Cloning, sequencing and characterization of the biosynthetic gene cluster of sanglifehrin A, a potent cyclophilin inhibitor

Abstract

Sanglifehrin A (SFA), a potent cyclophilin inhibitor produced by Streptomyces flaveolus DSM 9954, bears a unique [5.5] spirolactam moiety conjugated with a 22-membered, highly functionalized macrolide through a linear carbon chain. SFA displays a diverse range of biological activities and offers significant therapeutic potential. However, the structural complexity of SFA poses a tremendous challenge for new analogue development via chemical synthesis. Based on a rational prediction of its biosynthetic origin, herein we report the cloning, sequencing and characterization of the gene cluster responsible for SFA biosynthesis. Analysis of the 92 776 bp contiguous DNA region reveals a mixed polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) pathway which includes a variety of unique features for unusual PKS and NRPS building block formation. Our findings suggest that SFA biosynthesis requires a crotonyl-CoA reductase/carboxylase (CCR) for generation of the putative unusual PKS starter unit (2R)-2-ethylmalonamyl-CoA, an iterative type I PKS for the putative atypical extender unit (2S)-2-(2-oxo-butyl)malonyl-CoA and a phenylalanine hydroxylase for the NRPS extender unit (2S)-m-tyrosine. A spontaneous ketalization of significant note, may trigger spirolactam formation in a stereo-selective manner. This study provides a framework for the application of combinatorial biosynthesis methods in order to expand the structural diversity of SFA.

N-Carbamoylation of 2,4-Diaminobutyrate Reroutes the Outcome in Padanamide Biosynthesis

“…We constructed DpadO and its complemented mutant (Figure 2A; Figure S2C), and the HPLC results showed that the DpadO mutant abolished padanamide production, indicating that it probably acts as a pathway activator. PadO also has sequence identity of 51% to Orf4 of the kutzneride gene cluster (Fujimori et al, 2007) and 45% to SfaC of the sanglifehrin gene cluster (Qu et al, 2011). Both Orf4 and SfaC are annotated as putative regulators; however, no genetic evidence is available.…”

“…Accessory genes would be involved in the construction of the 2-methoxyacetyl group and might resemble the starter unit pathway for apoptolidins (Du et al, 2011). Additionally, an ornithine N-monooxygenase would likely generate N 5 -OH-ornithine, the precursor to Piz, as observed in the biosynthesis of the kutznerides (Fujimori et al, 2007), sanglifehrin A (Qu et al, 2011), himastatin (Ma et al, 2011), and other Piz-containing natural products. To find the padanamide gene cluster, we initially explored isolation of adenylation (A) domain and N-oxygenase genes from RJA2928 genomic DNA using PCR with degenerate primers.…”

“…The gene product of padN is homologous to various lysine/ ornithine monooxygenases, and its homologs have been found in the biosynthetic gene clusters of kutznerides, himastatin, and sanglifehrin, all of which contain Piz (Fujimori et al, 2007;Ma et al, 2011;Qu et al, 2011). KtzI, the homolog from the kutzneride gene cluster, has been recently defined as an ornithine N-hydroxylase, which catalyzes the conversion of ornithine to N 5 -OH-ornithine (Neumann et al, 2012).…”

Nichtproteinogene Aminosäurebausteine für Peptidgerüste aus nichtribosomalen Peptiden und hybriden Polyketiden

“…Rapamycine, die Verbindungen der FK506-Familie, [7,8] Sanglifehrin [9] und die Epothilone sind Beispiele für therapeutisch interessante Gruppen solcher molekularer NRP-PKHybride mit nichtproteinogenen Aminosäureeinheiten. [10,11] 2.…”

CDKN2B, SLC19A3 and DLEC1 promoter methylation alterations in the bone marrow of patients with acute myeloid leukemia during chemotherapy

“…Samples were determined to be methylated or unmethylated on the basis of the visible peaks generated by the Q-Analyzer. The methylated and unmethylated primer sequences of CDKN2B (27), SLC19A3 (28) and DLEC1 (29) genes are presented in Table II . Some of the DNA samples were sequenced using an ABI 3730 DNA Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the results indicated a successful bisulfite conversion and amplification (Fig.…”