Abstract
BackgroundBitespiramycin (BT) is produced by recombinant spiramycin (SP) producing strain Streptomyces spiramyceticus harboring a heterologous 4″-O-isovaleryltransferase gene (ist). Exogenous l-Leucine (l-Leu) could improve the production of BT. The orf2 gene found from the genomic sequence of S. spiramyceticus encodes a leucine-responsive regulatory protein (Lrp) family regulator named as SSP_Lrp. The functions of SSP_Lrp and l-Leu involved in the biosynthesis of spiramycin (SP) and BT were investigated in S. spiramyceticus.ResultsSSP_Lrp was a global regulator directly affecting the expression of three positive regulatory genes, bsm23, bsm42 and acyB2, in SP or BT biosynthesis. Inactivation of SSP_Lrp gene in S. spiramyceticus 1941 caused minor increase of SP production. However, SP production of the ΔSSP_Lrp-SP strain containing an SSP_Lrp deficient of putative l-Leu binding domain was higher than that of S. spiramyceticus 1941 (476.2 ± 3.1 μg/L versus 313.3 ± 25.2 μg/L, respectively), especially SP III increased remarkably. The yield of BT in ΔSSP_Lrp-BT strain was more than twice than that in 1941-BT. The fact that intracellular concentrations of branched-chain amino acids (BCAAs) decreased markedly in the ΔSSP_Lrp-SP demonstrated increasing catabolism of BCAAs provided more precursors for SP biosynthesis. Comparative analysis of transcriptome profiles of the ΔSSP_Lrp-SP and S. spiramyceticus 1941 found 12 genes with obvious differences in expression, including 6 up-regulated genes and 6 down-regulated genes. The up-regulated genes are related to PKS gene for SP biosynthesis, isoprenoid biosynthesis, a Sigma24 family factor, the metabolism of aspartic acid, pyruvate and acyl-CoA; and the down-regulated genes are associated with ribosomal proteins, an AcrR family regulator, and biosynthesis of terpenoid, glutamate and glutamine.ConclusionSSP_Lrp in S. spiramyceticus was a negative regulator involved in the SP and BT biosynthesis. The deletion of SSP_Lrp putative l-Leu binding domain was advantageous for production of BT and SP, especially their III components.
Highlights
Bitespiramycin (BT) is produced by recombinant spiramycin (SP) producing strain Streptomyces spiramyceticus harboring a heterologous 4′′-O-isovaleryltransferase gene
The leucine-responsive regulatory protein (Lrp) gene in S. spiramyceticus 1941 There are three positive regulatory genes to be found in the BT biosynthetic gene cluster (GenBank accession number: MH460451) including acyB2 (GenBank accession number: D31821.1 or KR818745), bsm42, and bsm23
Expression of SSP_Lrp in E. coli BL21 (DE3) and electrophoretic mobility shift assay (EMSA) His6-tagged SSP_Lrp was expressed in E. coli BL21 (DE3) (Fig. 2a), and its affinity for promoter sequences of regulatory genes involved in SP and BT biosynthesis was examined by EMSA
Summary
Bitespiramycin (BT) is produced by recombinant spiramycin (SP) producing strain Streptomyces spiramyceticus harboring a heterologous 4′′-O-isovaleryltransferase gene (ist). Exogenous l-Leucine (l-Leu) could improve the production of BT. The orf gene found from the genomic sequence of S. spiramyceticus encodes a leucine-responsive regulatory protein (Lrp) family regulator named as SSP_Lrp. The functions of SSP_Lrp and l-Leu involved in the biosynthesis of spiramycin (SP) and BT were investigated in S. spiramyceticus. An effective way to develop high-producing BT strain that produces more l-Leu is to eliminate the feedback regulation caused by l-Leu itself as end product. The engineering of relevant regulatory genes to modulate transcriptional activation or inhibition is an effective way to improve the yield of target secondary metabolites in Streptomyces [6, 7]
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