Contribution

To achieve antibiotic-free and low-antibiotic farming in livestock husbandry, our team sought out an antibiotic substitute and sought to limit the use of antibiotics. The shikimic acid synthesis route was enhanced and optimized, and the CRISPR-Cas9 gene-knockout technique was used. Finally, the entire product launch procedure is envisioned.

We have achieved the following contributions.

1 Genome-scale metabolic model (GSMM)

Throughout the initial stages of the project's technology pathway design, to improve shikimic acid yields, we utilize models to predict yields, modify genes, and improve knockout procedures. Our model is based on the genome-scale metabolic model (GSMM) of Escherichia coli (iML1515), which is used to perform an in silico screening of key gene targets for improved shikimic acid biosynthesis [1]. In the future, if other teams are required to conduct research on the yield of shikimic acid, they can use this model to forecast the yield and be better ready to plan their experiments in advance.

(1)Increase PEP production:
(i)Knocking out the ptsG, ptsH, and ptsI genes prevents PEP from being converted to pyruvate by blocking the phosphate transport pathway (PTS system).
(ii) By knocking out the poxB gene, one can prevent PEP branching pathways like those for lactate, acetate, and ethanol synthesis.

(2) Increase E4P production: tktA and talB genes are overexpressed.

(3) Build and improve the shikimic acid synthesis pathway:
(i) Increased metabolic flux of shikimic acid produced from PEP and E4P by overexpressing the aroG, aroB, aroD, and aroE genes.
(ii) The shikimic acid catabolic pathway is blocked by aroK and aroL gene knockout. Methods, Results and Discussion

2 Shikimic acid pathway construction in E. Coli MG1655

To improve the synthesis level of shikimic acid by E. Coli MG1655, we modified the shikimic acid pathway and totally constructed 26 parts. Our work can provide a reference for future igem teams working on related topics and pathways. Based on the above model predictions and literature search, it was determined that our final technology path is shown below (Figure 1):

(1) Increase PEP production：
(i) Knocking out the ptsG gene prevents PEP from being converted to pyruvate by blocking the phosphate transport pathway (PTS system).
ptsGup（BBa_K4891016）
ptsGdown（BBa_K4891017）
(ii) Increased glucose uptake by the heterologous introduction of the non-phosphorylation pathway through overexpression of the Zymomonas mobilis derived glk and glf genes (primarily through expression of plasmids and genomic integration into the ptsG). ptsGup-glk-glf-ptsGdown（BBa_K4891015）：
(iii)By knocking out the poxB, ldhA, adhE, and pta genes, one can prevent PEP branching pathways like those for lactate, acetate, and ethanol synthesis.
ldhA(BBa_K4891008)
adhE(BBa_K4891009)
poxB(BBa_K4891012)
pta(BBa_K4891013)

(2) Increase E4P production: tktA and talB genes are overexpressed.
tktA-talB（BBa_K4891025）
Build and improve the shikimic acid synthesis pathway:
(i) Increased metabolic flux of shikimic acid produced from PEP and E4P by overexpressing the aroG, aroB, aroD, and aroE genes.
aroGfbr-aroB-aroD-aroE （BBa_K4891005）
(ii) The shikimic acid catabolic pathway is blocked by aroK and aroL gene knockout.
aroK（BBa_K4891006）
aroL（BBa_K4891007）

3 Knockout genes by CRISPR-Cas9

The pCas/pTargetF dual vector technique was utilized in our CRISPR-Cas9 gene editing study to achieve gene knockout. The pCas/pTargetF dual vector system can be used to carry out gene editing in E. coli to accomplish gene knockout or knock-in. The pTargetF plasmid elimination, a crucial stage in the cellular expression system, is intended to drastically reduce or even totally remove the expression in the cell. The pTargetF plasmid elimination can be used for gene function studies, drug target screening, and efficacy evaluation. It can also be used to disclose the regulatory mechanism of target genes, enhance their expression, and improve biosynthetic pathways. It will provide more possibilities for analyzing gene functions and developing novel therapeutic methods.

The main steps are as follows:
1）Plasmid pTargetF, which targets the desired gene, is constructed by designing sgRNA sequences;
2）Construct a homologous recombinant plasmid containing upstream and downstream of the target gene;
3) Plasmid pCas9 containing the cleavage function was electrophoresed into the competent cells of E. coli MG1655, and the competent cells were incubated at 30℃.
4) Plasmid pTargetF containing specific sgRNA sequences and homologous gene fragments was transferred into the target strain and positive colonies were obtained by colony PCR.
5) Add IPTG to eliminate plasmid pTargetF;
6)The pCas plasmid is a heat-sensitive replicon, and plasmid pCas9 is removed by incubation at 42°C.

4 Product market access legislation

We have gained knowledge about the operational processes for listing genetically modified (GM) products added to feed from the pertinent legislation. Our project contends that the finalized product is premixed compound feed, which must pass the safety evaluation under the pertinent provisions of the Regulations on the Safety Administration of Agricultural Genetically Modified Organisms and obtain the safety certificate of such organisms, as well as the evaluation under the pertinent provisions of the Measures for the Administration of New Types of Feeds and New Types of Feed Additives. If other Chinese research teams need to study feed, agricultural genetically modified products, etc., and need to put their goods on the market for sale. They can produce and market their products using the regulations that our team is concerned about.

a.Catalog of Feed Additive Varieties (2013) (Ministry of Agriculture Announcement No. 2045)
b.Measures for the Administration of Production License of Feed and Feed Additives ( Revised by Decree of the Ministry of Agriculture and Rural Development No. 1 of 2022 on January 7, 2022)