Team:UESTC-China/Demonstrate

team

Demonstrate

Pathway construction

For efficient expression of multiple enzymes in E. coli, codon optimization of all target genes was performed before DNA synthesis. The obtained genes were subsequently cloned into different expression vectors by using Gibson Assembly and Golden Gate strategies. The resulting vectors piGEM2018-Module001, piGEM2018-Module002, and piGEM2018-Module003 are listed in Table 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 the desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful. (Fig. 1)

Fig.1 Double restriction enzyme digestion of three constructed vectors analyzed by using agarose gel electrophoresis.

(a) piGEM2018-Module001 digested by EcoR32Ⅰ+NcoⅠ(lane 1), piGEM2018-Module001 digested by NcoIⅠ+XhoⅠ(lane 2);

(b) piGEM2018-Module002 digested by PstⅠ+KpnⅠ(lane 1), piGEM2018-Module001 digested by HindⅢ+KpnⅠ(lane 2);

(c) piGEM2018-Module003 digested by EcoRⅠ+NcoⅠ(lane 1), piGEM2018-Module001 digested by BamHⅠ+BglⅡ(lane 2);

From straw to glucose

E. coli BL21(DE3) carrying piGEM2018-Module001 was used to degrade straw to glucose. Three extracellular expressed enzymes ferulic acid esterase, xylanase and cellulase are involved in the pathway. The expression of the three target protein were verified by enzyme activity using fermentation liquid and enzyme crude extract, respectively (Fig. 3). The results showed that the activities of all three enzymes in fermentation liquid fraction are too low to be detected, which could be due to the fact of low enzyme concentration. We also determined the enzyme activity using enzyme crude extract. The enzyme activity in fraction obtained from the strain carrying piGEM2018-Module001 was higher than that without the corresponding vector for all enzymes.

Fig.2 Enzyme activity detection using crude extract fraction of strain with or without. Module001. All sample were collected after fermentation for 24 h.

(a) The activity of xylanase. U definition:

(b)The activity of Ferulic Acid Esterase.

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

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

In order to further evaluate the extracellular expression of ferulic acid esterase, xylanase and cellulose, the formation of the corresponding intermediates were determined. GC-MS was used to detect ferulic acid. The ferulic acid production was monitored by periodically taking samples from the fermentation liquid of E. coli BL21(DE3) carrying piGEM2018-Module001. Carvacrol was used as an internal standard for GC measurements [5]. GC results showed that the peak of ferulic acid was appeared after fermentation for 24h with the strain carrying piGEM2018-Module001, while no ferulic acid was detected in the sample of E. coli BL21(DE3) without vector (Fig. 3). The ferulic acid peak was further analyzed by using GC-MS (Fig. 4).

Fig.3 The production of ferulic acid determined using GC.The supernatants of fermented liquid of E. coli BL21(DE3) with or without piGEM2018-Module001were used for the determination of ferulic acid, respectively. Samples were taken from reaction mixtureat 0h, 24h.

Fig.4 Mass spectrums of ferulic acid derivative. Sample was the supernatants of E. coli BL21(DE3) carrying piGEM2018-Module001after fermentation for 24h.

The activity of cellulase was determined by using Congo Red assay [4]. As shown in figure 6, the strains carrying piGEM2018-Module001 displayed a zone of clearance. Such a zone of clearance was also observed in the positive control carried out by using commercial cellulose, while no zone of clearance was observed with same E. coli strain carrying empty vector. The results indicated that cellulose was successfully extracellularly expressed in E. coli BL21(DE3) with piGEM2018-Module001.

Fig.5 Activity determination of cellulase using Congo Red assay. (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: commercial cellulose (Enzyme concentration: 0.2, 0.3, 0.4 mg/ml from left to right); Negative Control: BL21 (DE3) carrying empty vector (OD600: 1, 3, 5 from left to right)
Butanol production

E. coli BL21(DE3) carrying piGEM2018-Module002 was used to convert glucose to butanol. Five enzymes AtoB, Hbd, crt, ter and adhE2 are involved in the pathway, which was validated by monitoring the production of butanol using GC. 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.

The butanol production was determined by periodically taking samples from the supernatant of fermented liquid of BL21(DE3). Isobutanol was used as an internal standard [5]. The results showed that the peak of butanol was appeared after fermentation for 24h with the strain carrying piGEM2018-Module002, while no butanol peak was observed for the negative control (E. coli BL23(DE3) with empty vector(Fig.6).

Fig.6 The production of butanol determined using GC. The supernatants of fermented liquid of E. coli BL21(DE3) with or without piGEM2018-Module002 were used for the determination of ferulic acid, respectively. Samples were taken from reaction mixture at 0h, 24h.

To improve butanol production, the fermentation conditions were optimized by using orthogonal array design by designing 17 three factor analysis. 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 24h under the optimized conditions. (Fig. 7)And the final maximum yield of butanol is 0.37 g / L in 24 hours.

Fig.7 The butanol production curve at optimized conditions (temperature=34℃, initial OD=13, Initial pH=8)

Hydrogen production

To verify whether the piGEM-Module003 plasmid was working normally in E. coli DH5α, we set up an anaerobic fermentation unit. The produced gases flow through the following setup to remove carbon dioxide as much as possible to get hydrogen.

Fig.8 Picture of hydrogen-collecting device.

The collected gas is quantitatively detected by combustion test. As seen from Video 1, one can clearly hear the sound of boom, while no similar phenomenon was observed for the control group

Video 1. The video of gas combustion test. Samples were collected from the engineered E.coli DH5α carrying the plasmid piGEM2018-Module003 or wild type plasmid after shaking 48h in TB medium

Next, we used gas collection devices to quantitatively detect hydrogen production. The recombinant strain and the original DH5a strain were fermented continuously for 36 hours in a 200 ml 1% (w/v) glucose M9 medium.

Fig.9 Hydrogen is collected from the recombinant bacteria and the original bacteria in the gas collection device.

Apart from the experiments, here we also carried out some theoretical modeling to provide some guidance to optimize the experimental condition. As demonstrated as following, 1mol glucose can produce 2 mole hydrogen from the theoretical point of view [6]. Next, we measured the amount of hydrogen and formation rate following the 40hr anaerobic fermentation. The results showed that under the condition of 30°C, 1.13% (W/V), ph=6.8, our recombinant strain has the highest hydrogen production. Based on this, we compare the hydrogen production between us and the literature. The results are as follows.

Method	Yield	Reference
Our project	1.72 mol∙(mol glucose)-1
Knockout hyca and Overexpression fhla	0.95 mol∙(mol glucose)-1	Yoshida et al. 2006[7]
Knockout iscR+MCS2 and Overexpression YdbK+CpFdx+hydA+hydF+hydG+hydE	1.46 mol∙(mol glucose)-1	Akhtar and Jones 2009[8]
Overexpression Fe-hydrogenase coded gene from E. cloacae IIT-BT-08	3.12 mol∙(mol glucose)-1	Chittibabu et al. 2006[9]

Table 3, Comparison between our result and other references []
【1】Clark, D. P. (1989). The fermentation pathways of escherichia coli. Fems Microbiology Reviews, 63(3), 223-234.
【2】Yoshida A, Nishimura T, Kawaguchi H, Inui M, Yukawa H. 2006a. Enhanced hydrogen production from glucose using ldh- and frd-inactivated Escherichia coli strains. Appl. Microbiol. Biotechnol. 73: 67-72
【3】Akhtar MK, Jones PR. 2009. Construction of a synthetic YdbK-dependent pyruvate:H2 pathway in Escherichia coli BL21(DE3) Metab. Eng. 11: 139-147
【4】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.

Validation of integrate pathway

What we aim at is combing straw degradation with butanol production. Therefore, after Co-transformation piGEM2018-Module001 with piGEM2018-Module003, we get our super E. coli and cultivate it with straw and then transform it into anaerobic conditions. Using GC-MS to detect our production. We successfully detect the ferulic acid derivative and butanol (Fig. 11).

Fig.11 Mass spectrums of ferulic acid derivative and butanol. Sample was transgenic E. coli carrying piGEM2018-Module001 and piGEM2018-Module002, then taken from reaction mixture after 72h. 24h in aerobic condition and 28h in anaerobic condition.
Work going on

1. 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 [10]. Fig. 12 shows that GroESL have a good effect on butanol tolerance.

Fig.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.13). If we could knock out these competitive pathway, then our butanol production will be greatly improved.

Fig.13 E. coli self-metabolic pathway
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] Qiu-Yi, L. I., Gan, G. P., Wang, G. Z., & Liu, Y. W. (2007). Determination of ferulic acid in ligusticum chuanxiong hort. oil by gc. Lishizhen Medicine & Materia Medica Research.

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

[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.

[6] Clark, D. P. (1989). The fermentation pathways of escherichia coli. Fems Microbiology Reviews, 63(3), 223-234.

[7] Yoshida A, Nishimura T, Kawaguchi H, Inui M, Yukawa H. 2006a. Enhanced hydrogen production from glucose using ldh- and frd-inactivated Escherichia coli strains. Appl. Microbiol. Biotechnol. 73: 67-72

[8] Akhtar MK, Jones PR. 2009. Construction of a synthetic YdbK-dependent pyruvate:H2 pathway in Escherichia coli BL21(DE3) Metab. Eng. 11: 139-147

[9] 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.

[10] Abdelaal, A. S., Ageez, A. M., El, A. E. H. A. A., & Abdallah, N. A. (2015). Genetic improvement of n-butanol tolerance in Escherichia coli by heterologous overexpression of groESL operon from Clostridium acetobutylicum. 3 Biotech, 5(4), 401-410.