Optimize The Components Of Biotechmycin By Genetic Engineering

Biotechmycins(BT) is a group of 4"-acylated spiramycins with 4"-isovalerylspiramycin as the major component, produced by recombinant S. spiramyceticus F21 integrating a 4"-O-acyltransferase gene (ist) from S. mycarofaciens 1748.The components of biotechmycin are very complicated with two main reasons, first, biotechmycins were derived from spiramycins,which have three components; second, 4"-O-isovaleryltransferase poor recognition to its substrates. The aim of this dissertation is to simplify the components of the BT through reducing the components of spiramycin, therefore simplifying the extraction process and promoting biotechmycins' industrialization.In this study, degenerated primers were designed according to the conserved regions of two3-O-acyltransferase, MdmB and AcyA, in the medicamycin and carbomycin producers of S.mycarofaciens and S. thermotolerans, respectively, and an 878bp DNA fragment was amplifiedfrom spiramycin-producer of S. spiramyceticus F21. Blast analysis of the 878bp DNA fragmentsuggested that it encoded the 3-O-acyltransferase (3-O-AT) gene for spiramycin biosynthesis.The flanking regions of this 878bp DNA fragment were then amplified bysingle-oligonucleotide-nested PCR, and a total of 4.3kb DNA was obtained, covering the wholeputative 3-O-acyltransferase gene, sspA, for spiramycin biosynthesis. Then the sspA was deletedfrom the S. spiramyceticus F21 genome by double cross-over homologous recombination,mediated by temperature-sensitive plasmid pKC1139. Comparison of the components ofspiramycins produced by the sspA-ddetod mutant strain with that of the parent strain by HPLCanalysis showed that sspA-deleted mutant mainly produced SP I. Then mdmB (medicamycin's3-O-acyltransferase gene) was inserted, in the place of sspA, by double cross-over homologousrecombination in the genome of sspA-deleted mutant.The expected mutant strain that supposedto produce mainly SPIII produced still a substantial amount of SPI and SPII. At the same time,mutant strains with sspA deleted or replaced with mdmB decreased significantly in theirspiramycin production levels.Based on the above experimental results, the strategy to reduce the components of BT is changed to delete a DNA fragment in sspA gene's frame from the genome of biotechmycin bioengineerd strain to make the mutants mainly produce isovalerylspiramycin I but not to influence their potential to produce spiramycin. To achieve this purpose, we inframe-deleted an internal of 612bp, about 50% of the size of the of sspA gene, by double cross-over of homologous DNA recombination. HPLC analysis showed that mutants with shortened sspA gene produced only SPI and its 4"-O-acylation products, with SPII and SPIII and its 4"-O-acylation products disappeared, drastically reduced the component number. The main derivative of 4"-O-acylspiramycin produced by the mutants was determined to be 4"-O-isovalerylspiramycin I. Thus, we obtained genetically engineered new biotechmycin producer with reduced component number and with 4"-O-isovalerylspiramycin I as the major component.