Background

Considering antibiotics can effectively prevent human and animal plague and take precaution against bacterial infection, human began to use excessive antibiotics in daily life, even in the treatment of mild bacterial infection. However, high doses of antibiotic active substances (30% - 90%) cannot be degraded by organisms and flow into the soil or water resources through feces pollution after the intake of antibiotics. According to the research, as early as 2017, the global sales great amount of antibacterial drugs was nearly 93309 tons, and there is even statistical analysis estimate that the sales volume of antibacterial drugs will reach 104079 tons in the year of 2030. Among them, sulfonamides are one of the most widely used antibiotics in agriculture and domestic animal breeding industries in China.

Antibiotics that enter the soil with feces can stimulate the resistance of bacteria and screening antibiotic-resistance bacterial strain, which could be fatal in human populations. In 2019, about 700 thousand people died directly from antibiotic resistant bacterial infections. And according to data estimates, 10 million people worldwide will die from antibiotic resistant bacterial infections every year in the future. Additionally, the economy and stability of the society will also be greatly affected. The economic burden of the health sector in 2050 will be even greater $100 trillion according to statically analysis. In general, the abuse of antibiotics (especially sulfadiazine) is a global issue that brooks no delay, and mankind must pay attention to it and actively improve the precautions in order to prevent further epidemic infectious diseases.

Project inspiration

We first considered that sulfadiazine users were patients taking antibiotics, so we interviewed hospital staff. However, hospital staff indicated that the hospital now has very strict regulations to limit the abuse of antibiotics. Furthermore, Sulfonamides antibiotics has been proved that great amount of bacteria including the MRSA, has relative resistance on this type of drugs, which has been stopped using in clinical prescription. And because of the lack of knowledge on bacterial resistance decades ago, people took a large number of sulfa antibiotics to fight against diseases, so now the flora in human intestine has been immune to sulfa antibiotics. However, they also said that sulfa antibiotics were not fully immune to the bacteria in the intestines of livestock, so they were widely used in the breeding industry to effectively prevent diseases of livestock raised in high-density environments, and it’s economical affordable for farmers.

We followed the clues to find the workers of the farm, and through interviewing these employers, we have received precious information and learned that they were feeding sulfa antibiotics to their livestock, which their excreta would be discharged into the environment and causes severe pollution.

Existing solutions

At present, there are three main methods to treat sulfadiazine residues in animal husbandry: physical extraction, chemical Fenton advanced oxidation treatment technology, and biological aerobic activated sludge process. These treatment have several disadvantages including slow efficiency, incomplete removal, possible aggravation of pollution, and inability to exert the maximum potential of degrading bacteria. The physical extraction method needs to pretreat the raw materials at first, and it is extremely difficult to separate the solute after it is dissolved in the organic solvent. Fenton treatment is due to the difficulty of hydrogen peroxide operation, the high cost of hydrogen peroxide agent and the cost calculated by most enterprises now often does not include the increase of sludge, which is easy to reverse color, and difficult to control the reaction process. For advanced oxidation treatment technology Some of the treatment processes were too complex, the treatment cost extremely costly and the oxidant consumes large carbonate ions and suspended solids interfere with the reaction. This technology is only applicable to the treatment of wastewater with high concentration and small water flow, but it is difficult to apply wastewater with low concentration and large water flow. The disadvantages of aerobic activated sludge method are intensive energy consumption, high sludge output, foam, sludge bulking and other potential issues.

Fig 1. Table that displays the drawbacks of current methods.

laccase

Laccase is the general name of a series of enzymes widely used by human beings. The current applications of Laccase including food industry, paper industry, textile industry, biosensors, and environmental remediation. Nevertheless, we have discovered that Lac6, an enzyme in laccase, has the ability to degrade Sulfadiazine residues with high efficiency and high degree of accomplishment. Our group has extract the DNA fragment of Lac6 in a fungus called Pleurotus ostreatus, and used it to degrade sulfadiazine in the intestines of livestock. Further indications shows the requirement of living livestock is unnecessary to achieve our research.

Fig .Schematic of how Laccase enzymes work.

E.Coli Nissle1917

After we have selected our target protein, we need a qualified engineering bacterium as a constructing chassis to carry out biological transformation. E. ColiNissle1917 (EcN) is one of the few non pathogenic gram-negative bacteria, which could be consumed by livestock and is easy to be genetically modified. At the beginning of the project, it was designed to feed the strain directly to livestock to degrade sulfadiazine inside the intestine, so EcN was the engineering strain we decided to choose for genetic transformation and sulfadiazine degradation.

Ice Nucleoprotein (INP)

Ice crystal nuclear protein is our anchor protein. Since the C-terminal of ice crystal nucleoprotein is hydrophobic and the N-terminal is hydrophilic, INP will be actively embedded on the cell membrane of engineering bacterial strain. Through co-expression, INP and Lac6 could be linked together through anchoring and Lac6 will be exposed outside the cell membrane, so as to degrade sulfadiazine more efficiently.

Construction of whole cell catalyst

By double - enzyme digestion of EcN plasmid, laccase and INP genes were incorporated into the plasmid, and this plasmid was implanted into EcN, we assembled all gene elements together. Through the fusion expression of laccase and ice crystal nuclear protein, laccase can be exposed outside the EcN cell membrane, so as to more effectively degrade the residues of sulfadiazine antibiotics in feces or environmental residue.

Fig .The design of our engineered strain.

Outlook

We have identified a few potential changes to our design that would better improve the degradation rate of SDZ.

1.Instead of using EcN, DE3 should serve as the host cell
We initially selected EcN because of its advantages in the small intestine. Its probiotic features and strong adherence to the small intestine wall made it an ideal choice for performing degradation functions inside livestock. Having realized later that feeding the engineered strain to farm animals was neither useful nor caution-free, it was already too late for us to make changes to our design.

Fig .Testing with other host cells such as DE3.

DE3 has a few advantages over EcN when applied directly to the feces. DE3 is more active, so it reproduces more frequently. This will definitely increase the efficiency of SDZ degradation.

2.Test with different Laccase enzymes
There are many forms of Laccase enzymes, such as Lacc3, Lacc6, Lacc9, and Lacc10. Laccase enzymes have a broad substrate range, and different Laccases can bind to the same compound. Although we chose to use Lacc6 in our engineered strain, other Laccase enzymes might hold better effects in SDZ degradation. Through experimenting with all possible candidates, we can locate the optimal Laccase enzyme.

Fig .Running tests with other Laccase enzymes.

3.Conduct additional tests in SDZ-concentrated feces
The complicated environment in animal feces may have unexpected effects on the engineered strain. For example, the presence of other microorganisms might interfere with our engineered strain. Running tests in a more realistic environment will give us a better picture of the capabilities of the engineered strain when put to actual use.

4.Perform codon optimization
Codon optimization can significantly improve the protein expression level in organisms by increasing the translation efficiency of target gene fragments.

When the utilizing frequency of synonymous codons of the Lacc6 gene matches that of EcN-1917, the protein expression level will be significantly increased. In general, the combination of high-frequency and sub-high-frequency codons works more effectively after the optimization of Codons.

Project Description