Reverse metabolic engineering is a technology that uses reverse reasoning to perform gene knockout, knock-in and other operations on microorganisms based on known metabolic pathways and genomic information to change their metabolic pathways and products. In the field of breeding, reverse metabolic engineering can achieve breeding goals by genetically modifying microorganisms to improve traits such as yield, quality, or resistance.
The following are the basic steps for breeding using reverse metabolic engineering:
1.Determine the breeding goals: The first thing you need to do is to clarify the breeding goals, such as increasing the yield of a certain microorganism, improving its quality, or enhancing its resistance. These goals can be determined through an analysis of market demand, microbial production processes, and relevant scientific literature.
2.Identification of metabolic pathways: Depending on the breeding goals, the relevant metabolic pathways need to be determined. The metabolic pathways and regulatory mechanisms of microorganisms can be determined by consulting relevant scientific literature and using bioinformatics tools.
3.Gene knockout or knock-in: By performing gene knockout or knock-in operations on the target microorganism, its metabolic pathways and products can be altered. For example, if an enzyme encoded by a gene plays a key role in a metabolic pathway, that metabolic pathway can be blocked by knocking out the gene, thereby reducing the yield or quality of the microorganism. Conversely, if a gene encodes an enzyme that promotes a certain metabolic pathway, it is possible to increase the efficiency of that metabolic pathway by knocking in that gene, thereby increasing the yield or quality of the microbe.
4.Screening and identification: Strains with desired traits can be found by screening and identifying microorganisms after knockout or knock-in. The physiological and biochemical indexes, metabolites, etc. of the strain can be detected and analyzed to determine whether it meets the breeding goals.
5.Optimization and scale-up: If a strain with the desired trait is found, further optimization and scale-up experiments are required. The production efficiency and stability of the strain can be improved by optimizing the culture conditions, gene expression regulation and other factors. At the same time, the application effect of the strain in actual production can also be verified through amplification experiments.
It should be noted that the application of reverse metabolic engineering in the field of breeding requires a certain background in bioinformatics, molecular biology and microbiology. At the same time, because the technology involves genetic modification and manipulation, it is necessary to strictly comply with relevant laws, regulations and ethical norms.