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

How to integrate the two related pathways both straw degradation and the synthesis of biofuel? After reading many literature, especially the work of Dodd et al., (2009) Maeda et al., (2018) and Wen et al., (2016), we separately designed three module plasmids. Among them, we have created a bifunctional enzyme system(Fig.1), which can efficiently degrade straw. The other two modules completed the efficient utilization of straw products.This could endow our system with multiple functions by sequential combination of these three modules. In the end, our system could degrade straw successfully and transform the cellulose into valuable biofuel.

There are three modules in our systems, including straw degradation, hydrogen production and butanol production(Fig.2). To get a good expression of these genes in E. coli, we did codon optimization before DNA synthesis.

Complex structure of straw is the most difficult problem of straw utilization nowadays. The cellulose in straw is mainly used for the production of clean energy. Cellulose in straw is wrapped by hemicellulose, and interspersed lignin. Lignin is linked to hemicellulose and lignin by ferulate ester bond, which makes straw structure compact and difficult to decompose. Lignin's unique structure can be nonreactive binding with cellulase(Fig.3), thus reducing cellulase activity. Therefore, it is often necessary to pretreat the straw to separate the lignin. The most common pretreatment methods of today are physical method, chemical method and biological method. The physical method is not suitable for industrial production because of its complication and high cost. Chemical methods often cause environmental pollution due to the heavy use of acid and alkali. However, biological method has been unable to treat straw directly because of its low enzyme activity. Through reading literature, we learnt that ferulic acid esterase, xylanase and beta-glucosidase have synergistic effect. This greatly accelerated the rate of straw degradation. Thus, straw pretreatment can be avoided.

The enzymes Xyn10D-fae1A, Xyl3A, Cex and CenA are the key enzymes to degrade straw into glucose and other products(Fig.4a). Xyn10D-fae1A is the bifunctional enzyme, which has the activity of ferulic acid esterase and xylanase. Ferulic acid esterase can break the ferulic acid ester bond, which is responsible for attaching in complex cellular cell wall structures. Xylanase can hydrolyze plant cell wall component xylan. It can cut the β-1,4 glycosidic bond between the xylose residues in the xylan backbone[1]. From BioBrick, we found endoglucanase CenA, (BBa_K118023)and exoglucanase Cex, (BBa_K118022). Cellulose will be degraded into glucose with the help of Xyl3A, CenA and Cex[2]. Besides, to achieve extracellular expression, an extra signal peptide pelB together with five aspartate repeats(Fig.4b) were adopted to facilitate the extracellular expression of the four enzymes in E. coli (Kim et al., 2015)[3].

There is a butanol synthesis pathway in native Clostridium, in order to meet the needs of industrial production, we hope to achieve continuous production of butanol 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[4], which mainly uses six genes to construct a biosynthetic pathway from glucose to butanol(Fig.5a).

Meanwhile, FRE is the transcriptional and translational regulatory element upstream of these mixed acids fermentation genes, including the promoter, ribosome binding site (RBS) and the entire transcription factor (TF) binding site. Wen et al. have proved that using its original regulatory element of ackA and adhE (FREadhE, FREackA) has a good effect on butanol production, without adding revulsant, antibiotics and under anaerobic fermentation conditions[5]. 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 the production process. Thus, we will import GroESL gene from Clostridium acetobutylicum to allow E. coli expresses heat shock protein groESL chaperone, which has proved it could improve the yield of butanol[6](Fig.6).

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.7a), via an enzyme complex called formate hydrogenlyase (FHL).

On this basis, we decided to enhance the hydrogen production capacity of E. coli by overexpressing FhlA, an activating protein gene of the FHL system[7]. Meanwhile, we think about the hydrogenase in FHL system and find that it belongs to [Ni-Fe] hydrogenase with low efficiency. In green algae and some bacteria, there is another hydrogenase - [Fe] hydrogenase, whose catalytic center has a unique non-prion H cluster, so it is 100 times more active than other hydrogenases. Therefore, we decided to introduce the [Fe] hydrogenase gene Hyda of Enterobacter cloacae IIT-BT 08 into E. coli to further increase hydrogen production[8](Fig.7b).

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[9].

[1]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.

[2]Lakhundi, S. S. (2012). Synthetic biology approach to cellulose degradation.

[3]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.

[4] 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.

[5]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.

[6]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.

[7]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.

[8]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.

[9]Gerdes, K., et al., 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 the E. coli relB operon. Embo j, 1986. 5(8): p. 2023-9.