Engineering of leucine-responsive regulatory protein improves spiramycin and bitespiramycin biosynthesis

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 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.

SSP_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.

SSP_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.