Future works

　Cellulase experiment

In order to realize our idea of changing agricultural waste into biofuel, we figured that we could try to obtain glucose from cellulose by cellulase. This simple idea could be carried out by several procedures. First, we did a pre-experiment by using quantitative filter paper (Advantec) as cellulose source which was made with highest quality alpha cotton cellulose, and was ashless(<0.1%). We cut the filter paper into the size of 0.5 cm square. Next, we weighed and put 3 grams of the filter paper chips in a 50ml centrifuge tube filled up to 50ml with 0.1M Na-citrate (ph5.3). We then added 375mg cellulase, which came from Trichoderma reesei (Sigma-Aldrich) and put it in 40℃ incubation(200rpm) for 96 hours. After that, we could get milk-like filter paper mash. Finally, 3.6% of glucose was measured by doing DNS assay!

The next challenge would be use E. coli we modified to yield isobutanol with the glucose medium made by cellulose. We filtered and transferred the glucose medium to another flask, and added our M9T9 formula except of adding glucose into it. Then we inoculated E. coli in it for 24 to 42 hours, 42℃, 200rpm. The GC result of the isobutanol yield was 25ppm. Though it wasn’t prominent, it proved that the whole procedures (from cellulose degradation to isobutanol production) were practical. However, we could infer that underneath the toxicity tolerance, we still might acquire isobutanol as much as possible. So we must modify every procedure to enhance the yield of isobutanol. In the future, we will try rice stalks from a farm as cellulose source, hoping that the outcome will give everyone a big surprise!

　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 29, 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.

　Reference

James C Liao,Joachim Messing.(2012).Energy biotechnology Editorial overview. Current Opinion in Biotechnology, 23:287–289
Bao-Wei Wang, Ai-Qin Shi, Ran Tu, Xue-Li Zhang,Qin-Hong Wang and Feng-Wu Bai.(2011).Branched-Chain Higher Alcohols. Biochem Engin/Biotechnol,128: 101–118

Adrienne E Mckee ,et al.(2012).Manipulation of the Carbon Storage Regulator System for Metabolite Remodeling and Biofuel Production in Escherichia coli.Microbial Cell Factories,11:79 doi:10.1186/1475-2859-11-79

Cong T. Trinh.(2012).Elucidating and reprogramming Escherichia coli metabolisms for obligate anaerobic n-butanol and isobutanol production.Appl Microbiol Biotechnol,95:1083–1094 DOI 10.1007/s00253-012-4197-7

Erin Garza ,et al.(2012).Engineering a homobutanol fermentation pathway in Escherichia coli EG03.J Ind Microbiol Biotechnol ,39:1101–1107 DOI 10.1007/s10295-012-1151-8

Mark P Brynildsen and James C Liao.(2009).An integrated network approach identifies the isobutanol response network of Escherichia coli,Molecular Systems Biology,5:277

Machado, H.B., et al. (2012). A selection platform for carbon chain elongation using the CoA-dependent pathway to produce linear higher alcohols. Metab. Eng. (2012), http://dx.doi.org/10.1016/j.ymben.2012.07.002

Reyes, L.H., et al. (2012). Visualizing evolution in real time to determine the molecular mechanisms of n-butanol tolerance in Escherichia coli. Metab. Eng. (2012), http://dx.doi.org/10.1016/j.ymben.2012.05.002

Kevin M. Smith, James C. Liao.(2011).An evolutionary strategy for isobutanol production strain development in Escherichia coli. Metabolic Engineering,13:674–681

Bastian Blombach,and Bernhard J. Eikmanns.(2011).Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum,Bioengineered Bugs 2:6,1-5

Kevin Michael Smith,Kwang-Myung Cho,James C. Liao.(2010).Engineering Corynebacterium glutamicum for isobutanol production.Appl Microbiol Biotechnol,87:1045–1055

Shota Atsumi, et al.(2010).Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes.Appl Microbiol Biotechnol, 85:651–657

Shota Atsumi, et al.(2010)Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli.Molecular Systems Biology 6; Article number 449; doi:10.1038/msb.2010.98

Antonino Baez, Kwang-Myung Cho, James C. Liao.(2011).High-flux isobutanol production using engineered Escherichia coli: a bioreactor study with in situ product removal,Appl Microbiol Biotechnol, 90:1681–1690

Wendy Higashide,Yongchao Li,Yunfeng Yang,and James C. Liao.(2011).Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose,APPLIED AND ENVIRONMENTAL MICROBIOLOGY,p. 2727–2733

S.Atsumi, T. Hanai and J.C. Liao.(2008).Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels,Nature 451:86-90, doi:10.1038

Ekaterina A. Savrasova, Aleksander D. Kivero, Rustem S. Shakulov, Nataliya V. Stoynova.(2011).Use of the valine biosynthetic pathway to convert glucose into isobutanol,J Ind Microbiol Biotechnol,38:1287–1294