipped with a condenser. The three flasks were linked with pipes. One end of the pipe (air out) must be under the water level, so that the air would expose into water of the destined flask. We pump air to strip the isobutanol to the flask for product collection. If isobutanol could be transferred from the fermentation flask, we expected the production rate could extend tremendously and the following condensate collector will obtain higher concentration of isobutanol than the previous fermentation flask. By having this higher concentrated isobutanol, isobutanol purification will be much more favorable to be conducted.

　Conclusion

The main aim of our “E.coline” project is to generate isobutanol, a promising eco-fuel, in a productive and efficient way.

To produce isobutanol, at first we use four pyruvate catalytic enzyme genes: alsS, ilvC, ilvD, kivD all together. We then designed a temperature control system to allow E.coli to produce optimum isobutanol before being poisoned by isobutyaldehyde. According to our data, our temperature control system had been proven to work successfully.

To produce isobutanol more efficiently, we combined zinc fingers and our enzymes together and put the fusion proteins in catalytic pathway order, thus the isobutanol conversion process can be accelerated. in addition, We have also mutated the stop codons in our fusion proteins.

　Optimization

To maximize the isobutanol production, we optimize E coli strains, cultural medium, time, temperature and carbon source. Amazingly, our production surpass the published reference whose production is 6.8g/L for 24 hr by using modified JCL16 strain (Smith KM, Liao JC, An evolutionary strategy for isobutanol production strain development in Escherichia coli,2011).

　E coli strain optimization

First, we tested how much isobutanol would be produced by the four kinds of strains harboring our circuit.

According to the result, we knew that the DH5α produced most isobutanol,which produced by16 hours, 37℃ culture and then transfer to low temperature27℃,8 hours. So, we chose the DH5α as the main strain producing isobutanol in our upcoming experiments. By the way, we found that even if inducing these strains with IPTG, the yield of isobutanol wasn’t higher.

　Medium optimization

Next, we tried to find suitable medium for DH5α to produce isobutanol.

In addition, we cultured the DH5α strain in the common medium, M9, and other 2 kinds of mediums to see which medium is the best to produce isobutanol in the low temperature release system. Data showsthat M9TY medium is the most appropriate medium, which conforms to the journal（Smith KM, Liao JC,Anevolutionary strategy for isobutanol production strain development in Escherichia coli,2011.）As a result, we decided to use M9TY as the mediumin to our upcoming experiments.

(delveloping)

　Cultural time and temperature condition optimization

Since knowing the appropriate medium and strain, we tried to find out the best condition, including temperature and culture time, for our host cell to produce isobutanol.

The report indicates that changing from the 37゜C environment into the lower temperature environment did get more isobutanol. We could see that E.coli being in 37゜C environment for 20 hours and in 32゜C environment for 4 hours have the highest production quantity of isobutanol.

According to the data, we discovered that E.coli being in 42℃ environment for 24 hours would have higher production of isobutanol than being in 37℃ environment at the beginning. This result totally beyond our expectation, and the high production really surprised us! We pondered the possible reason might that the kivD enzyme being in the 37℃ environment had lower activity than being in the 42℃ environment. In addition,the toxic byproducts are absent because of low expression of kivD enzyme in the 42℃ environment.

　Carbon source optimization

Alternatively, we had also tested the production by using the different carbon source: glycerol.

After learning that glycerol is the redundant product of the petroleum pyrolysis, we wanted to reduce the useless byproduct of petroleum Industry on earth. We did another experiment to see whether glycerol was possible to be our E.coli‘s ingredient or not. We discovered that using glycerol as the ingredient could get the 1/3 of the yield of isobutanol produced by using glucose as the ingredient. In this result , the unwanted byproduct, glycerol, can also be digested by our host turning into the promising bio-fuel.

　Future works

　Ingredient Production

In order to realize our idea to change trash into fuel, we did some research. Therefore, the first thing we have to do is to figure out how to degrade the cellulose. First, we want to get xylose from cellulose through xylanase. Xylanase is a class of enzyme which degrades the linear polysaccharide beta-1,4-xylan into xylose, thus breaks down hemicellulose, one of the major components of plant cell walls. Xylose is a good carbon source. As such, xylanase plays a major role in micro-organisms thriving on plant sources (mammals, conversely, do not produce xylanase).

According to the Journal of Applied Microbiology ( Y.P. Chen et al. 2011), the cell-surface display of Cex, which encodes xylanase from Cellulomonas fimi, was constructed on E. coli using PgsA as the anchor protein. Through Fig??, it shows that Cex do have the activity to catalyze xylan into xylose.

Another benefit of using PgsA fusion enzyme is that it can lead isobutanol-producing enzymes catalyze through consolidated bioprocessing(CBP) , the CBP in converting Cellulose into isobutanol requires combinations of biological events(production of xylanases, hydrolysis of the polysaccharides in the biomass, temperature controlling, and production of isobutanol) in one reactor. CBP has gained recognition as a potential breakthrough for low-cost biomass processing. So, if we incubate E. coli with this mechanism with our isobutanol-synthesis E. coli, we can cost down the expenses of enzyme purification. Finally, the reactor as a whole will be more like a biofuel production line!

　Cellulose Degradation

Furthermore, we found another potential way on coverting cellulose into glucose by utilizing the Biobrick from 2008 and 2011 Edinburgh igem team. Edinburgh2008 iGEM team found out three Coding parts on cellulose degradation,cenA: BBa_K118023 (endoglucanase), cex: BBa_K118022 (exoglucanase), and bglX: BBa_K118028 (beta glucosidase). Edinburgh2011 iGEM team able to display bglX (a cryptic E. coli β-glucosidase gene) and the exoglucanase cex on cell surface. Therefore, through MUG assay and MUC assay, bglX and cex can be proven its effect. Because bglX is capable of degrading the substrate MUG, which has a β (1→4) bond, similar to that of cellobiose. So in the future work, we can use an INP-β-glucosidase fusion (BBa_K523008 + BBa_K523004),which INP(BBa_K523008, based on BBa_K265008), a carrier for displaying enzymes on cell surface, can be used to carry proteins to the cell surface, by constructing BBa_K523013 with a new β-glucosidase (bglX) BioBrick, BBa_K523002.

　Biofuel Industry

Next step, we will focus on researching the reaction rate, intermediate, and by-products of mechanisms. For example, the retention time for producing a certain concentration of 2-ketoisovalerate per 300 ml culture medium under different processing parameters!

With the data, we can optimize the Eco-line economic justification; design the flow rate, vessel capacity, the driving equipment and instrumentation for totally auto-controlled system. Thus, we can build a manufacturing automation technology to produce isobutanol inexhaustibly.

Furthermore, we wish we could apply our project in commercial way some other day.
We use the above introduced cellulase to produce xylose as ingredient(cheaper resource of raw material) in the first drum (preparation stage); The biosynthetic production of isobutanol generated on our project pre-reactor and reactor (reaction stage, R-301& R-302); The last section is to purify isobutanol by azeotropic distillation (separation stage, T-401, D-401＆ D-402). Hopefully the enormous production could be an alternative of gasoline for future green life.