Background and Inspiration
Ruminants make up the majority of our daily meals, but what exactly do "ruminants" mean? Elegantly speaking, "ruminant" is the process of re-digesting the food they eat. This function is because they have more than one stomach, which make them capable of digesting the cellulose in the grass.
Ruminants have played an integral part in human history. Humans can barely digest cellulose from agricultural and by-products. Meanwhile, ruminants can solve these problems since they can break down these hard-to-absorb things into monosaccharides and polysaccharides, which transform into beef and mutton that we eat. In the transformation process, the protein conversion rate (i.e., the ratio of the energy intake of ruminants to the amount of energy that humans get) is high.
According to professionals, the demand for ruminant feed in 2006 was 45.19 million tons, but the supply was only 6.52 million tons. Supply is nearly one-seventh of market demand. Such feed shortages have prevented ruminant populations from meeting market demand.
Silage is a kind of natural plant feed. It is made from chopped-up plant stems and leaves with a water content of 65%-75%, and is under the condition of closed hypoxia, using the fermentation effect of anaerobic lactic acid bacteria to inhibit the reproduction of various bacteria. Silage is mainly used to feed ruminants (cattle, sheep, alpacas, and deer). Silage can be more easily stored than fresh feed. It is also rich in nutrition, which makes it an excellent feed source for livestock.
Silage gradually occupies a more significant proportion of the market for ruminant feed due to its advantages. In 2015, the demand for silage was 65.4% in the cattle farming industry and 21.5% in the sheep farming industry. The total demand for the cattle and sheep farming industry is expected to be around 67.3% by 2022.
In spite of silage's inestimable advantages, it is not perfect. Silage contains a large amount of cellulose, the main structural component of plant cell walls, usually combined with hemicelluloses, pectin, and lignin, making them difficult to digest and absorb. Ruminants can decompose them into monosaccharide and disaccharide. These sugars can later be absorbed and go through body oxidation to produce the energy they need to supply metabolic activities or combine into fat storage.
In nature, fungi have the strong ability to degrade cellulose and produce enzymes throughout the process. But the enzyme (cellulase) produced is usually acid resistant, limiting its potential industrial production. Compared to fungi, bacteria grow more rapidly, are easier to have higher cellulase production, and with several other advantages. The cellulase produced would attach itself to the surface of the cellulose and degrade it from the inside. According to Lu et al., cellulase produced by PKC-001 strain of Pseudomonas aeruginosa were detected active under both alkaline and acidic environment, which allows its cellulase to be mass-produced and for commercial use.
Our team is working on an additive that could help degrade the cellulose in silage to make it easier to be absorbed so as to improve ruminants' ability to acquire monosaccharides. Besides, it will also enhance its taste as there would be more soluble and sweet monosaccharides less insoluble cellulose. With our special addictive, the improved silage can promote the appetite of livestock, supplement the content of various digestive enzymes in ruminants' bodies, which prevents some common gastrointestinal diseases in livestock. For consumers, all the above vastly improve the quality of livestock products.
In order to achieve our final goal, we target the gene in PKC-001 strain of Pseudomonas aeruginosa which could secret the high-quality cellulase to construct our engineered bacteria. Firstly, we optimized the PKC-001 gene for adapting to the recipient cell - E. coli BL21 and constructed a plasmid with the vector pET-25b which contains a pelB signal peptide to improve the protein expression. After being transformed into the E. coli BL21, several experiments including the protein-induced expression would be conducted to analyze the performance of this engineered strain.
1. MUCK R，NADEAU E，MCALLISTEＲ T，et al． Silage review: recent advances and future uses of silage additives ［J］． J Dairy Sci，
2. 刘海燕，张鹏举，王秀飞，等． 正交试验法优化玉米秸秆穰叶青贮发酵剂的研究 ［J］． 中国饲料，2018 ( 11) : 74－79．
3. Coward-Kelly G., Aiello-Mazzari C., Kim S., Granda C., and Holtzapple M., 2003, Suggested improvements to the standard filter paper assay used to measure cellulase activity,Biotechnology & Bioengineering, 82(6): 745-749.
4. Ghose T.K., 1987, Measurement of cellulase activities, Pure & Appl Chem, 59(2): 257-268.
5. Lu Z.L., Chen D., Zhang S.S., Lu Q., and Huang R.B., 2012, A Pseudomonas aeruginosa producing alkaline cellulase, China Patent, 2012103821716