Team:NCTU Formosa/Project-sub5

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 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, 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.

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 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

Figure 26.We hope to make an automatic control instrument in the future.

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.

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 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