Genetic Manipulation Of Streptomyces Fradiae And Streptomyces Thermotolerans

Tilmicosin is a semisynthetic macrolide antibiotic derived from tylosin. The traditional method of tilmicosin synthesis starts with the removal of L-mycarose from tylosin to get desmycosin. The disruption of tylCV gene from tylosin producer-Streptomyces fradiae would result in the accumulation of desmycosin, the direct precursor of tilmicosin. In this study, a new rapid gene disruption method of Streptomyces genes was estabolished by combination of fusion PCR and PCR-targeted system. The tylosin industrial producer BIB1028 was used as the start strain and the tylCV gene disruption cassette was constructed by the new method. The tylCV in-frame replacement strain of Streptomyces fradiae, BIB715 was obtained through intergeneric conjugation between Streptomyces fradiae and Eo coli. The mutant BIB715 was confirmed by nested-PCR and the fermentation results in the level of flask indicated the main fermentation product was desmycosin.The FLP recombinase acts on the FRT sites flanking the antibiotic resistance cassette and oriT_RK2 to generate the disruption structure of tylCV in-frame deletion. The disruption structure was inserted into the conjugal vector pBIB1015 to get pBIB1017. The tylCV in-frame deletion strain of Streptomyces fradiae, BIB1209 was obtained through intergeneric conjugation between BIB715 and E. coli. The mutant BIB1209 was confirmed by PCR, RT-PCR and the fermentation results in the level of flask indicated the main fermentation product was desmycosin. According the fermetaion procedure of tylosin, the yield of desmycosin reached 7.3 g 1^-1 in 5 1 jar fermentor after optimization of the culture conditions. Compared with the 9.1 g 1^-1 of the start strain, after conversion, the productivity reached 106±5% of the start strain, which could be scale-up for industrial fermentation to produce large amount of desmycosin for semisynthesis of tilmicosin directly.The tilmicosin synthesis method was also investigated in this study. Compared with the traditional method, desmycosin fermented by the obtained recombinant mutants only needed two or three simple treatment and over 90% desmycosin could be transferred into the organic level for synthesis of tilmicosin. Compared with the traditional recovery rate 70-80% of tylosin, the new method not only simplified the procedure, but also increased the recovery rate. The results of HPLC-MS assays showed the synthesized product was identical to the industrial product of tilmicosin.Acetyl-isovaleryl tylosin (AIV) is another important derivative of tylosin, which is obtained through biotransformation of Streptomyces thermotolerans, After optimization, the conjugal transfer system between S. thermotoIerans and E. coli was established. The genes responsible for the acylation, acyB1 and acyB2 were investigated through PCR-targeting system and the rapid gene disruption method applied in S. fradiae. The results showed that acyB2 should be a polyphonic factor, which at least positively control the expression of acyA, acyB1 and the mycarose transferase gene.There are two resistance genes, carA and carB in the genome of S. thermotolerans. CarA (similar to tlrC) encodes an ATP binding transporter protein and is responsible for the transmembrane of tylosin; CarB (similar to tlrB) encodes a ribosome monomethytransferase. ErmE derived from Saccharopolyspora erythraea, which encodes a ribosome dimethyltransferase, was expressed in S. thermotolerans, to increase the substance resistance during the biotransformation of tylosin to AIV. The resistance experiment showed the integrating expression of ermE could dramatically increase the resistance to tylosin of S. thermotolerans and the acylation activity was not affected. The negative regulator of Streptomyces differentiation (nsdA), the widespread gene in Streptomyces genome, was also investigated in S. thermotolerans. The disruption cassette of nsdA was constructed through PCR-targeting system. The nsdA mutant was obtained through intergeneric conjugal transfer between S. thermotolerans and E. coli. The fermentation results in the level of flask showed the disruption of nsdA gene could increase the acylation activity of S. thermotolerans.The substance tolerance of S. thermotolerans was also investigated. Two tylosin derivatives, acetyl-desmycosin and 20-Deoxy-20-(3, 5-dimethylpiperidin-1-yl)-tylosin was acquired.