Genetic Engineering in Combination with Semi-Synthesis Leads to a New Route for Gram-Scale Production of the Immunosuppressive Natural Product Brasilicardin†A.

Brasilicardin A (1) consists of an unusual anti/syn/anti-perhydrophenanthrene skeleton with a carbohydrate side chain and an amino acid moiety. It exhibits potent immunosuppressive activity, yet its mode of action differs from standard drugs that are currently in use. Further pre-clinical evaluation of this promising, biologically active natural product is hampered by restricted access to the ready material, as its synthesis requires both a low-yielding fermentation process using a pathogenic organism and an elaborate, multi-step total synthesis. Our semi-synthetic approach included a) the heterologous expression of the brasilicardin A gene cluster in different non-pathogenic bacterial strains producing brasilicardin A aglycone (5) in excellent yield and b) the chemical transformation of the aglycone 5 into the trifluoroacetic acid salt of brasilicardin A (1 a) via a short and straightforward five-steps synthetic route. Additionally, we report the first preclinical data for brasilicardin A.

Molecular genetics of naringenin biosynthesis, a typical plant secondary metabolite produced by Streptomyces clavuligerus.

BACKGROUND: Some types of flavonoid intermediates seemed to be restricted to plants. Naringenin is a typical plant metabolite, that has never been reported to be produced in prokariotes. Naringenin is formed by the action of a chalcone synthase using as starter 4-coumaroyl-CoA, which in dicotyledonous plants derives from phenylalanine by the action of a phenylalanine ammonia lyase. RESULTS: A compound produced by Streptomyces clavuligerus has been identified by LC-MS and NMR as naringenin and coelutes in HPLC with a naringenin standard. Genome mining of S. clavuligerus revealed the presence of a gene for a chalcone synthase (ncs), side by side to a gene encoding a P450 cytochrome (ncyP) and separated from a gene encoding a Pal/Tal ammonia lyase (tal). Deletion of any of these genes results in naringenin non producer mutants. Complementation with the deleted gene restores naringenin production in the transformants. Furthermore, naringenin production increases in cultures supplemented with phenylalanine or tyrosine. CONCLUSION: This is the first time that naringenin is reported to be produced naturally in a prokariote. Interestingly three non-clustered genes are involved in naringenin production, which is unusual for secondary metabolites. A tentative pathway for naringenin biosynthesis has been proposed.

A novel LysR-type regulator negatively affects biosynthesis of the immunosuppressant brasilicardin.

Brasilicardin A (BraA) is a promising immunosuppressive compound produced naturally by the pathogenic bacterium Nocardia terpenica IFM 0406. Heterologous host expression of brasilicardin gene cluster showed to be efficient to bypass the safety issues, low production levels and lack of genetic tools related with the use of native producer. Further improvement of production yields requires better understanding of gene expression regulation within the BraA biosynthetic gene cluster (Bra-BGC); however, the only so far known regulator of this gene cluster is Bra12. In this study, we discovered the protein LysRNt, a novel member of the LysR-type transcriptional regulator family, as a regulator of the Bra-BGC. Using in vitro approaches, we identified the gene promoters which are controlled by LysRNt within the Bra-BGC. Corresponding genes encode enzymes involved in BraA biosynthesis as well as the key Bra-BGC regulator Bra12. Importantly, we provide in vivo evidence that LysRNt negatively affects production of brasilicardin congeners in the heterologous host Amycolatopsis japonicum. Finally, we demonstrate that some of the pathway related metabolites, and their chemical analogs, can interact with LysRNt which in turn affects its DNA-binding activity.

ArgR of Streptomyces coelicolor Is a Pleiotropic Transcriptional Regulator: Effect on the Transcriptome, Antibiotic Production, and Differentiation in Liquid Cultures.

ArgR is a well-characterized transcriptional repressor controlling the expression of arginine and pyrimidine biosynthetic genes in bacteria. In this work, the biological role of Streptomyces coelicolor ArgR was analyzed by comparing the transcriptomes of S. coelicolor ΔargR and its parental strain, S. coelicolor M145, at five different times over a 66-h period. The effect of S. coelicolor ArgR was more widespread than that of the orthologous protein of Escherichia coli, affecting the expression of 1544 genes along the microarray time series. This S. coelicolor regulator repressed the expression of arginine and pyrimidine biosynthetic genes, but it also modulated the expression of genes not previously described to be regulated by ArgR: genes involved in nitrogen metabolism and nitrate utilization; the act, red, and cpk genes for antibiotic production; genes for the synthesis of the osmotic stress protector ectoine; genes related to hydrophobic cover formation and sporulation (chaplins, rodlins, ramR, and whi genes); all the cwg genes encoding proteins for glycan cell wall biosynthesis; and genes involved in gas vesicle formation. Many of these genes contain ARG boxes for ArgR binding. ArgR binding to seven new ARG boxes, located upstream or near the ectA-ectB, afsS, afsR, glnR, and redH genes, was tested by DNA band-shift assays. These data and those of previously assayed fragments permitted the construction of an improved model of the ArgR binding site. Interestingly, the overexpression of sporulation genes observed in the ΔargR mutant in our culture conditions correlated with a sporulation-like process, an uncommon phenotype.

High-Quality Draft Genome Sequence of the Actinobacterium Nocardia terpenica IFM 0406, Producer of the Immunosuppressant Brasilicardins, Using Illumina and PacBio Technologies.

The bacterium Nocardia terpenica IFM 0406 is known as the producer of the immunosuppressant brasilicardin A. Here, we report the completely sequenced genome of strain IFM 0406, which facilitates the heterologous expression of the brasilicardin biosynthetic gene cluster but also unveils the intriguing biosynthetic capacity of the strain to produce secondary metabolites.

ArgR of Streptomyces coelicolor is a versatile regulator.

ArgR is the regulator of arginine biosynthesis genes in Streptomyces species. Transcriptomic comparison by microarrays has been made between Streptomyces coelicolor M145 and its mutant S. coelicolor ΔargR under control, unsupplemented conditions, and in the presence of arginine. Expression of 459 genes was different in transcriptomic assays, but only 27 genes were affected by arginine supplementation. Arginine and pyrimidine biosynthesis genes were derepressed by the lack of ArgR, while no strong effect on expression resulted on arginine supplementation. Several nitrogen metabolism genes expression as glnK, glnA and glnII, were downregulated in S. coelicolor ΔargR. In addition, downregulation of genes for the yellow type I polyketide CPK antibiotic and for the antibiotic regulatory genes afsS and scbR was observed. The transcriptomic data were validated by either reverse transcription-PCR, expression of the gene-promoter coupled to the luciferase gene, proteomic or by electrophoresis mobility shift assay (EMSA) using pure Strep-tagged ArgR. Two ARG-boxes in the arginine operon genes suggest that these genes are more tightly controlled. Other genes, including genes encoding regulatory proteins, possess a DNA sequence formed by a single ARG-box which responds to ArgR, as validated by EMSA.