Description

Abstract

Antibody-free animal breeding protects human health, reduces the problem of resistance, and improves food safety. Shikimic acid (SA) is an important biochemical metabolite in plants and microorganisms that is known for antimicrobial and anti-inflammatory activities. In our project, we engineered the native shikimate pathway of E. coli to accumulate SA more efficiently. We plan to use it as a feed additive for preventive use in livestock breeding during seasons of high disease incidence. It will help improve the immunity of livestock and poultry, reduce the use of antibiotics, and potentially huge economic losses, and achieve the goal of sustainability.

1 Background

According to the World Health Organization (WHO), "antibiotic resistance is one of the biggest threats to global health, food security, and development today" (World Health Organization, 2016). In the United States, over 2 million illnesses and 23,000 deaths are estimated to be caused by infections of antibiotic-resistant pathogens annually (Centers for Disease Control and Prevention, 2017). In addition, antibiotic residues in food animals and the emergence of drug-resistant microorganisms pose a direct risk to human health. In the past, the dangers of antibiotic misuse have been ignored to improve economic efficiency, posing a hidden threat to food safety. China's Ministry of Agriculture 2021 has issued a guidance program for further reduction and replacement of veterinary antimicrobial drugs and supported the development and application of veterinary traditional Chinese medicine antimicrobial agents (Ministry of Agriculture and Rural Development, 2021). Before that, the effectiveness of the action to reduce the use of veterinary antimicrobials was obvious, the use of antimicrobials in livestock and poultry farming in 2020 than in 2017 decreased by 21. 4% (Ministry of Agriculture and Rural Development, 2021). Therefore, the development of veterinary herbal antimicrobials is in line with the national agricultural development program and is very promising for application. Shikimic acid is a compound that is well known as the main ingredient of Hypericum perforatum, Illicium verum, and Hypericum laricfolium, which are plants that are used in popular medicine (Bertelli et al., 2008). In addition, shikimic acid has been reported to have antibacterial, anti-inflammatory, gut flora-regulating, and immunity-enhancing effects, and has the potential to be used as an antimicrobial agent in veterinary herbal medicine (Li et al., 2023; Batory and Rotsztejn, 2021).

2 Shikimic Acid

2.1 What’s Shikimic Acid
SA is found in the dried, ripe fruit of anise (Illicium verum), a Chinese traditional medicine in the magnolia family. Chinese star anise has long been used in Traditional Chinese Medicine, and its fruit is listed in Chinese Pharmacopoeia (2010 edition) and used for treating vomiting, stomach aches, insomnia, skin inflammation, and rheumatic pain (Itoigawa et al., 2004) In addition to plants, SA is also widely found in microorganisms and can be extracted by microbial fermentation. Shikimic acid is antimicrobial (including bacteria and fungi), and anti-inflammatory, and improves intestinal immunity by regulating intestinal flora (Li et al., 2023; Batory and Rotsztejn, 2021). Its structural formula is shown below:

2.2 Properties of SA
a. Relatively safe: As a natural compound extracted from anise, which is used as a spice and herb in China, SA is relatively safe and non-toxic, harmless to humans and animals, and does not cause environmental pollution.
b. Intestinal Benefits: SA can improve the damaged micro-ecological environment of the animal intestinal tract, promote the growth and proliferation of beneficial bacteria, inhibit the growth of harmful bacteria, protect intestinal health, and improve animal productivity (Li et al., 2023).
c. Anti-bacteria: SA mainly affects gram-positive bacteria. SA is the main antimicrobial compound against Staphylococcus aureus in the aqueous extract obtained from Cedrus deodara pine needles. In general, the bactericidal mechanism consists of damaging the cell membrane and other cellular components of the bacteria (Bai et al., 2015; Zeng et al., 2012).
d. Anti-inflammatory effect: SA has exhibited anti-inflammatory properties. The current study shows that SA interferes with NF-κB and MAPK signaling pathways, protects against intestinal epithelial barrier dysfunction induced by inflammatory cytokines TNF-α, IL-1β, and IFN-γ, and enhances intestinal barrier function (Rabel et al., 2016) After researching, we found that shikimic acid has antibacterial, anti-inflammatory and protects the intestinal health of animals, these advantages can improve the resistance of animals to swine fever and other diseases, and at the same time help to reduce the demand for antibiotics in the livestock industry, realize the substitution and reduction of antibiotics, and ensure the stable development of the livestock industry. Therefore, SA can be added as a feed additive to the diets of poultry, swine, ruminants, and aquatic animals to improve their immune system and intestinal health and reduce the risk of disease.SA can also be used for animal or pet health to prevent bacterial infections in dogs, cats, and other companion animals. In summary, SA has a wide range of potential applications as a veterinary herbal antimicrobial and anti-inflammatory agent in a variety of animal species and feeding environments.

2.3 Industrial preparation of shikimic acid
At present, industrial production methods include the Diels-Alder method, which is the one in which a connected diene bonds with an alkene to produce a cyclohexene molecule. The other methods also include benzene and other hydrocarbon radical syntheses, quinic acid radical chiral synthesis, and sugar radical chiral synthesis. The disadvantage of these methods is limited raw materials, complicated steps, harsh reaction conditions, high production costs, and easy to cause environmental pollution. In microbial fermentation, Phosphoenolpyruvic acid (PEP) and Erythrose 4-phosphate (E4P) were used as precursors, which were transformed into Shikimic acid by DAHP synthase (AroG), 3-dehydronenenebc quinic acid synthase (AroB), 3-dehydronenenebd quinic acid dehydratase (AroD) and Shikimate dehydrogenase (AroE) [8] [9]. Compared with the traditional methods of extraction from plant fruits and chemical synthesis, the production of Shikimic acid by microbial fermentation has incomparable advantages such as a short production cycle, low cost, and less environmental pollution, and has gradually become a research hotspot at home and abroad. However, it has the disadvantage that the yield is not enough to meet the market demand, so how to increase the yield of Shikimic acid by means of genetic engineering is the problem we are committed to solving.

3. Chassis cell --- Escherichia coli (E.coli)

3.1 Introduction of E.coli
E. coli is a normal resident bacterium in the intestinal tract of humans and animals, which enters the intestinal tract with breastfeeding after the birth of infants and stays with people for life. Meanwhile, E. coli has been emphasized by genetic engineering experts due to its relative safety, clear genetic background, simple technical operation, simple culture conditions, and suitability for large-scale fermentation economy. Some studies have reported the use of Bacillus cereus, Bacillus subtilis, and Saccharomyces cerevisiae as chassis organisms for the production of Shikimic acid, but our investigations have led us to believe that Escherichia coli is expected to be used for the large-scale production of Shikimic acid by adjusting the expression of enzymes related to its metabolic pathways due to the advantages of its high genetic maneuverability, low requirements for growth conditions, and fast growth rate.

3.2 Advantages of E. coli for the preparation of Shikimic acid
Reasons why E. coli may be ideal as a chassis cell for shikimic acid production including
(1) There is a natural pathway to produce phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), the precursors for shikimic acid synthesis, in high yields using glucose as a substrate;
(2) the metabolic flow for shikimic acid synthesis can be made more efficient by simple genetic engineering modifications;
(3) E. coli is well tolerated with Shikimic acid.

4. Our Project

Our project this year was to use shikimic acid as an alternative to veterinary antibiotics to reduce the use of antibiotics in animal agriculture. We modified MG1655 to achieve the production of shikimic acid through metabolic engineering modifications and exploratory testing of a variety of independent variables that may affect shikimic acid yield. In order to be more in line with the industrial reality, we also tried the test of cheap substrates, you can check our project design and engineering.