Our team through experiments successfully constructed three kinds of cellulase fusion expression of S. cerevisiae strains, through error-prone PCR mutation breeding and purification isolated the high enzyme live xylan ase, and the promoter of S. cerevisiae xylose utilization optimization, various mutual combination, realize our expectations to improve the efficiency of straw degradation.

1 Part 1

1.1 Design and construction of the fusion genes

In this study, three cellulases from different sources, having similar reaction conditions, BG, CBH and EG were selected as shown in the Table 1-1. As predicted by the SignalP 4.1 tool (http://www.cbs.dtu.dk/services/SignalP/), two clear signal peptide cleavage sites are present at G15 of exoglucanase and A16 of endoglucanase; but no signal peptide in β-glucosidase. According to the analysis of the Carbohydrate Active Enzyme database (http://www.cazy.org/), the three cellulases are derived from three different glycoside hydrolase families: BG from the GH 1 family, CBH from the GH 7 family, and EG from the GH 12 family. According to the analysis of the Conserved Domain database (https://www.ncbi.nlm.nih.gov/cdd), none of the three cellulases has CBD regions.

In conclusion, the three selected enzymes have different sources, different classification, simple structure and compatible reaction conditions, which accord with the idea of the fusion strategy.

Table 1-1 Information tables of the three cellulases

The reverse coding sequence of the flexible adaptor (G4S) 3 (sequence GGGGSGGGGSGGGGS) was added to the reverse primers BG-L-Xba I-R and CBH-L-EcoR I-R for CBG and CBH. Two segments (G4S) 3 serve as the domain CD of the three cellulases. Meanwhile, appropriate restriction enzyme sites were added at the end of the primers to facilitate the fusion DNA with the cloned gene.The specific operation process is shown in Figure 1-1.

Figure 1-1 Flow chart of the fusion gene bce construction

PCR reactions were performed using primers BG-SnaB I-F and EG-Eco81 I-R using the enzyme ligation mixture as a template, and the results are shown in Figures 1-1. The PCR reaction mainly produces three bands, among which the main band with the highest electrophoresis brightness is about 3.0 kb, namely the fusion gene bce, while the band of about 2.4 kb is caused by the byproduct produced in the enzyme reaction as the template of the PCR reaction.

Figure 1-2 PCR-amplified bce electropherograms of the fusion gene

The resulting fusion gene bce from PCR was purified and digested with restriction enzymes SnaB I and Eco81 I and subjected to gel electrophoresis to recover the DNA fragments. The vector pHBM368-pgk-bg was also subjected to the same treatment. After ligation of the two recovered reactions, E. coli XL10-Gold competent cells were transformed. Single colonies were picked and inoculated in LB liquid medium containing ampicillin, and plasmids were extracted as described in Figures 1-3-a.

The restriction enzyme EcoR I was selected for enzyme digestion. The vector pHBM368-pgk and the fusion gene bce both contain a EcoR I restriction site, and the size of the two fragments is predicted to be 8.2 kb and 2.4 kb after the recombinant restriction enzyme, respectively. Transformants with slower electrophoretic rates than the control pHBM368-pgk-bg were picked, digested with EcoR I, and then examined by electrophoresis. The electrophoresis results are shown in Figures 1-3-b.

Figure 1-3 Verification plot of the fusion gene expression vector bce S. cerevisiae

Plasmids that met the predicted band size were selected for sequencing. The electrophoresis rate of plasmid 1 was slower than the control, and the size of EcoR I digestion met the prediction and the sequencing results met the design requirements. The recombinant vector was named pHBM368-pgk-bce. The schematic diagram of the vector is shown in Figure 1-4.

Figure 1-4 Schematic representation of the expression vector of the fusion gene bce Saccharomyces cerevisiae

1.2 Expression and identification of the fusion gene bce in S. cerevisiae

Saccharomyces cerevisiae INVSc1 competent cells were prepared and the expression vector pHBM368-pgk-bce was digested with the restriction enzyme HpaI and recovered. The fusion expression vector was then introduced into competent cells using electroconversion. SC media without uracil was used for screening. The Saccharomyces cerevisiae primitive INVSc1 is of the uracil-deficient type, while the expression vector contains the uracil screening marker URA 3. The recombinant strain could grow on SC medium, indicating that the genome of the strain was integrated with the recombinant vector.Single colonies of S. cerevisiae that could grow normally on SC solid medium were picked, activated on YPD solid medium, inoculated onto YPC-phenol blue solid medium and grown for 28℃ for approximately 4 days. The screening results of some recombinant strains are shown in Figure 1-5.

Figure 1-5 Plate screening plots of recombinant Saccharomyces cerevisiae strains

The original bacteria INVSc1 grew very poorly on YPC-Taiwan phenol blue solid medium with CMC-Na as the only carbon source, while the recombinant strains grew normally, indicating that the recombinant bacteria have cellulase (endoglucanase) activity. Because tryphenol blue can bind macromolecular polysaccharides and cannot bind small molecular polysaccharides, if the recombinant strain decomposes cellulose to produce small molecules of sugars, hydrolysis circle will appear around the colony. A clear hydrolysis circle can be seen around the colonies in the figure, indicating that these recombinant strains achieved the secreted expression of cellulase.

PCR was verified using total DNA from strains 7, the most prominent hydrolyzed ed, as template using primers BG-SnaB I-F and EG-Eco81 I-R, and the results are shown in Figures 1-6. Among them, the total DNA of strain 7 as the template and the predicted 3.0 kb band was observed in the products of PCR of the recombinant plasmid pHBM368-pgk-bce as the template. And not in the products of PCR using S. cerevisiae primitive INVSc1 total DNA as template. Therefore, strain 7 was selected as the S. cerevisiae expressing strain with single-gene multifunctional cellulase BCE and named INVSc1-BCE.

Figure 1-6 PCR validation plots of recombinant S. cerevisiae

1.3 Analysis of cellulase activity in recombinant strains of S. cerevisiae

Three activities of cellulase-specific substrate enzymes and total cellulase activity were determined, which were analyzed and compared.

Figure 1-7 Comparison of three cellulase-specific substrate enzyme activities and total cellulase activity

Figure 1-7 shows that the β -glucosidase activity of BCE reached 1144 U / L and the exoglucanase activity of BCE reached 950U / L, which was lower than the overall filter paper enzyme activity with an enzyme activity of 1278U / L. However, the endoglucanase activity of BCE was much higher than the overall filter paper enzyme activity, up to 4978U / L. The comparative results show that incorporating the expression of the CD region of the three cellulases can yield monopeptide proteins possessing the three cellulase activities. Moreover, the overall filter paper enzyme activity of fusion expression was higher than the specific substrates of two of the enzymes, suggesting a synergistic effect of the three enzymes.

2 Part 2

2.1 Mutation-optimized target genes

Mutation of the xylanase gene fragment using error-prone PCR results in Figure 2-1.

Figure 2-1 Validation plot of the mutated genes

The target band size was 717 bp. They appears in part of conditions. The target bands of 56,58, and 60℃ with 0.5% and 1.0% mutation rate were recovered, integrated with the vector, and often transferred into BL21 receptor. The substrate plate was continued to screen for strains producing high enzyme live xylanase.The target fragments of the obtained strains were tested in Figure Figure 2-2.

Figure 2-2 Validation of the target genes after the optimization

After the selected strains, the xylanase was isolated and purified, and the activity was up to 25980U / L.

3 Part 3

3.1 Construction of the recombinant S. cerevisiae expressing xylA

Five strong constitutive promoters were used to control the transcription of xylA. The recombinant INVSc1 bearing xylA driven by different promoters were named as INVSc1/pHM368-PPGK-xylA, INVSc1/pHM368-PADH1-xylA, INVSc1/pHM368-PGAPDH-xylA, INVSc1/pHM368-PPDC1-xylA and INVSc1/pHM368-PTEF1-xylA, respectively. The XI activities in engineered S. cerevisiae strains were evaluated. As shown in Figure 3-1, the XI activity of INVSc1/pHM368-PADH1-xylA reached 0.179 U at 48 h, which was 2.7-, 3.1- and 2.4-fold higher than INVSc1/pHM368-PPDC1-xylA (0.066 U), INVSc1/pHM368-PPGK-xylA (0.058 U) and INVSc1/pHM368-PTEF1-xylA (0.074 U), respectively. No XI activity was detected in INVSc1/pHM368-PGAPDH-xylA. XI activity of the engineered strains improved by promoter optimization strategy. According to this result, ADH1 promoter was the most efficient one for the expression of XI among these five promoters.

Figure 3-1 XI activity of S. cerevisiae strains INVSc1/pHM368-PADH1-xylA, INVSc1/pHM368-PGAPDH-xylA, INVSc1/pHM368-PPDC1-xylA, INVSc1/pHM368-PPGK-xylA and INVSc1/pHM368-PTEF1-xylA containing different promoters. Error bars indicate the average deviation.