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

Abstract

Background 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 . Results 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 Δ SSP_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 Δ SSP_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 Δ SSP_Lrp -SP demonstrated increasing catabolism of BCAAs provided more precursors for SP biosynthesis. Comparative analysis of transcriptome profiles of the Δ SSP_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. Conclusion 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. Electronic supplementary material The online version of this article (10.1186/s12934-019-1086-0) contains supplementary material, which is available to authorized users.

Enhancement of Carrimycin Production Via Traditional Mutagenesis with Metabolic Engineering in Streptomyces Spiramyceticus 54IA

“…The resulting fermentation broth was extracted with ethyl acetate (100 mL), and the solvent was removed in vacuum. The extracts were dissolved in 1 mL CH 3 OH and centrifuged at 13,000g for 10 min, and 10 μL of supernatant was subjected to HPLC analysis following the previously described system [15]. In order to calculate the yields of carrimycin in the mutants and the starting strain 54IA, a quantitative curve was established based on the relationship of integral area and the weight of carrimycin.…”

“…The titer of carrimycin was improved through the ribosome engineering, but it was far from the requirement in the scale-up fermentation. Many efforts have been made to improve the production of carrimycin in Streptomyces spiramyceticus WSJ−195 in recent years, including traditional random mutagenesis [9,10], medium optimization [11], exogenous feeding strategies [12,13], fermentation process control [14], and genetic engineering [15]. Among these approaches, traditional mutagenesis is a powerful and easily operated method for strain improvement in Streptomyces, especially for microbes with less understanding of genomic information and metabolic mechanisms.…”

Synthetic biology enabling access to designer polyketides

“…Exploring and optimizing polyketide biosynthesis can be completed within native production strains given that the BGC and necessary precursors are often already functionally expressed and produced. Typically, enhancing polyketide production focuses on the redirection and optimization of carbon flux via enhanced precursor availability [11,12], promoter and ribosome engineering [13], removal or overexpression of regulatory elements [14,15], deletion of competing biosynthetic pathways, and combinations thereof to significantly enhance titers above that of the wild-type pathway. Strategies including cooperative induction [9], co-culturing [16], and transcription factor decoys [17] have been explored for the activation of cryptic BGCs (cBGCs).…”

Anti-tumor effect of carrimycin on oral squamous cell carcinoma cells in vitro and in vivo

“…Carrimycin (trade name: Bite, formerly shengjimycin and bitespiramycin), a new genetic engineering 16-membered macrolide antibiotic, is a mixture of isovalerylspiramycin I, II and III, and a certain amount of (iso)butyryl/propionyl/acetylspiramycin III and(iso)butyryl/propionyl/acetylspiramycin II ( Fig. 1 a) [6] . Completed phase III clinical trial showed an excellent anti-bacterial effect of carrimycin, as well as good bioavailability and high biosafety.…”