The discovery of antibiotics for clinical use is perhaps the most important discovery in the history of therapeutic medicine. The application of antibiotics to the therapy of infectious diseases may conceivably have saved more lives than any other medical development. Penicillins are b-lactam antibiotics which have broad clinical utility. It began in 1929, when Alexander Fleming published his observation about the inhibition of growth of Staphylococcus aureus on an agar plate contaminated with Penicillium notatum. Three years later, it was shown that the growth inhibition was due to penicillin. The first clinical trials with penicillin were under-taken in 1941. In parallel with efforts to provide penicillin in large amounts, its structure was elucidated in 1945, when Hodgkin and Low showed by X-ray crystallography analysis that it is composed of a b-lactam structure.

The success of penicillin in the treatment of infectious disease is due to its high specificity and its low toxicity. The biosynthetic pathways of penicillin have been elucidated. The biosynthetic enzymes are often unstable and are present in the cell in only small amounts, making their purification difficult. Penicillins are produced by a number of microorganisms, including the several fungal species and several streptomycete species. Penicillium chrysogenum is an important industrial organism due to its capacity to produce penicillin which is still one of the main commercial antibiotics. Penicillin yields have been increased through development of better production strains by classical mutagenesis procedures and optimization of the growth conditions. Currently, the greatest progress in the molecular biology in fungi has been made, classical genetic techniques can be applied to P. chrysogenum and hence a detailed genetic map is available. Together with molecular techniques, this facilitated a thorough analysis of the genetic regulation of metabolic pathways. The great progress in elucidation of the molecular regulation of biosyntheses of penicillin has been made in the penicillin producer P. chrysogenum. Since the biochemistry of penicillin biosynthesis is rather well understood and recombinant techniques have been developed for some filamentous fungi, within the last few years, several studies have indicated that the penicillin biosynthesis genes are controlled by a complex regulatory network, the over expression of regulatory genes will lead to higher yields of penicillin in the respective production strains.

Penicillin biosynthesis is regulated by environmental factors such as the phosphate, carbon, nitrogen and oxygen content of the medium. The overall rate of penicillin synthesis is severely reduced under conditions of low oxygen. Reduction of oxygen supply leads to accumulation of penicillin N, a precursor of penicillin. The mechanism of oxygen control over penicillin synthesis is not well understood. Possibly, low oxygen levels directly affect the biosynthetic pathway of penicillin, which includes several oxidation reactions. It is also possible that a more efficient overall metabolism provided by higher oxygen levels indirectly results in higher penicillin yields. Regardless of the mechanism, technologies that improve aerobic metabolism in these organisms should have a positive effect on penicillin production.

Vitreoscilla hemoglobin (VHb) is so far the only one found in prokaryote. The expression of VHb is controlled by oxygen, and the amount of VHb expression increases greatly in lower oxygen environment. Possible mechanisms for VHb action include increasing the flux of oxygen to the respiratory apparatus, providing higher internal oxygen concentrations, altering the internal redox state, or functioning as an efficient terminal oxidase. VHb plays a major role in improving the conditions of bacterial growth, increasing the synthesis of protein and secondary metabolites, especially antibiotics, as well as promoting expression of heterogenous genes. The characteristic of VHb binding oxygen provides for us a possibility to enhance both the oxygen transfer and the efficiency of oxygen absorption in cells. Intracellular expression of a bacterial heme-binding protein (Vitreoscilla hemoglobin, or VHb) has resulted in higher productivity of a number of industrial cell types. For example, the expression of VHb in the filamentous bacterium Streptomyces coelicolor resulted in tenfold higher yields of the polyketide actinorhodin in bench scale batch fermentations run under reduced aeration. Also, the efficiency of cloned protein synthesis by oxygen-limited Escherichia coli cultures was increased in VHb-expressing strains. Several VHb-expressing transformants of Acremorium chrysogenum produced significantly higher yields of cephalosporion C than control strains in batch culture experiments. Therefore, intracellular expression of VHb in P. chrysogenum by recombinant DNA technology could provide a possibility that a productivity of penicillin was increased. Thus a basic requirement to establish a recombinant DNA system for the cloning of vgb gene (coding VHb) in P. chrysogenum is a gene transfer system. This includes a reproducible transformation procedure and proper selection markers. Several transformation techniques are available for filamentous fungi. Most protocols involve the transformation of protoplasts using electroporation or by a combination of CaCl₂ and polyethylene glycol (PEG). Filamentous fungi were transformed using two dominant selection markers, namely the bacterial resistance gene and complement gene of auxotrophic. Intact fungal cells have also been transformed by the transformation of protoplasts, but usually with a lower transformation frequency. Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, into plant cells during tumorigenesis. It is routinely used for the genetic modification of a wide range of plant species. For years, the transformation using A. tumefaciens has been applied to the yeast Saccharomyces cerevisiae and filamentous fungi, including Aspergillus niger, Fusarium venenatum, Trichoderma reesei, Colletotrichum gloeosporioides, Neurospora crassa, and the mushroom Agaricus bisporus.

This paper showed that Agrobacterium tumefaciens LBA4404 transfers part of its T-DNA with vgb and bleomycin resistance gene to P. chrysogenum cells. The T-DNA then integrates into the P. chrysogenum nuclear genome at a random position, so the vgb and bleomycin resistance gene were integrated into the P. chrysogenum genome.