Animal feed mainly includes silage feed and cereal feed. On the one hand, silage contains a lot of cellulose, which is usually combined with hemicellulose, pectin, and lignin, making it difficult to digest and absorb, and ruminants can decompose it into monosaccharides and disaccharides through revere vomiting. Therefore, cellulase, such as Pseudomonas aeruginosa strain PKC-001, can be added to silage, thereby improving the ability of ruminants to obtain monosaccharides. Xylanase and β-xylosidase are skeleton degrading enzymes of heterogeneous xylan. They can not only degrade the main chain of xylan to produce xylooligosaccharides or xylose with different degrees of polymerization but also play an important role in the degradation of hemicellulose. Strains secreting xylanase can generally produce β-xylosidase and work in combination with xylanase. More and more studies have shown that β-xylosidase plays an important role in the degradation of lignocellulose. During the hydrolysis of lignocellulose, β-xylosidase further hydrolyzes the hydrolysate of xylanase into xylose. It not only plays a key role in the complete degradation of xylan but also alleviates the inhibitory effect of xylooligosaccharides on xylanase and cellulase. Therefore, β-xylosidase has been considered one of the core enzymes in cellulase preparations.
Cereal energy feed has high potential nutritional value as feed for poultry. However, the high content of soluble non-starch polysaccharides hinders the utilization and absorption of poultry. Xylan is the main form of non-starch polysaccharides in plant feed, especially in cereals. The mutual entanglement of covalent bonds and non-covalent bonds between xylans makes it extremely sticky, and not easy to be digested by monogastric animals. In addition, it also hinders the contact between substrates and digestive enzymes and slows down the absorption and utilization of nutrients by the intestinal wall. Silage is a kind of natural plant feed, mainly used for feeding ruminants (such as cattle, sheep, alpacas, and deer, etc.), is an excellent source of livestock feed. Feed usually contains substantial cellulose, hemicellulose, lignin, and pectin, which are difficult for livestock to digest.
Multi-enzyme synergistic degradation is a method that can solve this issue. This tactic utilizes multiple enzymes to corporate and digests the nutrients in the feed, as shown in the following flowchart:

Xylanase has the characteristics of hydrolyzing hemicellulose and can cooperate with cellulase to promote the bioconversion of lignocellulose. To improve the utilization rate of silage and cereal mixed feed and the applicable varieties of livestock, we further added the xylanase xynA gene of Bacillus subtilis into the cloning recombinant vector, it could be expressed with cellulase PKC-01 and β-xylosidase at the same time.

This experiment aimed to increase the efficiency with which nutrition is absorbed from the feed using a multi-enzyme synergistic degradation strategy with 4 types of enzymes. The final product of the experiment is in the form of a dry powder additive or E.coli tablet.

The four enzymes used are:

To turn the desired genes into proteins, vectors play an important role. pET28a is a plasmid that contains the four target genes, and is the final plasmid from which our target proteins were synthesized. Therefore, inserting the gene sequences of the four enzymes was a major part of our experiment, as pET28a was originally an empty vector. Depending on the genes transferred onto pET28a, its suffix will also change. For example, after we put the gene encoding PKC onto pET28a, the resulting plasmid could be referred to as pET28a-PKC. The diagram below shows the structure of pET28a.

Casponson is a new archaeal and bacterial mobile element superfamily, which not only has the advantages of general transposons: including terminal reverse repeats and B-group DNA polymerase genes but also contains CASL endonuclease in this transposon, which is an enzyme that can integrate elements into the host genome. With this enzyme, genes can be integrated, and CASL can be used for integration and excision. This new casposase is the first self-synthesized transposon family found in prokaryotes. The casposase we used gave rise to the adaptation module of CRISPR-Cas systems.

During our experiments, we relied on two strains of bacteria: TOP10 competent cells and BL21(DE3). These are both competent cells[3], which are cells specifically engineered for easier transformation. During our experiment, we used thermal expansion to enlarge pores on the cell surface, allowing target plasmids to pass through and transform the cells. TOP10 competent cells are suitable for efficient DNA cloning and plasmid amplification, ensuring stable inheritance of plasmids. BL21(DE3) competent cells are suitable for the expression of non-toxic heterologous genes. This strain contains a λ DE3 pyrophosphate carrying the T7 RNA polymerase gene controlled by the lacUV5 promoter, enabling IPTG-induced expression of T7 RNA polymerase. BL21(DE3) is a strain of E. coli B that does not contain lon an protease. It also lacks the outer membrane protease OmpT. The absence of these two key proteases reduces the degradation of heterologous proteins expressed in cells.

Our experiment can be divided into three phases: construction of target plasmids, expression of genes, and purification of proteins.
First of all, we used PCR to increase the amount of the xyl3A gene fragment from its original plasmid. Then, restriction enzymes Xbal and BamHI were used to cut PKC and pET28a at the target sites. Restriction enzymes Nhel and HindIII were used to cut XynA and another sample of pET28a plasmid. Then, T4 DNA ligase was used to connect PKC, XynA, and xyl3A to their corresponding pET28a samples. Since the gene ccxynA was manufactured to be on pET28a, we didn’t need to use restriction enzymes or ligase. After target gene fragments were successfully connected to pET28a, we used gel electrophoresis to isolate the desired genes, and then retrieved them.
The four plasmids (pET28a-PKC, pET28a-xynA, pET28a-xyl3A, and pET28a-ccxynA) were then transformed into competent cells TOP10 amplify the sample. As the number of TOP10 cells increased, the target genes also replicated. Then, the cells were inoculated into a Luria Broth culture media with an antibiotic called Kanamycin added. This tests whether the plasmids within them successfully closed to form complete plasmids after being cut with restriction enzymes. Since a gene sequence on the pET28a plasmid can resist Kanamycin, cells with complete pET28a plasmids would survive on the cultural media.
The recombination plasmids were extracted from TOP10 and transformed into BL21(DE3). Again, the BL21 cells grew on an LB culture media that contained Kanamycin. after filtration, we added isopropylthio-β-galactoside (IPTG) into the cells, allowing BL21(DE3) to start expressing the target genes. Then, over twelve hours, the proteins accumulated within the BL21(DE3) cells. Afterward, we extracted the proteins using Ni-NTA beads and filtration.
Lastly, we used a process called sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE ) to test whether the protein we extracted was correct. We also used an enzyme biopsy test to further test the proteins, observing the change in color to determine whether the protein we produced was functional.

3.1 Successfully construct plasmid SIP403-PUS-xynA-xyl3A.
3.2 Successful expression of proteins (PKC, xynA, CcxynA, xyl3A).
3.3 Multi-enzyme synergistic degradation effect is obvious.

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