Construction Of Novel Strains For FK506 (Tacrolimus) Production

FK506 (Tacrolimus) is a well-known immunosuppressant that has been effectively used for the treatment of organ transplantation and auto-immune diseases. However, the production titer of FK506 is still very low, which restricted its economic production. Therefore, improvement of the production titer of FK506 is of great importance.In this study, heterologous expression of the entire biosynthetic gene cluster of FK506 was first attempted in the host strains that have clear genetic backgrounds. Through this approach, novel FK506 producing strain that can be manipulated by rational regulation can be constructed. The BAC (Bacteria Artificial Chromosome) library of the FK506 producer Streptomyces sp. KCCM 11116P was constructed, and a BAC vector containing the entire FK506 biosynthetic gene cluster was obtained. Using designed artificial fragments, BAC expression plasmid was constructed quickly via In-fusion and ?-RED mediated recombination. Heterologous expression strain based on S. albus J1074 was obtained and the transcription of both the antibiotic resistance gene and FK506 biosynthetic genes were confirmed. However, no FK506 production was detected, which indicates that the expression of FK506 biosynthetic gene cluster may be restricted by the characteristics of heterologous hosts.Subsequently, the possibility of enhancing the FK506 production by overexpression of novel transcriptional regulators was explored. Transcriptional regulators SidR, Sx5140 and SinR from marine actinomycetes, as well as two SARP family regulators BulZ and BulY that was isolated from FK506 producing strain were selected. Overexpression of these regulators was performed in Streptomyces sp. KCCM 11116P to construct FK506 producing strains. The fermentation results showed that overexpressing Sx5140 enhanced the production of FK506 in the fermentation prophase, but at late fermentation stage the FK506 titer was almost the same as the control strain. Overexpression of BulZ and BulY in Streptomyces sp. KCCM 11116P was also investigated. The fermentation results showed enhanced production of FK506 throughout the fermentation, the highest increase in bluZ overexpression strain is up to 1.6 fold of that in the control level. In case of other transcription regulators, no enhancement of FK506 production titer was detected.In order to explore the molecular mechanism of SARP family regulator on FK506 production, real time RT-PCR analysis was performed to detect the expression level of several key genes involved in the biosynthesis of FK506. The results showed that the expression levels of several genes were up-regulated, together with the gamma-butyrolactone receptor protein gene tsuRl. The results in this study revealed novel global regulatory mechanisms in FK506 biosynthesis, which added new aspects in addition to the pathway-specific regulation in the previous reports. The results in this study pave the way for further studying the regulatory mechanism of FK506 biosynthetic production, as well as exploration of novel regulators from marine actinomycetes to construct novel producers with improved production titier of antibiotics.