The Establishment Of High Throughput Screening On High Penicillin-Productivity

Penicillin is an important group of Beta-lactam antibiotics. Breeding of high penicillins-producing strains is the most effective way to improve the yield of penicillins. There are two main strategies in strain breeding: one is the rational strategy which is aimed to modify the metabolic pathway and (or) the genes on the basis of the known metabolic pathways and regulatory mechanisms. The other is the non-rational strategy including the completely random strategy or semi-rational strategy, namely that it modifies the pathway and (or) the genes of the strains through creating randomly a large number of mutants though the knowledge about the metabolic pathways and regulatory mechanisms are unknown or not fully known.Penicillin is one of the secondary metabolites which are regulated by complicated signal systems. Moreover, as far as the existing excellence strains are concerned, many genetically modified methods will lead to negative rather than positive mutations. Therefore, the non-rational strategy to obtain high yield strains seems to be more suitable than the rational strategy. The primary task in the present paper is to establish a platform for screening a large number of mutants, namely high-throughput screening platform.High-throughput screening platform inculdes two technical systems: one is the high-throughput cultivation technique, and the other is the high-throughput screening technique. The two techniques supplement each other to form a complete screening system.In this paper, the high throughput cultivating technique of Penicillium chrysogenum was established by using floating static culture technology on 24-well plates, which solved successfully the problem of the lack of dissolved oxygen in culture media for filamentous fungi. In addition, the technique system is simpler and more stable. Compared to the traditional shake flask cultivation, it improved the throughput at least 50 times, that is to say, at least 5 000 strains can be cultivated synchronously every day, and also the asexual spores of high-yield strain can be obtained.In this thesis, the high throughput testing technique of P. chrysogenum was also established. Using the method of penicillinase’s special degradation to penicillin, which was supported by the indicating system of starch and iodine, an accurate, rapid, and simple testing technique was established. By this means, it not only can accomplish the high throughput quantitative analysis, i.e. completing more than 100 samples of penicillin determination within 10 mins, but also can finish the high throughput screening, i.e. 500 samples can be compared within 10 mins and 90% of low-yield strains can be eliminated, which improved the thoughput at least 100 times than HPLC did.Operating and verifying the established high throughput screening platform: by using the methods of UV-mutation and NaNO2- mutation, 480 mutants were obtained. Finally a high-yield strain was obtaianed through the high throughput screeing platform, and the strain grew more rapidly and produced spores earlierly. Futhermore, the yield of penicillin produced by micro-training system increased to 10457 U/mL and to 21442 U/mL by shake flask, and fermentation cycles terminated 10h earlier but the fermentation unit growed 7.90% averagely.The factors affecting spores production of P. chrysogenum and the relationship between factors and the yield of penicillin were also studied by using the high-throughput screening platform.In conclusion, the innovation in this study is listed as follows: a high-throughput cultivation technique and screening technique about P. chrysogenum were established, and a completed high-throughput screening platform was formed. All the works lay a foundation for non-rational strain selection where a large number of mutants need to be screened rapidly.