Genomic characterization of a new endophytic Streptomyces kebangsaanensis identifies biosynthetic pathway gene clusters for novel phenazine antibiotic production

Introduction

Streptomyces are Gram positive, filamentous saprophytes known for their roles in producing various secondary metabolites important for medicinal therapies (Hopwood, 2007; Kieser, 2000). Despite the continuous efforts to isolate novel drug compounds from soil-dwelling Streptomyces, the numbers of newly identified compounds have been dwindling over the years (Palaez, 2006). One strategy to increase the chances of identifying new bioactive compounds as well as to combat the scourge of antimicrobial resistance is to investigate microorganism sources. Recently, it has since become clear that some Streptomyces sp. also exist as endophytes that dwell within the tissues of certain plants (Castillo et al., 2006; Ezra et al., 2004; Strobel & Daisy, 2003). The possibility that this unique living environment of endophytes may be the niche of many other unidentified species or strains of bacteria has gained our attention for its potential in unravelling new sources of biologically active compounds with industrial or medicinal applications (Ghadin et al., 2008; Strobel & Daisy, 2003).

In particular, Streptomyces kebangsaanensis represents a novel endophyte isolated from the ethnomedical plant, Portulaca oleracea Linn, known in Malaysia as ‘Gelang pasir’, that was demonstrated to have medicinal and pharmaceutical properties such as antiseptic and anti-inflammatory activities (Lim & Quah, 2007; Sarmin et al., 2013). When cultured on International Streptomyces Project 2 agar, the formation of greenish-yellow substrate mycelia and greenish-grey aerial hyphae were readily visible (Sarmin et al., 2013). S. kebangsaanensis (Streptomyces SUK12^T; GenBank accession number: HM449824) is a Gram-positive bacterium (Family: Streptomycetaceae; Class: Actinobacteria) (Sarmin et al., 2013). Recently, it has been found to produce the bioactive compound phenazine-1-carboxylic acid (known as tubermycin B), which was shown to have antibacterial, anticancer, antiparasitic, and antiviral properties (Laursen & Nielsen, 2004; Sarmin et al., 2013). This suggests that S. kebangsaanensis represents an untapped source of bioactive compounds such as phenazines that might be potentially further utilised. However, more studies are needed to characterise other bioactive compounds from this species as well as to elucidate the genes responsible for the biosynthesis of these metabolites.

Phenazines are a group of nitrogen-containing heterocyclic compounds known for their antibacterial, antifungal, antiviral, and anticancer functions (Laursen & Nielsen, 2004; McDonald et al., 2001). These compounds are derived from bacteria of diverse genera such as Pseudomonas, Streptomyces, Vibrio and Pelagiobacter (Mavrodi et al., 2010). Notably, while more complex phenazines are biosynthesized by Streptomyces, less complex derivatives are normally obtained from Pseudomonas (Laursen & Nielsen, 2004). The first known phenazine isolated from Streptomyces was the antibiotic griseolutien (Umezawa et al., 1950) and subsequently many Streptomyces sp. have been shown to produce numerous diverse and complex phenazines including lomofungin from Streptomyces lomondensis (Johnson & Dietz, 1969) and endophenazines from Streptomyces anulatus (Gebhardt et al., 2002; Krastel et al., 2002). Although the phenazine biosynthesis core structure has already been described (Haagen et al., 2006; Mavrodi et al., 1998), the formation of more complex phenazine structures are still hypothetical and largely unknown (Mentel et al., 2009). Therefore, more research in elucidating the biosynthetic pathway of complex phenazines from Streptomyces species such as S. kebangsaanensis is crucial.

In the current study, we successfully isolated a novel phenazine compound termed 6-((2-hydroxy-4-metoxyphenoxy) carbonyl) phenazine-1-carboxylic acid (HCPCA) from S. kebangsaanensis. Its molecular structure was elucidated using nuclear magnetic resonance spectroscopy (NMR). This structure was then compared against other known phenazine compounds available in public databases to search for closely related compounds.

In order to discover the biosynthesis of this novel compound as well as other metabolites from S. kebangsaanensis, genome sequencing was carried out using IIlumina Hiseq2000. Several gene clusters including a phenazine biosynthetic gene cluster were identified and used to further elucidate the biosynthesis pathway of HCPCA.

Material and Methods

Results

Isolation and structural determination of HCPCA

Results of ¹HNMR, ¹³C-APT¹H-¹H correlation spectroscopy (COSY), and ¹H-¹³C heteronuclear multiple bond coherence (HMBC) are shown in Fig. 1. C₂₁H₁₄N₂O₆ (390), UV in MeOH, λmax/nm (log ε): 246, 363.8; EI-MS m/z (relative intensity) 390 (26.4, Na⁺); ¹H-NMR (MeOD, 600 MHz) δ 8.86 (1 H, dd, J = 7:08, 1.5, H-4), 8.47 (1 H, dd, J = 8.64, 1.5, H-2), 8:08 (1 H, m, H-3), 8.03 (1 H, m, H-7), 8.06 (1 H, m, H-8), 8.38 (1 H, m, H-9), 15.61 (1 H, s, 1′-OOH), 7.14 (1 H, m, 4′-OH), 7:40 (1 H, m, H-5′), 7.22 (1 H, m, H-7′), 7.45 (1 H, m, H-8′), and 1.34 (3 H, s, H-9′). ¹³C NMR (MeOD, 600 MHz) detected 21 carbon signals. We observed 9 carbon methines that were absorbed at δ 133.68 (C-2), 129.83 (C-3), 134.94 (C-4), 131.51 (C-7), 131.96 (C-8), 128.91 (C-9), 118.84 (C5), 123.61 (C7) and 124.52 (C-8′). The carbon quaternary signals were absorbed at δ 126.22 (C-1), 142.50 (C-4a), 143.06 (C-5a), 129.02 (C-6), 140.49 (C-9a), 140.18 (C-10A), and 138.38 (C-3′). The aromatic quaternary carbon signal was absorbed at δ 163.90 (C-1′) whereas the methyl carbon signal was absorbed at δ 29.63 (C-9) (Table S3). The obtained structure (Fig. 1) exhibits a phenazine core structure with additional functional groups.

This HCPCA structure was then compared against all known phenazine structures in the NCBI database without detecting any similarities. The most closely related compound was saphenamycin with an additional methyl group and a different functional group [6-(1-hydroxyethyl)1-phenazinecarboxylic acid instead of 4-methoxybenzene-1,2-diol in HCPCA] (Fig. S2).

Genomic Study of S. kebangsaanensis

To elucidate the biosynthetic genes and related pathways of phenazines in S. kebangsaanensis, whole genome sequencing followed by bioinformatics analysis was performed. Whole genome sequencing using HiSeq2000 (Illumina, San Diego, CA, USA) resulted in 2.6 Gbp raw reads. Reads pre-processing was performed to remove adaptors as well as low quality and ambiguous bases. The sequences were then assembled using de-novo assembly, which produced 560 contigs and 170 scaffolds. The longest scaffold contained 453,879 base pair (bp) whereas the shortest was 1,072 bp, with the median (N50) and mean length being 110,454 bp and 48,992 bp, respectively (Table 1). The draft genome sequence of S. kebangsaanensis consisted of 8,328,719 bp, with an average GC content of 71.35% (Table 1). Gene sequence annotation predicted 8,001 open reading frames including 7,558 genes with known function; the remainder (443 genes) did not result in any significant BLAST hits (Table 2). The S. kebangsaanensis genome also contained high numbers of tRNA gene sequences (80), one sequence of tmRNA, and 12 operons of rRNA (16S-23S-5S), which was comparable to other Streptomyces (Table 2).

Table 1:

Genomic data for Streptomyces kebangsaanensis.

Assembled scaffolds	170
Predicted gene	8,001
Total size	8,328,719
Longest	453,879
Shortest	1,072
N50	110,454
Mean size	48,992
% A	14.16
% C	35.58
% G	35.77
% T	14.19
% N	0.30

DOI: 10.7717/peerj.3738/table-1

Table 2:

Genomic feature comparison of S. kebangsaanensis with other Streptomyces.

No.	Species	Length (Mbp)	Avg. G + C content (%)	No. of protein coding genes	No. of rRNA (16S-23S-5S)	No. of tRNA genes	No. of other RNAs	References
1.	Streptomyces kebangsaanensis	8.32	71.35	7558	12	80	1	–
2.	Streptomyces griseus	8.54	72.2	7138	6	66	1	Ohnishi et al. (2008)
3.	Streptomyces coelicolor A3(2)	8. 67	72.2	7825	6	63	1	Bentley et al. (2002)
4.	Streptomyces avertimilis	9.03	70.7	7583	6	68	1	Ōmura et al. (2001)
5.	Streptomyces albus J1074	6.84	73.3	5746	21	66	1	Zaburannyi et al. (2014)
6.	Streptomyces bingchenggensis BCW-1	11.94	70.8	9309	18	66	3	Wang et al. (2010)
7.	Streptomyces cattleya NRRL 8057	6.28	72.9	5360	18	64	1	Barbe et al. (2011)
8.	Streptomyces davawensis JCM4913	9.47	70.6	8174	18	70	3	Jankowitsch et al. (2012)
9.	Streptomyces fulvissimus DSM40593	7.91	71.5	6729	18	73	3	Myronovskyi et al. (2013)
10.	Streptomyces hygroscopicus subsp jingangensis 5008	10.15	71.9	8673	18	70	5	Wu et al. (2012)
11.	Streptomyces scabiei 87.22	10.15	71.5	8440	18	75	3	Yaxley (2009)
12.	Streptomyces sp. PAMC 26508	7.53	71.06	6345	18	68	3	Data deposited in NCBI without publication
13.	Streptomyces sp. Sirex AA-E	7.41	71.7	6331	19	64	3	Data deposited in NCBI without publication
14.	Streptomyces venezuelae ATCC 10712	8.23	72.4	7080	12	66	1	Pullan et al. (2011)
15.	Streptomyces violacuesniger Tu 4113	10.66	71.0	8264	18	64	3	Data deposited in NCBI without publication

DOI: 10.7717/peerj.3738/table-2

The predicted genes/open reading frames were functionally categorized using Gene Ontology (GO) annotations (Consortium, 2013) of which 3,238 genes were predicted to be involved in numerous biological processes, 1,402 genes in cell components, and 6551 in molecular functions (Fig. 2). The neighbour-joining phylogenetic tree generated based on 16S rRNA gene sequences (1,599 nt) specified the evolutionary relationship between S. kebangsaanensis with other members of the Streptomyces (Fig. S3).

Phenazine genes and predicted phenazine gene clusters

We hypothesized that 31 genes might be responsible for phenazine biosynthesis in S. kebangsaanensis. These include phenazine modification genes, resistance genes, as well as regulatory genes (Table 3). The majority of these genes (27) were located in scaffold 110 including genes for the phenazine core structure (putative anthranilate synthase, phzE; phenazine biosynthesis protein, phzD; 2,3-dihydroxybenzoate-2,3-dehydrogenase, phzA; and 3-deoxy-7-phosphoheptulonate synthase, phzC) and were therefore referred to as the phenazine cluster. Notably, other predicted phenazine genes were also found located in scaffold 120 (phenazine biosynthesis protein; phzF), scaffold 163 (phenazine biosynthesis phzC/phzF protein), and scaffold 167 (2-dehydro-3-deoxyphosphoheptonate aldolase, phzC; and putative anthranilate synthase, phzE) (Table 3). In comparison, the Streptomyces cinnamonensis DSM1042 genome also showed a slightly similar gene distribution profile, wherein all the genes essential for phenazine biosynthesis were found to be located within two different loci (Seeger et al., 2011).

However, an additional nine genes were present in the phenazine cluster that were determined by exhaustive comparison against other previous studies as not having been previously annotated as representing phenazine biosynthetic genes. These genes were identified as N-acetyltransferases (two genes), putative carboxylesterase, 3-carboxy-cis,cis-muconate cycloisomerase, hemerythrin HHE cation binding domain protein, 3-hydroxyacyl CoA dehydrogenase, putative F420 dependent oxidase, NAD binding protein 3-hydroxylacyl-CoA dehydrogenase, and a putative uncharacterized protein (Table 3).

Furthermore, the S. kebangsaanensis phenazine gene cluster was also compared against 14 other complete genomes of Streptomyces to investigate whether the gene cluster was present in other Streptomyces as well (Fig. 4). It was clear that the phenazine gene cluster was mostly well conserved within all of these genomes, suggesting the existence of common genes and potentially pathways in the biosynthesis of phenazine, in particular of its backbone structure. However, several differences were also observed between these clusters suggesting that each species may produce different phenazine derivatives.

Discussion

Endophytes are ubiquitous and are very likely to be found in all plant species (Rosenblueth & Martinez-Romero, 2006). In this mutual relationship, the host serves the microbes a protective niche for the microbes to live and in return, these microbes help the plant in their growth and development. Microbial secondary metabolites are low molecular weight, which usually produced during the late growth phase of microorganisms. They are not vital for the growth of the producing cultures but provide many survival functions in nature for the host (Ruiz et al., 2010). Actinomycete bacteria, especially those of the genus Streptomyces, are one of the most interesting bacteria that produce secondary metabolites with promising biological activity. These bacteria produce many classes of secondary metabolites with antibacteria, anticancer, antifungus, and antiinflammation activity, including polyletides and terpenes. For instant, analysis of secondary metabolites gene cluster in marine Streptomyces sp. MP131-18 showed that, six gene clusters with type 1 polyketide synthase, and five gene clusters for terpene biosynthesis were found within the genome of the bacteria (Paulus et al., 2017).

In this study, we found that S. kebangsaanensis produces a novel phenazine derivative termed HCPCA (Fig. 1), which is structurally dissimilar to any of the 11,609 phenazine structure compounds found in NCBI database (http://www.ncbi.nlm.nih.gov/). The closest related compounds was identified as saphenamycin (Fig. S2), which was isolated from S. canaries MG314-hF8 (Kitahara et al., 1982) and S. antibiotics (Geiger et al., 1988). However, several differences were noted between the functional groups of HCPCA compared to those of saphenamycin, such as the locations of hydroxyl, methyl, and phenol groups (Fig. S2). Saphenamycin displayed a broad spectrum of biological activities namely, antibacterial (Geiger et al., 1988), antitumour (Kitahara et al., 1982), and larvacidal activities as well as free radical scavenger (Laursen & Nielsen, 2004). Whereas, HCPCA exhibited strong antibacterial activity towards Bacillus subtilis ATCC 6633 (Sarmin, 2012).

Although Streptomyces are known to produce many phenazine derivatives only two gene clusters have been identified to date in S. anulatus (Saleh et al., 2009) and S. cinnamonensis (Haagen et al., 2006). Furthermore, out of 14 complete operons of phenazine biosynthesis documented in NCBI; nine were from Pseudomonas sp. and the remainder were from Streptomyces sp. (S. anulatus, S. tendae, S. cinnamonensis, S. iakyrus, and S. griseoluteus). Although the gene clusters share high similarities in operons, our study indicates that there were nine different genes from S. kebangsaanensis with no sharing homologous compared to other genome. The genes are; 3-oxoacyl-[acyl-carrier-protein] synthase III (gene 3268), N-acetyltransferase (gene 3274), polyketide cyclase (gene 3278), putative acyl-CoA synthetase (gene 3275), putative monooxygenase (gene 3276), while gene 3283, 3284, 3286, 3287 encoding for hemerythrin HHE, oxidoreductase, 3-oxoacyl-(Acyl-carrier-protein) synthase III, and 3-hydroxyacyl-CoA dehydrogenase, respectively. Given that HCPCA (Fig. 1) differs from all other known phenazine derivatives, unique gene sets or biochemical pathways may be required for its biosynthesis in S. kebangsaanensis. Hereby, we proposed a putative biosynthetic pathway of phenazine in this species by referring to the previously reported pathway (Blankenfeldt, 2013; Haagen et al., 2006; Mavrodi et al., 1998; McDonald et al., 2001) and the genome data that we obtained.

Within S. kebangsaanensis, the HCPCA phenazine structure is hypothesised to be derived from the combination of two biosynthetic pathways, phenazine-1,6-dicarboxylic acid (PDC) and 4-methoxybenzene-1,2-diol (MBD) (Fig. 5). Both pathways are proposed to have originated from the shikimate pathway. Genes involved in the PDC and PCA pathways including phzE, phzD, phzF, phzB, and phzG, sequentially (Blankenfeldt, 2013; Mentel et al., 2009), all of which were found in the predicted phenazine cluster as proposed previously by McDonald et al. (2001), with the exception of phzG. phzG constitutes the final enzyme in PDC biosynthesis and converts 1,2,5,5a,6,7-hexahydrophenazine-1,6-dicarboxylic acid (HHPDC) to 5,10-dihydrophenazine-1,6-dicarboxylic acid (5,10-DHPDC), which is subsequently converted to PDC through a reduction process. Previous findings showed that phzG is similar to flavin mononucleotide-dependent pyridoxamine oxidases, which oxidize 6-amino-5-hydroxycyclohexane-1,3-dienecarboxylic acid to the respective 3-keto compound to form a tricyclic phenazine precursor (Mentel et al., 2009; Pierson et al., 1995). phzG was known to encode a protein exhibiting homodimeric flavin enzyme similar to pyridoxine-5′-phosphate oxidase. Notably, gene 3288 from our study was found to share 85% sequence identity to the LLM class F420-dependent oxidoreductase of Streptomyces sp. FxanaA7, a flavonoid cofactor dependent enzyme-like pyridoxine-5′-phosphate oxidase (Selengut & Haft, 2010). Therefore, it is possible that gene 3288 may assume the function of phzG to oxidize the HHPDC in the S. kebangsaanensis phenazine biosynthesis pathway (Fig. 5). Additionally, all the other eight genes mentioned previously may also individually or collectively play an important role in the modification of phenazine structure in S. kebangsaanensis. However, to confirm the proposed pathway, gene knock-out experiment will be needed to provide functional evidence for the genes that encoded in the cluster in phenazine biosynthesis pathway. Moreover, this will also help in the identification of the gene products that are currently unknown.

Conversely, the MBD pathway is proposed to branch off from chorismic acid to form 3,4-dihydoxybenzoic acid, followed by benzene-1,2-4-triol and finally MBD. However, specific genes (genes W, X, and Y) involved in the reactions of this pathway are still unknown. We speculate that gene W could be involved in the removal of one hydroxyl group from shikimic acid through a dehydration process, while gene X is proposed to be involved in dehydration at a carboxylic acid (COOH) functional group, and gene Y is involved in the addition of one methyl group (methylation) at a hydroxyl group. Furthermore, another gene (gene Z) that is involved in the dehydration process between the MBD hydroxyl and PDC carboxylic acid functional groups to form HCPCA is also unknown (red functional groups in Fig. 5). Additional studies utilizing genetic manipulation are likely required to verify the function of these individual genes in the biosynthetic pathway.

Whole genome sequencing of S. kebangsaanensis followed by bioinformatics analysis led to the discovery of different gene clusters believed to be involved in the production of numerous secondary metabolites. In particular, the S. kebangsaanensis genome was suggested to comprise a linear structure which was found in other species of Streptomyces, i.e., S. lividans (Lin et al., 1993) and S. coelicolor (Bentley et al., 2002). Nevertheless, the S. kebangsaanensis genome (8.3 Mbp) was noted to be shorter than other Streptomyces i.e., S. coelicolor (8.7 Mbp) (Bentley et al., 2002), S. avermitilis ATCC 31267 (8.7 Mbp) (Ōmura et al., 2001), and S. griseus IFO 13350 (8.5 Mbp) (Ohnishi et al., 2008) (Table 2).

The genomic data also showed the presence of 24 biosynthetic gene clusters potentially involved in the production of secondary metabolites (Fig. 6). These gene clusters were comparable to those in other Streptomyces sp. such as S. coelicolor, S. cattleya NRRL 8057, and S. flavogriseus, which have 25, 27, and 28 gene clusters, respectively. As these Streptomyces come from similar genera, almost all genomes contained the same classes of secondary metabolite gene clusters such as terpenes, siderophores, NRPSs, butyrolactones, lantipeptides, PKSs, and melanins.

The production of different types of terpenes by S. kebangsaanensis was predicted based on the presence of four gene clusters. It is worth noting that, the anticancer drug paclitaxel (Taxol^®) and the antimalarial drug artemisinin are among several terpenes with established medical applications (Paddon & Keasling, 2014). Terpene backbones are synthesized by two enzymes: isopentenyl-diphosphate and dimethylallyltransferase. The genes encoding these enzymes were also found to be present in S. kebangsaanensis (Fig. 3). Further analysis revealed the possibility of one of the terpene biosynthesis gene clusters being involved in producing an albaflavenone compound (with 50% identity to S. viridochromogenes DSM 40736). Albaflavenone is a novel sesquiterpene antibiotic first isolated from S. coelicolor that belongs to the phylum of actinobacteria (Zhao et al., 2008). Subsequently, genes encoding this metabolite including terpene synthases were found to be ubiquitous in bacteria, especially among Streptomyces (Yamada et al., 2015).

Siderophore biosynthetic genes were also predicted from the genome of S. kebangsaanensis. Over 10 distinct species of Streptomyces have been identified thus far to have the capability to produce desferrioxamine siderophores, such as desferrioxamine G, B, and E (Challis & Hopwood, 2003; Wang et al., 2014). In particular, our study pointed to the presence of one biosynthesis gene cluster involved in the production of desferrioxamine B (Fig. 3). The potential of its therapeutic application is reflected by the use of S. pilosus derived Desferrioxamine B, used for the treatment of iron intoxication (Nakouti, Sihanonth & Hobbs, 2012) and Plasmodium falciparum infection (Miethke & Marahiel, 2007). Furthermore, siderophores produced by endophytes have previously been given more attention due to their role in controlling soil borne plant pathogens (Loper & Buyer, 1991). For example, siderophores isolated from the endophyte Streptomyces sp. strain S96 were involved in inhibition of Fusarium oxysporum f. sp. cubense while also showed plant growth-promoting property (Cao et al., 2005). However, some siderophores from actinobacteria are also known to carry Fe molecule to Rhizobium including Streptomyces lydicus WYEC108, which colonizes roots and affects the nodulation of pear tree roots (Tokala et al., 2002). Therefore, siderophore biosynthetic gene clusters that are present in S. kebangsaanensis might hold key information pertaining to the growth promotion and inhibition of plant pathogens as well as towards its own survival in the plant.

In addition, all Streptomyces genomes have been shown to carry a single ectoine biosynthesis gene cluster (Fig. 6). In the S. kebangsaanensis genome, this biosynthesis gene cluster is located at scaffold 167 with a length of 10,408 bp (Fig. 3). The genes involved are ectoine/hydroxyectoine ABC transporter, L-ectoine synthase, and a putative ectoine hydroxylase, which pointed to the presence of the conventional route of ectoine production in S. kebangsaanensis (Pastor et al., 2010). Ectoine comprises one of the most extensively found compatible solutes throughout different halotolerant and halophilic microorganisms including actinobacteria from the Brevibacterium and Streptomyces genera (Pastor et al., 2010). Despite living in high ionic and hyperosmotic habitats, halophilic microorganisms are able to maintain proper osmotic balance to prevent cell leakage (Roeßler & Müller, 2001). Thus, the discovery of ectoines in nature may indicate significant applications including as protective agents for cellular components, in addition to their potential therapeutic uses (Pastor et al., 2010).

Furthermore, the genomic analysis also revealed that S. kebangsaanensis carries two biosynthetic gene clusters that are important in producing different types of antibiotics such as PKS Type II, as well as one cluster of PKS Type III and three clusters of NRPS biosynthetic genes (Fig. 3). PKS and NRPS comprise two classes of natural products with valuable biological activities (antimicrobial, antifungal, antiparasitic, antitumour, and cholesterol lowering agents as well as immunosuppressive agents), which are found mainly in bacteria (Du & Lou, 2010). The presence of PKS and NRPS are also common in other bacteria such as S. coelicolor (Bentley et al., 2002) and S. avermitilis (Ōmura et al., 2001).

Finally, S. kebangsaanensis might also produce different types of bacteriocin. For example, an informatipeptin pathway has been predicted in S. kebangsaanensis based on S. gancidicus BKS 13-15 and S. prunicolor NBRC 13075 gene clusters (Fig. 3). Bacteriocin has been isolated from most bacteria and archaea, each of which exhibited different structure, size, and mode of action as well as mechanism (Farris et al., 2011; Nes, Yoon & Diep, 2007). The presence of bacteriocin genes in S. kebangsaanensis in different scaffolds thus suggests the potential for this strain to produce different types of bacteriocin.

Overall, the genome of S. kebangsaanensis has revealed its potential for producing bioactive metabolites based on the 24 identified biosynthetic gene clusters. Therefore, future studies should be focused on specific metabolite identification and purification to shed light on new bioactive molecule discovery.

Conclusion

S. kebangsaanensis represents a new endophyte that produces a novel compound, HCPCA. This structure has been elucidated using NMR and its novelty was demonstrated by structural comparison. Subsequently, genome sequencing of S. kebangsaanensis allowed the proposal of the phenazine biosynthetic pathway for this organism. We also identified several genes that are unique to S. kebangsaanensis in the phenazine cluster, which might be involved in the biosynthesis of HCPCA. The genome sequence also revealed numerous secondary metabolite gene clusters in S. kebangsaanensis, further analysis of which may lead to new and potentially bioactive secondary metabolites/antibiotics.

Supplemental Information