Biosynthesis Of Echosides In Streptomyces Sp. LZ35

The para-terphenyl natural products have unique structural features and diverse biological activities, including antioxidant, antitumor and immunosuppressive activities. Most of these compounds were isolated from fungi, and only a dozen members have been reported from Streptomyces species. The biosynthesis mechanism of this group of metabolites has not been reported until recent years. Therefore, the studies on the biosynthesis of p-terphenyls will lead to the discovery of novel enzymatic mechanism, and facilitate bioengineering novel p-terphenyls with better properties.Previously, five novel p-terphenyls bearing glucuronic acid moiety, namely echosides A-E, were isolated from Streptomyces sp. LZ35. Base on the biosynthetic genes of p-terphenyls in fungi, two putative p-terphenyls biosynthetic gene clusters (SM4and SM25) were identified from the genome of strain LZ35. Both of them have a single-module NRPS-like enzyme, which is proposed to be responsible for the biosynthesis of the core carbon skeleton of p-terphenyls. In order to identify which one is involved in the formation of echosides, the gene disruption experiments were performed. The HPLC analysis of the metabolites from the mutants and wild type revealed that SM25is responsible for the biosynthesis of echosides. The SM25cluster consists of a single-module NRPS gene (echA), which lacks a condensation (C) domain, seven redox genes (echB, echC, orf4-6,9, and12), and a methyltransferase gene (echD). In addition, the ech cluster harbors genes postulated to encode transporter genes (orf1-3) and regulatory factor genes (orf7,10,11, and13). Surprisingly, there is no gene encoding aminotransferase, which is responsible for the formation of phenylpyruvic acid from L-phenylalanine, and a gene encoding glycosyltransferase, which is responsible for the addition of the sugar residue to the echosides, located in the gene cluster. Both of them could be located elsewhere in the genome of strain LZ35. In this study, the "core" genes in SM25, echABC, were characterized by genetic and biochemical means.The gene disruption experiments indicated that only three genes in SM25, echABC, were involved in the biosynthesis of echosides. The metabolic profiling of single and double mutant of echB and echC revealed that the reaction catalyzed by EchB is before that by EchC. In addition, the intermediates accumulated in the echB and echC mutant were identified. Base on the above results, the biosynthetic pathway of echosides was proposed: two molecules of phenylpyruvate were condensed into polyporic acid by EchA, which was then reduced to DP-A, in the presence of NAD(P)H, by EchB, followed by dehydration catalyzed by EchC to produce the aglycone of echoside C, which was further processed by glycosylation, methylation and heterocyclization to give echosides.The quinone synthetase EchA is a single tri-domain (adenylation-thiolation-thioesterase, A-T-TE) NRPS module, which lacks the condensation domain for peptide bond formation. The a-aromatic keto acid was activated and loaded by A domain, followed by Dieckmann condensation to form C-C bond and released from the NRPS catalyzed by TE domain, which presents a novel catalytic mechanism of NRPS. Previously, the TE domain of NRPS can only release linear polypeptide chain by forming C-O bond, or cyclized polypeptide chain by forming C-N bond. According to the studies on TdiA and AtrA, we prepared holo-EchA through enzymatic phosphopantetheinyl (Ppant) transfer, catalyzed by the Ppant transferase Sfp. When the holo-EchA was incubated with phenylpyruvate and ATP, it was able to convert phenylpyruvate to polyporic acid. By measuring the conversion of phenylpyruvate to polyporic acid, the maintenance of good activity of EchA was presented in a linear reaction phase, and we determined that EchA had a KM of4.5±1.1μM and a kcat of0.35±0.02min-1. We therefore conclude that EchA represents the first quinone synthetase of the Streptomyces genus that has been biochemically characterized. Since this is the first report that a quinone synthetase can use phenylpyruvate as a native substrate, EchA was named polyporic acid synthetase.Bioinformation analysis revealed that EchB belongs to short-chain dehydrogenases/reductases (SDRs) superfamily, which is one of largest protein superfamily widely distributed in the biosphere. Members of SDR superfamily typically shared only15-30%sequence identity, and the most conserved feature is a classical Rossmann fold for nucleotide binding. SDRs are different from other dehydrogenase by using NAD(H) or NADP(H) as co-factor, and no metal is required. When EchB was incubated with polyporic acid, in the presence of NADPH or NADH, DP-A was produced in the reaction mixture, which is the intermediate accumulated in the echC disruption mutant. Therefore, EchB was named polyporic acid reductase.Bioinformation analysis revealed that EchB has a SnoaL-like domain, which belongs to NTF2-like superfamily. Members of this superfamily include isomerase, hydrolase, dehydratase and many signal transduction related proteins. According to the intermediate identified from echC disruption mutant, we proposed that EchC is a dehydratase. However, the in vitro enzymatic assay got no positive results. In order to identify the reason, an experimental three dimensional structure of EchC will be much helpful. The crystal structure of EchC is a dimer, which is similar to other members of its family. Base on the electron density map, we observed that an unknown ligand occupied the active site pocket of EchC, which may affect the catalytic activity of EchC. Therefore, we will remove the ligand by means of degeneration/dialysis/refolding or express EchC in strain LZ35in the subsequent experiment, and then test the activity of EchC.In conclusion, the echosides (ech) biosynthetic gene cluster was identified and characterized. The gene knockout and intermediates identification experiments revealed that EchA, EchB and EchC were responsible for the formation of echosides from phenylpyruvate. In addition, the functions of EchA and EchB were biochemically characterized in vitro. Finally, the crystal structure of EchC has been resolved, which will facilitate the subsequent biochemically study of EchC.