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Keywords: Natural Sciences
Issue Date: 2014
Publisher: Quaid-i-Azam University Islamabad, Pakistan
Abstract: Tylosin is a macrolide antibiotic used as veterinary drug and growth promoter. Attempts were made for over expression of tylosin through random mutagenesis of Streptomyces fradiae NRRL-2702 using ultraviolet (UV) and gamma (γ) irradiations. After 100 and 120 seconds exposure of UV (λ=300 nm), spore suspension of wild type strain exhibited 5% and 8% survival rate, respectively and these exposure times were found suitable to isolate the most potent mutant colonies. 300 colonies appeared on the agar plates at above mentioned exposure times including six morphologically altered colony types. Initially, all these colonies were screened for tylosin production using Bacillus subtilis bioassay and it was found that only morphologically altered colonies (UV-1, UV-2, UV-3, UV-4, UV-5 and UV-6) indicated tylosin production. Furthermore, HPLC analysis revealed an increase of 2.7 fold in tylosin yield (1500 mg/l) by one of the morphological mutants i.e., UV-2 in complex medium which was improved to 1750 mg/l as compared to the wild type strain (620 mg/l) by optimizing fermentation conditions in chemically defined media. The values of different kinetic parameters such as dry cell mass (21 g/l), maximum specific growth rate, μmax (0.052 h-1) and specific tylosin productivity qp (1.36 mg/g/h) were also higher in mutant UV-2 as compared to wild type strain. To further improve the stability and productivity of mutant UV-2, it was subjected to another round of mutagenesis by gamma irradiation using 60 Co source. As a result of this treatment, another morphologically altered mutant (γ-1) with an enhanced expression of tylosin up to 2500 mg/l in complex medium was obtained. Use of chemically defined media promoted tylosin production up to 3800 mg/l by this mutant γ-1 in the presence of glucose+lactose (45 g/l) and sodium glutamate (12.5 g/l) as carbon and nitrogen sources, respectively. In addition, much higher value of qp (3.34 mg/gh) was observed in case of mutant γ-1 as compared to wild strain (0.81 mg/gh). Moreover, UV irradiation associated changes were observed to be unstable with loss of tylosin expression whereas, mutant γ-1 displayed high stability on subsequent culturing even after 3 years of storage at -72°C. Furthermore, solid state fermentation system (SSF) was also developed for tylosin production with mutant γ-1 and wild type strains by using various economically cheaper and easily available agro-industrial wastes. Wheat bran as solid substrate gave highest production (2500 μg of tylosin/g substrate) by mutant γ-1 against wild type strain (300 μg tylosin/g substrate). The tylosin yield was further improved to 4500 μg/g substrate at optimized conditions of 70% moisture level, 10% inoculum (v/w), pH 9.2, temperature 30°C, supplemented with lactose and sodium glutamate on 9th day. Wild type strain displayed reduced production of tylosin (655 μg tylosin/g substrate) in SSF even after optimization of process parameters. This study has shown that solid state fermentation system significantly enhanced the tylosin yield by mutant γ-1. In this way, an overall increase of 6.87 fold in tylosin yield was achieved through a combination of UV and gamma irradiation mutagenesis and fermentation screening by mutant γ-1. Additionally, to acquire some more knowledge about enhanced expression of tylosin in mutant γ-1, molecular studies were attempted to explore any change in the regulatory genes (tylQ, tylP, tylS, tylR & tylT) of tyl cluster. Expression analysis by RT-PCR revealed that tylQ was switched off earlier in mutant γ-1 as compared to wild type strain, although there was no change in the sequence of tylQ gene from both the strains. On the other hand, the analysis of tylP indicated no change in expression pattern between wild type and mutant γ-1 strains but there was difference of a single base and a substitution mutation of T A was recorded at position 214 in 420bp product of tylP gene. Moreover, the deduced protein sequences of tylP gene from wild type and mutant γ-1 indicated that the point mutation resulted in the change of single amino acid i.e., serine to threonine (S T) at position 72. Furthermore, RT-PCR remained unsuccessful to detect the expression of three other regulatory genes i.e., tylS, tylR and tylT. So, routine PCR was used to amplify these genes, which revealed that there was no change in the nucleotide sequence of these genes either from mutant γ-1 or wild type strain. However, the sequences of these genes could be a valuable addition in databases because these were not available previously for this particular strain of Streptomyces fradiae NRRL-2702.
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