Study On Efficient Biosynthesis Of Tacrolimus And Activation Of The Tsukabarubucin Gene Cluster

Tacrolimus(FK506)is the preferred anti-rejection drug used for organ transplantation and the international market has reached 2.3 billion dollars.But its fermentation level is relatively low and the costs for production of raw materials is high,so we can use synthetic biology technology to systematically carry out high-yield modifications.Genome sequencing has revealed that Streptomyces contains a wide range of biosynthetic gene clusters(BGCs).However,most gene clusters are cryptic under normal growth conditions.Activation of cryptic gene clusters can discover new active compounds from them,which can provide candidate compounds for the creation of new drugs In this paper,using the industrial tacrolimus-producing Streptomyces tsukubaensis L19 as experimental materials,we focused on systematically refactoring the biosynthesis system for tacrolimus using synthetic biology technology and activating the cryptic biosynthetic gene clusters.The results are illustrated as follows:From the aspects of competitive pathways,regulatory pathway remodeling and enhancing the supply of precursors,we refactored the biosynthesis system of tacrolimus.(1)Basing on RT-PCR analysis,three putative competitive pathways were screened and blocked,yielding the mutant strain L19-2 with increased tacrolimus yield from 140.3 to 170.3 mg/L and decreased non-desired secondary metabolites.(2)By replacing the intergenic rigon between the gene tcs6 and tcs7 in tacrolimus biosynthetic gene cluster with the constructed bidirectional promoter casettes in situ,the regulatory pathway of tacrolimus biosynthesis was reshaped.In the resultant strain L19-9,the transcriptional levels of all biosynthetic genes in the tacrolimus gene cluster were significantly increased and the tacrolimus yield increased dramatically from 170.3 mg/L to410.3 mg/L in shake-flask culture.(3)For enhancing the supply of precursors,the tacrolimus production in strain L19-9 was further increased from 410.3 to 603 mg/L in shake-flask culture by adding L-isoleucine at a final concentration of 6 g/L into Fm mediumin.Moreover,the tacrolimus titer of the strain L19-9 could reach 830.3 mg/L when fermented in a 15-L fermenter using the optimized meidium,showing great potential for industrial application.Meanwhile,through activation of cryptic gene clusters in strain L19,we identified a novel anthracycline compound with potent bioactivity.(1)Combining overexpression of the putative SARPs(Streptomyces antibiotic regulatory proteins)and bioactivity-guided screening,a novel anthracycline(tsukubarubicin)was further isolated and identified.(2)Bioactivity experiments showed that the promising tsukubarubicin exhibited more potent antitumor activity than clinically used doxorubicin against lung cancer cell line,colon cancer cell line,breast cancer cell line and gastric cancer cell line.(3)According to the structure of tsukubarubicin and bioinformatics analysis,the gene cluster tsu for biosynthesis of tsukubarubicin was first characterized and the biosynthetic mechanisms of tsukubarubicin was proposed.In summary,we realized the efficient biosynthesis of tacrolimus and at the same time,we discovered the novel compound tsukubarubicin with potent antitumor activity.The strategies for hyper-production and activation of cryptic gene cluster presented in this paper are generally applicable and lay an important foundation for carrying out related works in industrial Streptomyces.