Published by the University of Cape Town (UCT) under a non-exclusive license granted to UCT by the author. Financial support from the National Research Foundation (NRF) for this research is acknowledged. The opinions and conclusions expressed are those of the author and are not necessarily attributed to NRF.
At this stage, the model cannot account for the source of benzoic acid, because in vivo gene disruption experiments have disproved both hypotheses about how benzoic acid is synthesized in S.
General introduction
However, the biosynthetic machinery involved in the synthesis of spoxazomycins has not yet been elucidated (Inahashi et al., 2011). The thioesterase (TE) domain, in the termination module, catalyzes the release (and in some cases oligomerization and/or cyclization) of the mature peptide product bound to the NRPS (Figure 1.6) (Bloudoff et al., 2013). A carrier protein variant commonly found in NRPS siderophore systems is the aryl carrier protein (ArCP) (Qiao et al., 2007).
Moreover, this condensation reaction is strictly unidirectional, leading to a downstream synthesis of the NRPS product (Samel et al., 2007). The catalytic core motif, HHxxxDGxS, of the C domain has been modified to DxxxxDxxS in the Cy domain. Both sides are separated by the tryptophan indole ring at position 239 (Trp239), which is located at the bottom of the pocket (Stachelhaus et al., 1999).
Early attempts at isolating the gene cluster responsible for DPO
The decarboxylation of the oxazole unit will lead to the formation of DPO as the final product (Figure 2.1E). The formation of the amide bond and subsequent heterocyclization reaction is assumed to be catalyzed by an NRPS. The presence of an ortholog of the encP gene, encoding phenylalanine ammonialase (PAL), in the genome of S.
All degenerate primers used in the initial attempts to isolate the NRPS gene cluster are listed in Table 2.1, along with their sequences and binding positions. Two more Cy domain forward primers, VVFTS CycF and QTPQV CycF, were designed based on a multi-sequence alignment of the amino acid sequences used in the initial primer design (Table 2.2) plus three new Cy domain sequences from S. The primer set, PALHAL_F and PALHAL_R were designed using DNAMAN from the consensus sequence of a multi-sequence alignment of the PAL amino acid sequence.
Cycling conditions for amplification of the DPO gene array using primer sets A3F/Paak_AveR, Paak_AveF/A7R, VVFTS CycF/A7R, and QTPQV CycF/A7R were performed according to the manufacturer's instructions. The presence of the desired inserts was determined by performing colony PCR (section 2.3.4.6) using the M13F and M13R primer set (Table 2.1). POLYANTIBIOTICUS SPRT The identification of an A domain in the SPR strain genome with a binding pocket substrate specificity for phenylalanine or 3-hydroxyphenylalanine may indicate that the A domain is part of the NRPS responsible for DPO biosynthesis.
The identification of the genes surrounding the phenylalanine A domain, as well as the A domains specific for serine, will help to elucidate the DPO biosynthetic strategy. An amplification product of the correct size was observed using the VVFTS CycF/A7R primer set, but sequencing revealed that a non-target product was amplified. It has been shown that the presence of the PAL-encoding gene, encP, is absolutely necessary for benzoyl-CoA formation in 'S.
Organization of the biosynthetic gene cluster for the macrolide anthelmintic polyketide avermectin in Streptomyces avermitilis.
Streptomyces polyantibioticus SPR T genome exploration
114 3.4.3 Identification of the putative DPO biosynthetic gene cluster 117 3.4.4 Examination of the putative DPO gene cluster. The gene content and arrangement of the genes in the proposed DPO gene cluster was identical to the biosynthetic congocidin gene cluster identified in Streptomyces ambofaciens ATCC 23877T. Analysis of the NRPS domains within the putative DPO biosynthetic gene cluster revealed a non-linear NRPS initiation module, two solitary C domains, a single PCP domain and a solitary A domain encoded by an acyl-CoA synthetase.
Initially, the genes in each of the individual contigs obtained from the 454 genome sequencing were predicted using the Rapid Annotation Using Subsystem Technology (RAST) 2.0 server database (Overbeek et al., 2014). In addition, the conserved NRPS signature motifs, as described by Schwarzer et al. 2003), was examined in the multiple sequence alignment of the putative S. The secondary metabolite gene cluster spanning the region located from nucleotide addition A) in the draft S.
Furthermore, all the genes that make up this group share homology with the genes that make up the coelichelin biosynthetic group identified in S. All genes in this group share homology with the congocidin biosynthetic gene group identified in S. Interestingly, the composition gene clusters and gene arrangement were most similar to the pyrrole-amide congocidin (also known as netropsin) biosynthetic gene cluster (Figure 3.2) identified in S.
In addition, the organization of the only complete NRPS module, A-PCP-C, differs from the canonical C-A-PCP organization, and the remaining NRPS-related domains are independent (Juguet et al., 2009). Both domain A specificity prediction results obtained by the LSI algorithm were based on an LSI score below 0.66, which is classified as an unreliable prediction (Baranasic et al., 2013). Ox domains have been identified as members of the McbC-like oxidoreductase superfamily and are commonly associated with heterocyclization modules (Marchler-Bauer et al., 2011).
A similar atypical positioning of the Ox domain may also occur in genes for DPO biosynthesis in S. The SPR_53090 gene was identified by antiSMASH to encode a TE domain.
Development of a transformation protocol for Streptomyces
The reason for the different response to protoplast transformation of taxonomically related species may be related to the variation in bacterial cell wall composition and density. In contrast to this conserved conjugation mechanism observed in most of the bacterial domain, conjugative plasmids of Streptomyces origin use a different mode of DNA transfer (Grohmann et al., 2003). The transfer of the plasmid was dependent on the origin of the transfer. oriT) of the incompatibility group P (IncP) broad host sequence E.
A double crossover results in a mutant allele that replaces the chromosomal copy of the target gene via two crossovers that cannot revert and are therefore more stable than a single crossover mutant (Keiser et al., 2000). All the other plasmids used in this study are derivatives of pOJ260 and pJN100 carrying the PCR-amplified target genes used in the gene disruption experiments (section 4.3.7). A colony PCR protocol was used to confirm the presence and correct size of the amplification product in transformants harboring recombinant constructs.
The ligation reaction was incubated at 22 °C for 14 h after which 10 ng of the reaction was transformed into E. Each of the inserts was ligated into dephosphorylated pOJ260, which had been subjected to restriction endonuclease digestion with EcoRI and converted into chemical competent E. Ligation was performed at 4 °C for 18 h and then 10 ng of the ligation product was converted to chemically competent E.
A 50 µl aliquot was then thawed on ice and 10 ng to 1 µg of plasmid DNA (pJN100 or pJNHYG) was added before the mixture was transferred to a 2 mm gap electrocuvette (Bio-Rad Laboratories Inc., USA). ) and exposed to an electrical pulse ranging from 0 to 12.5 kV/cm using a Gene Pulser (Bio-Rad Laboratories Inc., USA), which was connected to a pulse controller (25 µF capacitor) with a parallel resistance setting or 800 Ω . The pulsed mycelium was immediately diluted with ice-cold 0.75 mL CRM and incubated on a rotary shaker at 220 rpm for 3 h at 30 °C. The test bacterium was applied to the surface of a TLC plate (containing the DPO test sample, commercial sample DPO and toluene), the TLC plate was placed in a resealable plastic container lined with a moist paper towel and incubated overnight at 37 °C (approximately 18 h).
General discussion
Antibiotic resistance
It was reported in 2004 that more than 70% of disease-causing bacteria were predicted to be resistant to at least one of the currently available antibiotics. About 480,000 people developed MDR-TB globally in 2013, an increase of almost 7% from 2012, of which more than half of the cases were in developing countries such as India, China and China. It was also estimated that 9% of MDR-TB cases had XDR-TB (World Health Organization, 2014).
MDR-TB strains are defined as resistant to at least two of the first-line anti-tuberculosis drugs, isoniazid and rifampicin.
The phylum Actinobacteria
Among actinomycetes such as Streptomyces, it is estimated that more than 60% of the secondary metabolites produced are synthesized by enzyme systems known as non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), or mixed NRPS-PKS pathways (Baltz). , 2014). For example, the sequencing of the first antibiotic-producing actinomycete genomes, Streptomyces coelicolor strain A3(2) (8.6 Mb) and Streptomyces avermitilis strain MA-4680T (9.02 Mb), revealed the potential of both organisms to produce a large number of secondary metabolites, most of which were not synthesized under standard growth conditions (Baltz, 2008). The sequenced actinobacterial genomes (currently 4059) (GenBank, 2015; . Land et al., 2015) are an invaluable resource that has revealed that the more bacterial genomes are sequenced, the more new gene families are discovered, thereby discovering new genetic diversity and thus new biosynthetic capabilities (Wu et al., 2009).
This genetic diversity is directly enabled by multifunctional NRPS enzymatic systems capable of producing structurally diverse natural products with a remarkable variety of useful/significant pharmacological activities (Ashforth et al., 2010; Wu et al., 2009).
Natural product discovery
- Oxazole-containing natural prdoucts
They are produced in nature by dramatic chemical modifications performed on elongating peptide chains, mostly catalyzed by NRPS assembly lines (Walsh et al., 2001). The anticonvulsant alkaloid, pimprinine, was one of the earliest oxazole-containing compounds to be discovered, isolated from 'Streptomyces pimprina' in 1960 (Bhate et al., 1960). Furthermore, in 1966, the macrocyclic streptogramin antibiotic accommodating a 2,4-disubstituted oxazole ring, virginiamycin M2, was isolated from Streptomyces virginiae (Kondo et al., 1989).
Furthermore, in 1998, Rastogi and colleagues reported the antimycobacterial activity of the oxazole alkaloid, texaline, isolated from the plants Amyris texana and Amyris elemifera (Rastogi et al., 1998).
Non-ribosomal peptide synthetases (NRPSs)
- Assembly logic of NRP synthesis
- Activation by the Adenylation domain
- Transport of substrates and intermediates to the catalytic
- Peptide elongation by the Condensation domain
- Peptide release by the Thioesterase domain
- Editing/tailoring domains
- NRPS substrate specificity prediction and the non-ribosomal code
- NRPS biosynthetic strategies
- Combinatorial biosynthesis and intermolecular communication
A fourth domain, a thioesterase (TE), is often found in the C-terminus of the NRPS termination module and catalyzes the release of the peptide from the NRPS (Felnagle et al., 2008). The PPTase stimulates the nucleophilic attack of the PCP serine hydroxyl group on the pyrophosphate bridge of CoA, resulting in the transfer of 4'-PP to the PCP domain and the release of 3',5'-ADP (Lambalot et al., 1996). The C domain is approx. 450 amino acids long and is responsible for the elongation of the peptidyl chain.
Three Dhb-Ser-S-ppant intermediates are produced on the two modules of the enterobactin NRPS and oligomerized and circularized on the TE domain (Mootz et al., 2002).
Research aims
The overarching aim of this study was to confirm the production of DPO by S. polyantibioticus SPRT and then to focus on the elucidation of the biosynthetic pathway involved in its synthesis. This consisted of the identification and partial characterization of the genes involved in the production of DPO to determine whether an NRPS is involved. In order to determine whether the proposed biosynthetic pathway is correct, the genes coding for benzoic acid synthesis and the DPO NRPS had to be identified in the S. The objectives of the study were therefore:
To identify the genes involved in linking benzoic acid and phenylalanine/3-hydroxyphenylalanine in S. polyantibioticus SPRT to create a 2,5-disubstituted oxazole. ii).
