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, as well as ethanol, 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. As what we want, to get 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 would kill the host E.coli . In this study, we introduced two innovative and brilliant solutions to solve this serious problem. Now, let’s take a deeper look in our new 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.

　Temperature control system

To allow E.coli to produce isobutanol efficiently,we introduced the low temperature releasing system (Figure 3.) 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.

Figure 4 shows 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. (Figure 5.)

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. (Figure 6.)

　Result

We did an experiment to prove the isobutanol is truly toxic to the E.coli. The data shows that the higher concentration of the isobutanol was in the medium, the lower OD₆₀₀ value could be obtained. (Figure 7.)

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

According to Figure 8, our low temperature release system do truly work !

　Zinc finger

Figure 9 shows the whole circuit of our project. We encoded four zinc fingers(show as blue Cylinder) in front of each enzyme(show as orange). Besides, we encoded DNA program in the second circuit.

Zinc finger proteins contain a DNA binding domain and a functional domain. DNA binding domain could recognize specific DNA sequence, which called 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. (Figure 10.) With the zinc finger's "hand", the enzyme could bind to the specific DNA program. 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. (Figure 11.) 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.

We found that there are only five nucleotides between HIVC and ilvD genes. (ATG are the first three nucleotides of the ilvD gene.) (Firgure 12.) 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.

In order to assemble a production line in E.coli, we have to transform DNA program as well as our fusion protein genes in to E.coli. The design of our biobricks has the same order of zinc finger as 2010 Slovenia iGEM team, so we decided to use their DNA program (BBa_K323066) instead of synthesizing one.

After cloning, we sent it to Genomics BioSci & Tech Co., Ltd. for sequencing. We found that there is a deletion of a base pair in the zif268 biding site. (Figure 13.)

So, we designed a complementary primer to insert the lost base pair. After PCR of point mutation, we sequence it again. Figure 14 shows the outcome.

We submit this corrected DNA program as BBa_K887011. You can find out more information in its part wiki page.

　Instrument

(1)37°C

At the first step, we transformed the plasmid we designed into the E.coli ,and inoculated it in M9 medium- which 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 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 control system would initiate expression of kivd which would convert 2-ketoisovalerate to isobutyraldehyde. Then, isobutyraldehyde would be converted into isobutanol by E.coli's own alcohol dehydrogenase(ADH).

(3) preliminary distillation

After incubating the “E.coline” in 30°C environment for three days, the concentration of isobutanol is high enough to be collected. We prepared two flasks which contained 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 pumped 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. We then designed a temperature control system to allow E.coli to produce optimum isobutanol before being poisoned by isobutyaldehyde. According to our data(Figure 8), 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.

　Optimization

To maximize the isobutanol production, we optimize E.coli strains, culture medium, time, temperature and carbon source. Amazingly, our production surpasses 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.）.

　Medium optimization

First, we tried to find the most suitable medium for DH5α to produce isobutanol.

We cultured the DH5α strain in the common medium, M9,M9T(M9+ trace metal mix) and M9TY(M9+ trace metal mix+ yeast extract) in the low temperature release system. Data shows that when we changed M9 into M9T medium, the yield increased 10 times from 0.05% to 0.5%. Furthermore, when changed M9T into M9TY, the yield increased more than 50% from 0.5% to 0.8%! Consequently, M9TY is the most appropriate medium. And, this medium conforms to the journal we refers to（Smith KM, Liao JC, An evolutionary strategy for isobutanol production strain development in Escherichia coli,2011.）, so we decided to use M9TY as our medium in our upcoming experiments.

　E.coli strain optimization

Next, we wanted to know that if other strains could have great productivity of isobutanol. So, we tested the following five strains: DH5α,DH10b,JM109,MG1655,EPI300.

According to the result (Figure 17.), we knew that DH5α produced the most isobutanol. So, we choose DH5α to produce isobutanol on our upcoming experiments.

　How do we change our culture conditions?

After activating overnight in 37℃,we transferred 1/100 of volume to the new medium then cultured in 37℃ until OD₆₀₀ reached 0.2, we set this point as 0 hour and transferred each tube to different condition at specific time. (Figure 18.)

　Culture time and temperature optimization

After knowing the most 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 37℃ environment into lower temperature environment did produce more isobutanol. (Figure 19.) We could see that E.coli being in 37℃ environment for 20 hours and in 32℃ environment for 4 hours have the highest production quantity of isobutanol.

According to the data (Figure 20.), we discovered that E.coli under 42℃ environment for 24 hours would have higher production of isobutanol than under 37℃ environment at the beginning. This result is totally out of our expectation, and the high production really surprised us! There are several factors that influence the production rate of isobutanol as follows:

(1) Concentration of enzymes

A higher concentration of enzymes leads to more effective collisions per unit time, which leads to an increasing reaction rate. However, the higher protein expression level is a metabolic load of host cells and decrease the growth rate.

(2) Temperature

Usually, an increase in temperature is accompanied by an increase in the reaction rate. Temperature is a measure of the kinetic energy of a system, so higher temperature implies higher average kinetic energy of molecules and more collisions per unit time. But the enzyme has its suitable reaction temperature, higher temperature may decrease the enzyme activity.

(3) Presence of the byproducts

he α-ketoisovalerate decarboxylase (Kivd) is a unique lactococcal key enzyme in the decarboxylation of branched-chain α-keto acids derived from branched-chain amino acids transamination into aldehydes. The promiscuous nature of kivd decarboxylase does not allow good selectivity in the decarboxylation step. Intermediate byproducts such as isobutyrate were present in the fermentation broth. The byproducts decreas the production rate of isobutanol.

Because of these factors affect the final isobutanol concentration,many versions of synthetic circuits which have different protein expression levels are needed to created to test performance in reliability and consistency, but this process is both tedious and time consuming. To overcome this problem, we develop a temperature control method to construct a isobutanol production circuit that can use culture temperature shifts to control the expression levels of a series of metabolic proteins at the precise times. The experimental data in Fig. 7 reveal the design method works successfully, and E.coli under 42℃ environment for 24 hours have higher production of isobutanol (~0.75%).

　Carbon source optimization

Knowing that glycerol is the redundant product of the petroleum pyrolysis, we wanted to reduce the useless byproduct of petroleum Industry on earth. We fed our host with glycerol to see whether it is possible to be our E.coli‘s carbon source or not.

We discovered that using glycerol as carbon source could get the 1/3 of the yield of isobutanol produced by using glucose as carbon source. In this result (Figure 21.), the industrial byproduct, glycerol, can also be digested by our host and turned into the promising bio-fuel.

Furthermore, we also compared the productivity of different temperature.

By comparing 37℃ & 30℃ to 42℃, we found out that the best temperature to produce isobutanol is 42℃. From this figure, the productivity of all temperature decreased obviously after 24 hours. Thus, we conjectured that the isobutanol effused out to the air or the intermediate products of isobutanol pathway were converted into other byproducts.

　Future works

　Ingredient Production

In order to realize our idea to change trash into fuel, we did some research. Therefore, what 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.In Figure 24, it shows that Cex do have the activity to catalyze xylan into xylose.

Another advantage of using PgsA fusion enzyme is that it can lead isobutanol-producing enzymes to catalysis 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, 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 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.