Study On The Genetic Engineering Of Sulphur Containing Peptide Antibiotics

Penicillin was discovered by Fleming in 1932. It was widely used in clinic because of its anti-bacterial with high effect and broad-spectrum. However, concomitance with its excessive use, it has been resulted in the emergence of anti- drug pathogen. The only way to solve this problem is to use the semisynthetic-antibioctis. Penicillin G acylase (PGA, EC 3.5.1.11) catalyzes the hydrolysis of the acyl group of penicillin G yielding 6-aminopenicillanic acid (6-APA), a key intermediate in the production of semisynthetic β-lactam antibiotics. It is one of the most widely used industrial scale enzymes. In addition, PGA is useful as a biocatalyst in many potentially valuable reactions such as protection of amino and hydroxyl groups in peptide synthesis and resolution of racemic mixtures of chiral compounds. As an intracellular enzyme, PGA is produced by several microorganisms, including various bacteria, actinomycetes, fungi and yeasts. Although E.coli PGA is the most common source for industrial applications, the applicability of other bacterial PGAs have been evaluated with promising results. Compared to E.coli PGA, Kluyvera.citrophila PGA(KcPGA) has attracted interest due to many features suitable for industrial application, such as ease of immobilization and greater stability with respect to heat, pH and organic solvents. However, the previous reports on penicillin G acylase from Kluyvera citrophilamainly focused on its molecular characterization and substrate specificity. Few studies have been done with this enzyme regarding systematically genetic engineering including cloning and expression of recombinant enzyme. So in this study, the DNA fragment encoding KcPGA was amplified and cloned into the vector pET28b to obtain a C-terminus His-tagged fusion expression plasmid pETKcPGA. The fusion protein KcPGA was successfully overexpressed in E.coli BL21(DE3) induced by 5μM isopropylthio -beta -D-galactoside (IPTG) in LB medium. The fusion protein was purified in a single step by Ni-IDA affinity chromatograph to a specific activity of 35.3U/mg protein with a final yield of 89% representing a 23-fold purification. The data presented here suggest that the purified fusion protein is stable with respect to pH and temperature. The optimal pH and temperature of recombmant KcPGA are 8.5 and55°C, respectively. The Km and Vmax are 17.6uM. and 23.8U/mg, respectively. The KcPGA yield was significantly and conveniently increased by more than 2.4-fold in TB medium compared to that in LB medium, which need inducing by isopropylthio-beta-D-galactoside (IPTG). The results shown the lower temperature (28 °C) was more feasible for the overproduction of KcPGA compared to the higher temperature(37°C).The highest enzyme activity(15,000U/L) was obtained at 28°C in TB medium only when culture pH was closed to its original pH value and the content of glycerol is 2.5g/L, as well as culturing for about 24 hours.Nosiheptide(multithiomycin) is a sulphur containing polypeptide antibiotics, produced by Streptomyces.actuosus (S.actuosus). It was discovered by Rhonepoulenc S.A in France in 1961. After that, a lot of study was carried out on its production, biosynthesis and application by American, French and Japanese researchers. Nosiheptide is a tiny-yellow crystal, poor water solubility. It has obviously the inhibitory function to Gram-positive bacteria. However, it has not been applied to clinic because of its poor water solubility. On the other hand, it is so far one of the best feedstuff additive for animals, in which it can promote the animal's growth, enhancing the utilizing efficiency of the feedstuff, increasing the anti-virus ability. What's more, it has no residue in animal's body. It had been developed as animal feedstuff additive in France and Japan. The study was carried out on this strain by our lab in 1988. The Illveterinary certificate was obtained in 1998 and nosiheptide was put into industrial production. But its yield was not high. Mycelium combined and formed sphere shape during S.actuosus fermentation process and became self-dissolved because of insufficient oxygen supply in the late stage of fermentation, in which resulted in low fermentation product and badly influence on the output of nosiheptide. In order to conquer such disadvantages, oxygen was supplied continuously by air compressor in industrial production. However, it would expended 50% cost. So it is very important to solve the problem of oxygen supply and reducing the power comsumption.Vitreoscilla hemoglobin(VHb) is so far the only one found in prokaryote. There are much homology and similarity in gene structure, spectrum characteristic, and CO binding dynamics et al, between VHb and the other hemoglobins found in eukaryote. The expression of VHb is controlled by oxygen. The amount of VHb expression increases greatly in lower oxygen environment. The characteristic of VHb binding oxygen provides the possibility to enhance both the oxygen transfer and the efficiency of oxygen absorption cells. VHb plays a major role in improving the conditions of bacterial growth, increasing the synthesis of protein and secondary metablites,especially antibiotics, as well as promoting expression of heterogenous genes. Introduction of VHb gene into S.actuosus by genetic engineering technologies will have benefit to the decrease of energy consumption in fermentation industry. In this study VHb gene was cloned into vectors containing erm promoter and was introduced into S.actuosus by protoplast-plasmid transformation and conjugation methods. The successful expression of VHb gene was verified by the analysis of carbon monoxide-binding difference spectra. The effect of VHb expression on the production of recombinant S.actuosus was also discussed. Through the oxygen-limited experiment, the output of nosiheptide of the recombinant S.actuosu constructed by the method of protoplast-plasmid transfer was 3.1 times against the original strain, and 3.63 times against original strain constructed by conjugation. It is the first report that the genetic engineering technologies was utilized to introduce VHb gene into S.actuosus at home and abroad and the product of nosiheptide was greatly increased. The recombinant strains was stable and suitable for the industrial application. A new method was practically provided to reducing the cost and increasing the product of nosiheptide accordingly.