Method for constructing saccharopolyspora erythraea expression plasmid (pBlueV) containing vitreoscilla hemoglobin gene (vgb)

Abstract

The invention provides a method for cloning a saccharopolyspora erythraea expression plasmid (pBlueV) containing a vitreoscilla hemoglobin gene (vgb), belonging to the technical field of gene engineering. In the method, based on the fact that VHb (hemoglobin) can be combined with oxygen to form an oxygenated state, the VHb intervenes the oxygen related metabolic pathway of cells, thereby changing the original biological metabolism mode of the cells under oxygen-limited conditions, promoting the cell growth and protein synthesis under oxygen-deficient conditions, directly or indirectly affecting the action principles of primary metabolism and secondary metabolism of aerobes, firstly constructing an Escherichia coli expression plasmid pQEV, expressing the VHb with biological activity, and further constructing the saccharopolyspora erythraea expression plasmid (pBlueV). In order to realize high-density culture of cells and high-yield fermentation of metabolic products under oxygen-limited conditions, the invention provides an important pathway for homologous recombination of vgb gene elements and saccharopolyspora erythraea chromosome genomes.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering, and relates to a method for constructing a saccharopolyspora red mold expression plasmid (pBlueV) containing a hyaline hemoglobin gene (vgb). Background technique: [0002] The invention comes from the Liaoning Provincial Department of Education Youth Talent Fund Project "Research No.05L433 on Transforming Saccharopolyspora Erythromycetes to Increase Erythromycin Production and Reduce Costs". Saccharopolyspora erythromycetes is an engineered strain that produces the macrolide antibiotic erythromycin, which is widely used clinically, and its annual demand continues to increase. The fermentative production of erythromycin requires sufficient oxygen supply. Due to the low solubility of oxygen in water, it is far from the oxygen demand for the normal growth of Saccharopolyspora erythromycetes. Insufficient oxygen supply will lead to poor cell growth and erythromycetosis. The prime yield...

AI Technical Summary

Problems solved by technology

At present, industrial production uses methods such as increasing stirring speed and increasing ventilation to solve the problem of insufficient oxygen, but this often requires high equipment, consumes a lot of energy, and increases production costs.

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