Difference between revisions of "Team:UESTC-China/Design"

How to integrate the two related pathways which is involved in the straw degradation and the synthesis of butanol and hydrogen? Accroding to some references, we separately designed three module vectors in the present work. The first module piGEM2018-Module001 is designed to achieve the transform from straw to glucose, and it performed the function of bifunctional enzyme. The other two modules piGEM2018-Module002 and piGEM2018-Module003 were designed to produce two types of clean energy, butanol and hydrogen from glucose (Fig.1). After we checked if these three modules could work independently, we aimed to combine them together to achieve efficient conversion of straw to butanol and hydrogen in the near future.

There are three modules synthesized in our energy conversion systems, including straw degradation, butanol production and hydrogen production (Fig. 2 ). Prior to the DNA synthesis, we carried out the codon optimization for the purpose to gain better expression for the target genes in E. coli.

As shown in figure 2, the structure of straw is compact and is difficult to be decomposed, because cellulose is wrapped by hemicellulose, and is interspersed with lignin via ferulate ester bond (Fig. 3 ). It has been found that the presence of lignin would give rise to the reduced cellulase activity. Therefore, it is necessary and significant to pretreat the straw to make it separate with the lignin. The commonly used methods included physical method, chemical method and biological method. The physical method is not suitable for the industrial production because of its complication and high cost. Chemical methods often cause environmental pollution due to the usage of large amount of acid or alkali. Biological method has been unable to treat straw directly because of its low enzyme activity with the presence of lignin. Nevertheless, we know from some literatures that ferulic acid esterase, xylanase and beta-glucosidase have synergistic effect, which greatly accelerated the degradation rate of straw[1].

The enzymes Xyn10D-fae1A, Xyl3A, Cex and CenA are the key enzymes to degrade straw into glucose and other products shown in Fig. 4a. Xyn10D-fae1A is the bifunctional enzyme, which has the activity of both ferulic acid esterase and xylanase. Ferulic acid esterase (abbreviated as fae1A) can break the ferulic acid ester bond, which is responsible for attaching the complex cellular cell wall structures. Xylanase can hydrolyze xylan existing in plant cell wall, in which it can cut the β-1,4 glycosidic bond between the xylose residues in the xylan backbone [2]. From BioBrick, we found the endoglucanase CenA, (BBa_K118023) and exoglucanase Cex, (BBa_K118022). Cellulose will be degraded into glucose with the help of Xyl3A, CenA and Cex [3]. Besides, an extra signal peptide pelB followed by five aspartate repeats (peLB+5D seen in Fig. 4b) were introduced to facilitate the extracellular expression of the four enzymes inE. coli[4]

In order to meet the needs of industrial production of butanol, a butanol synthesis pathway existed in native Clostridium was rebuilt in E. coli. According to literature which studies the screening of the same enzymes of different species, we finally tried to assemble a relatively superior combination [5], which mainly uses six genes to construct a biosynthetic pathway from glucose to butanol (Fig.5a).

FRE, is a transcriptional and translational regulatory element that has been adopted by Wen et al. to enhance the production of butanol inE. coliwithout adding revulsant, antibiotics and under anaerobic fermentation conditions [6]. In order to improve They proved that to enhance the expression of ackA and adhE. The FRE element contains, Therefore, We assemble these parts together and construct our plasmid of butanol production(Fig. 5b).

The accumulation of butanol exerts a greatly inhibitory effect on cell growth during butanol production process. Thus, we will import GroESL gene from Clostridium acetobutylicum to allowE. coliexpresses heat shock protein groESL chaperone, which has shown the function for improving the yield of butanol by [7] (Fig. 5c).

E. coli is a microorganism capable of producing hydrogen under anaerobic conditions, which can convert the methyl formate to hydrogen gas and carbon dioxide (Fig. 6a) via an enzyme complex called formate hydrogenlyase (FHL) [8]. It has been reported that FhlA, an activating protein of FHL, could enhance the production capacity of hydrogen [9]. Additionally, structural analysis showed that FHL from E.coli belongs to [Ni-Fe] hydrogenase family. Its enzymatic activity is about 100 times lower than another hydrogenase from green algae and some bacteria. Therefore we introduced the gene encoding the hydrogenase from Enterobacter cloacae IIT-BT 08 into E. coli to further increase hydrogen production [10](Fig. 6b).

We plan to apply super E. coli to real life in the future, and for this purpose we should take full account of biosafety and maneuverability.
Like the design of TU_Darmstadt in 2014 iGEM project, we transferred hokD gene into our cells [11].

[1] Liu Pan. Study on the degradation of corn stover by biological method and the heat resistance modification of ferulic acid esterase [D]. Qilu University of Technology, 2016.
[2]Dodd, D., Kocherginskaya, S. A., Spies, M. A., Beery, K. E., Abbas, C. A., Mackie, R. I., & Cann, I. K. 2009. Biochemical analysis of a β-D-xylosidase and a bifunctional xylanase-ferulic acid esterase from a xylanolytic gene cluster in Prevotella ruminicola 23. Journal of bacteriology, 191(10), 3328-3338.
[3]Lakhundi, S. S. 2012. Synthetic biology approach to cellulose degradation.
[4]Kim, S. K., Park, Y. C., Lee, H. H., Jeon, S. T., Min, W. K., & Seo, J. H. 2015. Simple amino acid tags improve both expression and secretion of Candida antarctica lipase B in recombinant Escherichia coli. Biotechnology and bioengineering, 112(2), 346-355.
[5] Atsumi, S., Cann, A. F., Connor, M. R., Shen, C. R., Smith, K. M., Brynildsen, M. P., & Liao, J. C. 2008. Metabolic engineering of Escherichia coli for 1-butanol production. Metabolic engineering, 10(6), 305-311.
[6]Wen, R. C., & Shen, C. R. 2016. Self-regulated 1-butanol production in Escherichia coli based on the endogenous fermentative control. Biotechnology for biofuels, 9(1), 267.
[7]Zingaro, K. A., & Papoutsakis, E. T. 2013. GroESL overexpression imparts Escherichia coli tolerance to i-, n-, and 2-butanol, 1, 2, 4-butanetriol and ethanol with complex and unpredictable patterns. Metabolic engineering, 15, 196-205.
[8]Yoshida, A., Nishimura, T., Kawaguchi, H., Inui, M., & Yukawa, H. (2005). Enhanced hydrogen production from formic acid by formate hydrogen lyase-overexpressing escherichia coli strains. Appl Environ Microbiol, 71(11), 6762-8.
[9]Yoshida, A., Nishimura, T., Kawaguchi, H., Inui, M., & Yukawa, H. 2005. Enhanced hydrogen production from formic acid by formate hydrogen lyase-overexpressing Escherichia coli strains. Applied and Environmental Microbiology, 71(11), 6762-6768.
[10]Chittibabu, G., Nath, K., & Das, D. 2006. Feasibility studies on the fermentative hydrogen production by recombinant Escherichia coli BL-21. Process Biochemistry, 41(3), 682-688.
[11]Gerdes, K., et al. 1986. Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of theE. colirelB operon. Embo j, 1986. 5(8): p. 2023-9.