In the parts that we used -- laccase, leghemoglobin, nirB, ArcA, we optimized the laccase and leghemoglobin part. As a part of our function module, the efficiency of Laccase and leghemoglobin are directly related to the quality of the project.
As a reductive enzyme, laccase can catalyze the reaction of oxygen with reducing substances to achieve the effect of consuming intracellular oxygen. But during the experiment, we found that the promoters used were not very ideal. Because laccase can only function in the cellular periplasm, so it can not consume a lot of oxygen inside the cell. For the problem that laccase will be localized to the cellular periplasm, we found that the main reason is that the laccase amino acid residues contain 28 signal peptides. For this problem, we reconstructed the laccase plasmid to remove the signal peptide sequences contained in the laccase. The final reconstructed laccase plasmid also achieved the effect we expected.
leghemoglobin can directional transport oxygen adjacent to the cell periplasm specifically to provide energy for cell growth, realizing the purpose of reducing the intracellular oxygen concentration by transporting oxygen. However, the intron in the leghemoglobin gene prevented it from functioning in E. coli, we redesigned the leghemoglobin. We removed the introns to allow leghemoglobin to work properly in E. coli.
Beyond that, heme in leghemoglobin is the main reason it can work. But bean heme synthesis is highly conserved within organisms, and is a rate-limiting step in E. coli. Therefore, the heme content in leghemoglobin is not very much; it's delivery capacity of oxygen is limited. Therefore, we can improve the heme content by adding heme. Since it is difficult to order heme on the market, we use hemin as a substitute, which can change into heme and have the function.