Heterologous Expression Of Blasticidin S In S. Lividans Strain And Genetic Study Of The Key Genes In The Blasticidin S Biosynthetic Gene Cluster

Blasticidin S (BS) belongs to the peptidyl nucleoside family. This compound is very important in the protection of rice from rice blast and in the screening of the transgenic mutant in eukaryotic genetic engineering. Although the biosynthetic gene cluster of blasticidin S was reported in2003, the integrality of the gene cluster was still being assessed. Furthermore, the tough genetic manipulation barrier of the blasticidin S native producer S. griseochromogenes has circumvented the in vivo research on the BS biosynthesis. Through chromosome walking, a cosmid7D11containing the known BS biosynthetic gene cluster along with8.6kb downstream sequence was obtained from the genomic library of S. griseochromogenes. Bioinformatic analysis of the8.6kb sequence showed that it was irrelevant to the biosynthesis of blasticidin S.To further investigate the biosynthesis of blasticidin S, S. lividans LL2was engrafted with the insertion of7D11through two rounds of double crossovers. To our surprise, the bioassay of LL2showed that it had no inhibition effect on indicator strain Rhodotorula rubra AS2.166. To look into the reason why BS failed to be produce by LL2, the fermentation broth of LL2was analyzed by LC‐MS. The LC‐MS result showed that LL2could only produce non‐cytotoic deaminohydroxyblasticidin S(OH‐BS) instead of blasticidin S. Through bioinformatic analysis, a homolog of blasticidin S deaminase from Aspergillus terreus was revealed in the genome of S. lividians HXY16, and was named as SLBSD. His‐tagged SLBSD from E. coli was able to catalyze the transformation of blasticidin S to OH‐BS in vitro. Moreover, SLBSD also showed the deaminase activity toward two nucleosides, mildiomycin and CGA. These results were surprising as both mildiomycin and CGA had been successfully heterologously expressed in the Streptomyces lividans. Compared to wild type S. lividans HXY16, SLBSD disruption mutant S. lividans WJ1becomes more sensitive to blasticidin S at a concentration of100μg/ml.The SLBSD gene was disrupted in S. lividans LL2for the sake of the heterologous expression of BS. LC‐MS analysis of the fermentation broth of the resulted mutant S. lividans WJ2showed that S. lividans WJ2could successfully produce blasticidin S, which implied that S. lividans WJ2contained all necessary genes for BS biosynthesis. To test the effect of ions in the medium on BS production, S. lividans WJ2was inoculated in seed/fermentation broth and meassured by LC‐MS. LC‐MS assay revealed a new metabolite in the fermentation broth that was confirmed as demethylblasticidin S through MS and MS/MS analysis.For further investigation of BS biosynthesis, several genes in the gene cluster were studied in vivo. Bioinformatic prediction showed that BlsL was a N‐methyl transferase that might be involved in the formation of demethylblasticidin S. Production of BS was abolished in blsL mutant S. lividans WJ3. However, a large amount of demethylblasticidin S was accumulated in the resulted mutant. To exclude the possible polar effect caused by gene replacement, blsL was recomplemented to mutant S. lividans WJ3and it could restore the mutant to the ability of BS production, indicating that BlsL alone catalyzed the tranformation of demethylblasticidin S to blasticidin S. The previous proposed pathway by Cone et al should be reevaluated.In BS gene cluster, the function of blsF was unknown. Sequence alignment showed that BlsF shared homology to MilL, which was located in the mildiomycin biosynthesis gene cluster which was unnecessary to mildiomycin biosynthesis. To elucidate the function of BlsF, blsF was disrupted to construct S. lividans WJ4. LC‐MS analysis of the fermentation broth of S. lividans WJ4showed that the production of BS was increased in S. lividans WJ4. Moreover, a large amount of CGA, the intermediate of the BS biosynthetic pathway, was accumulated in the unpurified fermentation broth of S. lividans WJ4. These results implied that blsF could be a pathway specific regulatory gene in the BS gene cluster.In BS gene cluster, blsE encodes a Radical SAM family protein and its function was unkown.To identify the substrate of Blse, blsE was disrupted in S. lividans WJ2. The blsE disruption mutant S. lividans WJ5abolished the production of blasticidin S while a large amount of BS biosynthesis intermediate CGA was accumulated in the unpurified fermentation broth. Disruption of milG, A homolog of blsE in mildiomycin biosynthesis gene cluster showed similar result, in which huge amounts of hydroxymethyl‐CGA was accumulated. Together, CGA was confirmed as the substrate of BlsE.Recently, it proved that a decarboxylation‐recarboxylation process was exited in the BS biosynthesis. However, in the published cluster, none product of the gene was predicted as an carboxylase. Deeply researching into the gene cluster revealed that BlsI show homology to biotin carboxylase. To testify the function of BlsI, blsI was replaced by an aadA cassette to construct the mutant S. lividans WJ6. LC‐MS analaysis of the fermentation broth of WJ6showed that S. lividans WJ6abolished the production of blasticidin S. However, a metabolite with a mass of227 was detected in the unpurified fermentation broth of S. lividans WJ6. MS/MS of this metabolite indicated it contained a cytosine moiety and thus it was recognized as an intermediate in the BS biosynthetic pathway. This result exploded the former predictioted function of BlsI, including activating β‐arginine or leucyl‐β‐arginine for coupling with an amino deoxynucleoside and activating leucine for attachment to β‐arginine or demethylblasticidin S to give the dipeptide or demethylleucylblasticidin S.In summary, an artificial BS producing strain was obtained by successful heterologous expression of BS. With the help of the easy genetic manipulation of S. lividans, several key genes in the BS gene cluster was studied in vivo for the first time, which layed a solid foundation for future research of enzyme assay and biosynthetic pathway.