Team:UESTC-China/Demonstrate

team

Demonstrate

Pathway construction

In order to express multiple enzymes in E. coli, we firstly did the codon-optimized of the enzymes. And then these genes were commercially synthesized. Finally, with the application of Gibson Assembly and Golden Gate strategy, we successfully introduced the target gene into different E. coli expression vector. (Fig. 1)

No.	Vector	E. coli resistance	Description
1	piGEM2018-Module001	Amp	BBa_J23100-RBS-pelB+5D-Xyn10D-fae1A-RBS-pelB+5D-Xyl3A-RBS-pelB+5D-cex-RBS-pelB+5D-cenA-Ter
2	piGEM2018-Module002	Kan	Ter-Ter-RBS-Fdh-RBS-FRE_adhE-FRE_ackA-RBS-AtoB-RBS-AdhE2-RBS-Crt-RBS-Hbd-Ter
3	piGEM2018-Module003	Kan	BBa_J23100-RBS-FhlA-RBS-HydA-

Table 1.The introduction of piGEM2018-Module001 to piGEM2018-Module003.

Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful. (Fig. 2)

Figure 2.The image of agarose gel electrophoresis by double enzyme digestion.

(a)piGEM2018-Module001 (Line1,enzyme digested by EcoR32Ⅰ+NcoⅠ; Line2,enzyme digested by NcoIⅠ+XhoⅠ)

(b) piGEM2018-Module002 (Line1,enzyme digested by PstⅠ+KpnⅠ; Line2,enzyme digested by HindⅢ+KpnⅠ)

(c)piGEM2018-Module003 (Line1,enzyme digested by EcoRⅠ+NcoⅠ；Line2, enzyme digested by BamHⅠ+BglⅡ)

Straw degradation and glucose production

In order to verify whether E. coli BL21(DE3) with piGEM2018-Module001 has taken effect. We detect enzyme activity separately. (Fig. 3) Comparing with wild type, our E. coli carrying piGEM2018-Moudlu001 take effect. BL21(DE3) with piGEM2018-Module001 has the activity of ferulic acid esterase, xylanase and cellulase.

Figure 3. Enzyme activity detection. Module001: broken bacteria of BL21(DE3) with piGEM-Module001. WT: Broken bacteria of wild type.

(a)The activity of xylanase. Using the changes of absorbance value under 540nm as an indicator of changes in xylose concentration.

(b)The activity of Ferulic Acid Esterase. Using the changes of absorbance value under 410nm as an indicator of changes in ferulic acid concentration.

(c) The activity of cellulase. Using filter paper assay method. [1]

(d) The activity of CenA. Using CMC assay method. [2]

Meanwhile, we also use GC-MS to detect ferulic acid. The ferulic acid production was monitored by periodically taking samples from the supernatant of E. coli carrying piGEM2018-Module001. The ferulic acid production was monitored using the method of gas chromatography by periodically taking samples from the supernatant of fermented liquid, and the internal standard is carvacrol [3]. Ferulic acid will decompose into 9 carbon compounds in the case of ferulic acid in gas chromatography with nitrogen as carrier gas [4]. Fig.4 shows that the peak of ferulic acid derivative increased within 20 hours while ferulic acid derivative was not found in sample of wild-type E. coli. Fig.5 demonstrate that our product is ferulic acid by GC-MS.

Figure 4. Chromatogram of Ferulic Acid derivatives and Internal Standard carvarol in supernatant of transgenic E. coli carrying piGEM2018-Module001 and wild-type E. coli. Samples were taken from reaction mixture at 0h, 24h.

Figure 5. Mass spectrums of ferulic acid derivative. Sample was transgenic E. coli carrying piGEM2018-Module001 and taken from reaction mixture at 24h.

To further find whether the cellulase had been expressed successfully in BL21 (DE3), the method of Congo Red assay was performed. The results are shown in Fig.6. The strains carrying piGEM2018-Module001 showed a zone of clearance created by hydrolysis of CMC showed that cellulase was successfully transcribed and translated by BL21(DE3). The empty vector control didn't show any zone of clearance around the colonies.

Figure 6. (a) CMC agar plate before staining with Congo Red. (b)CMC agar plate after staining with Congo Red. Module001: BL21(DE3) carrying piGEM2018-Module001（OD600=1, 3, 5 from left to right) Positive Control: Enzyme (concentration=0.2, 0.3, 0.4 mg/ml from left to right) Negative Control: BL21 (DE3) carrying empty vector control (OD600=1, 3, 5 from left to right)

Butanol production

Work validation of multi-enzyme system in BL21(DE3) carrying piGEM2018-Module002. AtoB, Hbd, crt, ter and adhE2 catalyzed six-step reactions converting glucose to butanol. To test whether this multi-enzyme conversion system could work in our E. coli successfully or not, we detected the time production curve of butanol of BL21(DE3) carrying piGEM2018-Module002 under the anaerobic condition.

Figure 7. Chromatogram of butanol and Internal iosbutanol of transgenic E. coli carrying piGEM2018-Module002 and wild-type E. coli. Samples were taken from reaction mixture at 0h, 24h.

Besides, after a serious of butanol-production detection under different cultivating condition, we finally provided the necessary data for modeling. Then detecting the butanol production in 48h under the optimized conditions. (Fig. 8)And the final maximum yield of butanol is 0.37 g / L in 24 hours.

Figure 8. The butanol production curve at optimized conditions (temperature=34℃, initial OD=13, Initial pH=8)

Furthermore, comparing with the production of butanol from other paper, our butanol production has reached a medium level. (Tab 1)

Source	butanol titer (g/L · h)	knocking other pathway
[7]	0.012 g/L · h	No
Our Design	0.018 g/L · h	No
[8]
[9]

Table 2, Comparing with relevant paper in butanol titer. All paper are using E. coli.

Hydrogen production

To verify whether the piGEM-Module003 plasmid was working normally in E. coli DH5α. We set up an anaerobic fermentation unit. Because E. coli produces only carbon dioxide and hydrogen under anaerobic conditions, the removal of carbon dioxide theoretically allows us to produce relatively pure hydrogen. Then we test the collecting gas.

Figure 9. Overall setup of the hydrogen collection device.

Then we test the collecting gas (Video 1). We can see from this video. The gas produced in turn a makes a sharp explosion when it meets the burning stick, while the gas produced in the wild makes the stick extinguish directly.

Video 1, The video of gas test. Sample were collected from DH5α with piGEM2018-Module003 and wild type after 48h, shaking with TB medium.

For quantitative assay, we measure hydrogen using gas chromatography under different cultivating condition to provide data for Modeling. Then detecting the butanol production in 48h under the optimized conditions. (Fig. 8)And the final maximum yield of hydrogen is xxx g / L in 48 hours.

Figure 10. Gas production of wild type DH5a and DH5a transfected into piGEM-Module 003 plasmid was measured in this gas collection unit for 36 hours.

The theoretical maximum yield of facultative anaerobic bacteria is 2 moles H2 per mole of glucose. Based on the optimum reaction conditions for the modeling guidelines, we tested the hydrogen content and rate of production in the 40-hour anaerobic fermentation gas. The end result is x moles of hydrogen per gram of glucose.

Table 3, Comparing with relevant paper in hydrogen production. All paper are using E. coli.
Validation of straw degradation and butanol production pathway

In the future, we will try to integrate the gene, which control butanol and hydrogen production. Therefore, our super E. coli could produce both butanol and hydrogen. [Fig. 11]

Figure 11. awdawdawdwadaw
Work going on
All gene in one plasmid

In the future, we will try to integrate the gene which control butanol and hydrogen production. Therefore, our super E. coli could produce both butanol and hydrogen.
Import resistance gene

During the production process, the accumulation of butanol will exert a great inhibitory effect on cell growth. Therefore, we will introduce the GroEL gene and GroES gene from clostridium acetone butanol to make E. coli heterologous expression of heat shock protein -- a molecular chaperone of GroESL, which has been shown to increase butanol production. Fig. 18 shows that GroESL have a good effect on butanol tolerance.

Figure 12. After adding 0.3ml butanol in 30ml LB medium, the measurement of optical density (A600) deference between DH5α with GroESL and Negative Control in 24 hour.
Knock out relevant gene

Due to limitations of experimental conditions, we are not able to knock out competitive pathway. However, many by-products will produce in the process of butanol production, namely frdABCD for succinate, ldhA for lactate, pta-ack for acetate, and adhE for ethanol (Fig. ). If we could knock out these competitive pathway, then our butanol production will be greatly improved.

Figure 13. After adding 0.3ml butanol in 30ml LB medium, the measurement of optical density (A600) deference between DH5α with GroESL and Negative Control in 24 hour.
References

[1] Silveira, M. H. L., Rau, M., da Silva Bon, E. P., & Andreaus, J. (2012). A simple and fast method for the determination of endo-and exo-cellulase activity in cellulase preparations using filter paper. Enzyme and microbial technology, 51(5), 280-285.

[2] Wood, T. M., & Bhat, K. M. (1988). Methods for measuring cellulase activities. In Methods in enzymology (Vol. 160, pp. 87-112). Academic Press.

[3] http://en.cnki.com.cn/Article_en/CJFDTOTAL-SZGY200707082.htm

[4]Fiddler, W., Parker, W. E., Wasserman, A. E., & Doerr, R. C. (1967). Thermal decomposition of ferulic acid. Journal of Agricultural and Food Chemistry, 15(5), 757-761.

[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 E. coli for 1-butanol production. Metabolic engineering, 10(6), 305-311.