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Team:NCTU Formosa/Project
From 2012.igem.org
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. 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 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.
Figure 1.Isobutanol synthesis pathway.
Figure 2.We cloned four gene, ilvC, ilvD, AlsS, kivd. The figure above are the names, strains, length and point mutation of four genes.
Temperature control system
To allow E.coli to produce isobutanol efficiently,we introduced the low temperature releasing 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.
Figure 3.The idea of our low temperature release 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.Two circuits of our biobrick of temperature control system.
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.
Figure 5.The system works in the 37℃ environment.
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 6.The system works in the 30℃ environment.
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
Figure 7.Activate our E.coli overnight. Then, transfer it into the new medium with the microaerobic environment until OD600 reached 0.2. After that, measure the OD600 every 4 hours.
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.
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 the report, our low temperature release system do truly work !
Figure 8.Mark the second circuit with the fluorescent protein to test the expression of kivD enzyme.
Zinc finger
Figure 9.Mark the second circuit with the fluorescent protein to test the expression of kivD enzyme.
This is the whole circuit in 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.
Figure 10.This is the whole circuit in our project.
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. 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.
Figure 11.The intermediate and enzyme in the pathway.
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)
Figure 12.Point mutation for avoiding frame shift.
We found that there are only five nucleotides between HIVC and ilvD genes. (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.
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.Comparing with the sequence form the part wiki page, there is a missing base (pair) in the zif268 biding site, to be specific a deletion of 25 G. Image: NCBI BLAST.
So, we designed a complementary primer to insert the lost base pair. After PCR of point mutation, we sent it to sequence again.
Figure 14.There is a correct zif268 biding site in this DNA program. Image: NCBI BLAST.
We submit this corrected DNA program as BBa_K887011. You can find out more information in its part wiki page.
Instrument
Figure 15.Our project is applied to the simple instrument. Besides, we also add a simple way to collect isobutanol.
(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 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).
Medium optimization
First, we tried to find suitable medium for DH5α to produce isobutanol.
Figure 16.Medium test: Incubated host cell (DH5α)in M9,M9T(M9+ trace metal mix) and M9TY(M9+ trace metal mix+ yeast extract)medium in 37℃.Until OD 600 up to 0.5, we transfer to 27℃ environment(blue) to see which medium is the best 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 base on a journal（Smith KM, Liao JC, Anevolutionary 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.
Figure 17.Comparison of isobutanol production obtained with DH5α, DH10b,JM109,MG1655,EPI300, incubating in 37℃,3.6% glucose, M9TY medium. Until OD600 reached 0.2(set as 0 hr), strains were cultured for 24 hours then measure the production by GC.
According to the result, we knew that DH5α produced the highest amount of isobutanol. So, we choose DH5α to produce isobutanol for our upcoming experiments.
How do we change our culture conditions?
Figure 18.
This is our main idea in designing the experiment of the temperature control system. We cultured our E.coli in 37゜C environment which the most suitable for E.coli growing at the beginning. Then, we would change the temperature after being 0 hr or 4hrs or other times showed.
Culture time and temperature 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.
Figure 19.Transfer to different temperature at different timing. We inoculated our E.coli in the 37℃ environment until OD600 up to 0.2 , which we set as 0 hour. Then, E.coli was transferred into 32℃ 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 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.
Figure 20.Transfer to different temperature at different timing. We inoculated our E.coli in the 37℃ environment until OD 600 up to 0.2 , which we set as 0 hour. Then, E.coli was transferred into 42℃ 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℃ 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 because of low expression of kivD enzyme in the 42℃ environment.
Carbon source optimization
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 fed our host with glycerol to see whether it is possible to be our E.coli‘s ingredient or not.
Figure 21.Culture our DH5α host cell in the medium of 3.6% glucose or 5% glycerol for three days in 30℃,37℃,and42℃. Test the sample by GC every 24 hours.
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.
Figure 22.After activating our host in the medium overnight, we transferred it to new medium, and continued culturing in 37℃. Until OD600 reached 0.2, we set this point as 0 hour, and cultured tubes in three different temperature. To analyze the production rate, we collected 1ml of the broth every 24 hours and measured the yield by GC.
Future works
Ingredient Production
Figure 23.Cell-surface display of Cex by means of PgsA anchor protein
Figure 24.
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.
Figure 25.Isobutanol production through consolidated bioprocessing(CBP). There’re 2 types of E.coli in the reactor. One contains Cex-PgsA, which degrades hemicellulose and produces xylose. Another contains BBa_K887002, which turns xylose to isobutanol.
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
Figure 26.We hope to make an automatic control instrument in the future.
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.
Figure 27.The primary thought about our project on an industrial scale.
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.
