Transformation Of Vgb Into β-lactam Antibiotics-producing Industrial Strains Mediated By Agrobacterium Tumefaciens

In large-scale production of antibiotics by fermentation, it is a key problem to maintain sufficient dissolved oxygen concentration (DOC) in order to ensure the normal growth of microorganisms and effective antibiotics production. So far the common methods used to increase DOC in fermentation medium are aeration and agitation. On one hand, these methods can improve DOC in fermentation medium, but on the other hand, the cost of energy consumption in fermentation production should be increased owing to the requirement of the special equipments and the increasing energy.Semi-synthetic cephalosporins belong to p-lactam antibiotics and are one of the antiinfective drugs used in clinic. Their market share among anti-infective drugs has been steadily increasing year by year. Cephalosporin C (CPC) is used as a raw material for the production of 7-amino cephalosporanic acid (7-ACA), which is one of the most important intermediates of semi-synthetic cephalosporins. Production of CPC with low cost and high-efficiency has been pursued in industry. There are three reactions (the cyclization of tripeptide, ring expansion, and hydroxylation of deacetoxycephalosporin C) involved in the biosynthesis pathway of cephalosporin C, which are all oxygen-requiring processes. In order to acquire the high production of CPC, the sufficient oxygen should be supplied, which can cause higher energy consumption. It is estimated that only the cost of energy for oxygen supply during fermentation accounts for up to about 30 percent of total costs. Thus, introduction of VHb gene into some industrial microbes by genetic engineering techniques will have benefit in the decrease of energy consumption in fermentation industry.Vitreoscilla hemoglobin (VHb) is so far the only one found in prokaryote. There are higher homologies or similarity in gene structure, spectrum characteristic, and CO-binding dynamics etc. among VHb and the other hemoglobins found in eukaryote. The research work performed in Escherichia coll showed that the expression of VHb was modulated by oxygen if vgb gene was placed under the control of its natural promoters, and the amount of expressed VHb was greatly increased in poor oxygen conditions. Some other research work on the cloning of vgb gene in some microbes demonstrated that VHb could apparently improve the transport and uptake of oxygen in genetically engineered bacteria so as to make them growbetter and stimulate the expression of heterogenous genes and the synthesis of secondary metabolites, especially antibiotics.Recombinant DNA technology has been developed and applied to the engineering of filamentous fungi so as to improve their production. However, lack of efficient transformation system represents a major impediment to carry out the study of genetic engineering in this important industrial filamentous fungus-Cephalosporium acremonium. A method for genetic transformation mediated by Agrobacterium tumefaciens has been most widely used in gene cloning of plant. Besides its natural plant hosts, Agrobacterium has been shown to be able to transfer DNA to other microorganisms such as yeast, several species of fungi. However, whether Agrobacterium can be used to transform gene into C.acremonium remains unknown. In this work, an efficient and stable method for introduction of vgb genes into an industrial C. acremonium mediated by A. tumefaciens and the effects of vgb on the fermentation of cephalosporic C were reported.The summary of this research work is as follows:1. Construction of the binary vector plasmid pYG306This plasmid contains both a phleomycin-resistance gene and a Vitreoscilla hemoglobin gene, which are located downstream of the trpC gene promoter from Aspergillus nidulans and placed between the left border and right border repeats of the binary vector pBI121.2. The establishment of donor Agrobacterium clones bearing plasmid pYG306Plasmid pYG306 was then introduced by either electroporation or triparental mating into the A. tumefaciens LBA4404, which carries the vir gene responsible...