<p>At present, 5-ALA is mainly synthesized by chemical synthesis and microbial fermentation. The study of chemical synthesis began in the 50s of the 20th century, and was most active in the 90s. The researchers synthesized 5-ALA [9] using raw materials such as hippuric acid, succinic acid, tetrahydrofurfuramine, and levulinic acid. However, the chemical synthesis method has the disadvantages of high price, difficult to obtain, high toxicity, low production yield and harsh reaction conditions, and the synthetic products contain impurities that are difficult to determine, which may cause potential harm to agricultural and forestry [8]. <strong>In this project, <i>E. coli</i> was used as the chassis to screen strains that can produce stable and high yields of 5-ALA through enzyme modification, CRISPR-associated transposons (CASTs) system, and microfluidic high-throughput screening</strong>. Finally, the efficient and sustainable production of 5-ALA will be realized, thereby reducing the cost of biopesticides and contributing to the alleviation of the global food crisis. </p>
<p>At present, 5-ALA is mainly synthesized by chemical synthesis and microbial fermentation. The study of chemical synthesis began in the 50s of the 20th century, and was most active in the 90s. The researchers synthesized 5-ALA [9] using raw materials such as hippuric acid, succinic acid, tetrahydrofurfuramine, and levulinic acid. However, the chemical synthesis method has the disadvantages of high price, difficult to obtain, high toxicity, low production yield and harsh reaction conditions, and the synthetic products contain impurities that are difficult to determine, which may cause potential harm to agricultural and forestry [8]. <strong>In this project, <i>E. coli</i> was used as the chassis to screen strains that can produce stable and high yields of 5-ALA through enzyme modification, CRISPR-associated transposon (CAST) system, and microfluidic high-throughput screening</strong>. Finally, the efficient and sustainable production of 5-ALA will be realized, thereby reducing the cost of biopesticides and contributing to the alleviation of the global food crisis. </p>
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<h4style="text-align: left;"><b>(1) Enzyme modification and CRISPR-associated transposon system</b></h4>
<p>In order to improve the activity of ALAS enzymes, we decided to enzymatically modify ALAS and introduce <i>E. coli</i> BL21 (DE3) for fermentation validation.</p>
<p>When the plasmid enters the strain through transformation, the plasmid in the strain is often lost to varying degrees with the progress of fermentation, which seriously affects the stable synthesis of the product. Therefore, there is a need to adopt new strategies to improve the genetic stability of ALAS expression plasmids.</p>
<p>The CRISPR-Cas system has attracted extensive attention due to its excellent gene editing ability, but the limited homologous recombination efficiency has limited the application of the CRISPR-Cas system. We were inspired by two articles on CRISPR transposons published in Science and Nature in 2019. <strong>CRISPR-associated transposons (CASTs) are a unique system of mobile genetic elements that bind transposable proteins to Cas proteins lacking nuclease activity to catalyze the targeted integration of exogenous DNA in the genome</strong>. In this system, the cascade complex formed by the Cas protein is guided by the crRNA to a specific site in the DNA, which subsequently recruits the transposase,Integrate a donor DNA fragment with a transposase end recognition sequence downstream of the target site.Without relying on host DNA repair mechanisms,The CAST system allows for precise targeting of inserted DNA in the host genome, so that the exogenous genes are stable in the strain [10, 11]. We used this technique to integrate the ALAS gene into the <i>E. coli</i> chromosome, allowing it to be stably expressed, thus avoiding the negative impact of plasmid loss on yield.</p>
<p>The CRISPR-Cas system has attracted extensive attention due to its excellent gene editing ability, but the limited homologous recombination efficiency has limited the application of the CRISPR-Cas system. We were inspired by two articles on CRISPR transposons published in Science and Nature in 2019. <strong>CRISPR-associated transposon (CAST) system is a unique system of mobile genetic elements that bind transposable proteins to Cas proteins lacking nuclease activity to catalyze the targeted integration of exogenous DNA in the genome</strong>. In this system, the cascade complex formed by the Cas protein is guided by the crRNA to a specific site in the DNA, which subsequently recruits the transposase,Integrate a donor DNA fragment with a transposase end recognition sequence downstream of the target site.Without relying on host DNA repair mechanisms,The CAST system allows for precise targeting of inserted DNA in the host genome, so that the exogenous genes are stable in the strain [10, 11]. We used this technique to integrate the ALAS gene into the <i>E. coli</i> chromosome, allowing it to be stably expressed, thus avoiding the negative impact of plasmid loss on yield.</p>
