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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 ?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 ?g/L versus 313.3?±?25.2 ?g/L, respectively), especially SP III increased remarkably. The yield of BT in ?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 ?SSP_Lrp-SP demonstrated increasing catabolism of BCAAs provided more precursors for SP biosynthesis. Comparative analysis of transcriptome profiles of the ?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.

SUBMITTER: Lu Z

PROVIDER: S-EPMC6379999 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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

Lu Zhili Z Zhang Xiaoting X Dai Jianlu J Wang Yiguang Y He Weiqing W

Microbial cell factories 20190219 1

<h4>Background</h4>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 ...[more]

PMID: 30782164

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Project description:Feast-famine response proteins are a widely conserved class of global regulators in prokaryotes, the most highly studied of which is the Escherichia coli leucine-responsive regulatory protein (Lrp). Lrp senses the environmental nutrition status and subsequently regulates up to one-third of the genes in E. coli, either directly or indirectly. Lrp exists predominantly as octamers and hexadecamers (16mers), where leucine is believed to shift the equilibrium toward the octameric state. In this study, we analyzed the effects of three oligomerization state mutants of Lrp in terms of their ability to bind to DNA and regulate gene expression in response to exogenous leucine. We find that oligomerization beyond dimers is required for Lrp's regulatory activity and that, contrary to previous speculation, exogenous leucine modulates Lrp activity at its target promoters exclusively by inhibiting Lrp binding to DNA. We also show evidence that Lrp binding bridges DNA over length scales of multiple kilobases, revealing a new range of mechanisms for Lrp-mediated transcriptional regulation. IMPORTANCE Leucine-responsive regulatory protein (Lrp) is one of the most impactful regulators in E. coli and other bacteria. Lrp senses nutrient conditions and responds by controlling strategies for virulence, cellular motility, and nutrient acquisition. Despite its importance and being evolutionarily highly conserved across bacteria and archaea, several mysteries remain regarding Lrp, including how it actually responds to leucine to change its regulation of targets. Previous studies have led to the hypothesis that Lrp switches between two states, an octamer (8 Lrp molecules together) and a hexadecamer (16 Lrp molecules together), upon exposure to leucine; these are referred to as different oligomerization states. Here, we show that contrary to previous expectations, it is Lrp's propensity to bind DNA, rather than its oligomerization state, that is directly affected by leucine in the cell's environment. Our new understanding of Lrp activity will aid in identifying and disrupting pathways used by bacteria to cause disease.

Dataset Information

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

Publications

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

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