Huanglongmycin A-C, Cytotoxic Polyketides Biosynthesized by a Putative Type II Polyketide Synthase From Streptomyces sp. CB09001

Three natural products of nonaketide biosynthetic origin, probably biosynthesized from nine molecules of malonyl-CoA, have been isolated. Herein we described the isolation and structure elucidation of huanglongmycin (HLM) A-C and identification of the putative hlm biosynthetic gene cluster from Streptomyces sp. CB09001, isolated from a karstic cave in Xiangxi, China. Albeit previously isolated, HLM A was reported for the first time to exhibit moderate cytotoxicity against A549 lung cancer cell line (IC50 = 13.8 ± 1.5 μM) and weak antibacterial activity against gram-negative clinical isolates. A putative biosynthetic pathway for HLM A, featuring a nonaketide-specific type II polyketide synthase, was proposed. It would be consistent with the isolation of HLM B and C, which are two new natural products and likely shunt metabolites during HLM A biosynthesis.


INTRODUCTION
Aromatic polyketides of microbial origin are a large family of natural products with important biological activities, including anticancer agents doxorubicin and mithramycin, and antibiotics tetracyclines. Aromatic polyketides are typically biosynthesized by type II polyketide synthases (PKSs), minimally consisting of two ketosynthases KS α and KS β (also named "chain length factor") and an acyl carrier protein (ACP), that are associated with additional ketoreductases and cylases/aromatases (Hopwood, 1997;Shen, 2000;Hertweck et al., 2007;Das and Khosla, 2009;Zhou et al., 2010;Zhang et al., 2017). The KS α , KS β , and ACP are often clustered together and called minimal PKSs. Extensive biosynthetic studies of these polyketides, such as actinorhodin (1), frenolicin (2), and tetracenomycin C (3), have helped establish certain "design rules" to make designer analogs, such as DMAC (5) and SEK26 (6), by a biotechnology platform now often termed "combinatorial biosynthesis" (Figure 1) (McDaniel et al., 1993(McDaniel et al., , 1995Kramer et al., 1997;Yu et al., 1998). However, only a few natural nonaketide-derived aromatic polyketides, including the biaryl compounds julichromes (11), setomimycin and spectomycins, have been proposed to be biosynthesized by the Claisen-like condensation of nine malonyl-CoA (Marti et al., 2000;Zhang et al., 2008;Präg et al., 2014). Interestingly, the nonaketide precursors could form homo-or heterodimers through oxidative phenol coupling (Präg et al., 2014). We have been interested in collecting diverse microbial strains for natural product discovery and recently isolated several new natural products with antibacterial and anticancer activities (Luo et al., 2014;Ma et al., 2015Ma et al., , 2017Yan et al., 2016;Pan et al., 2017). The western part of Hunan province (also named "Xiangxi") in China is on the east of Yunnan-Kweichow plateau, which was geologically formed through epeirogeny uplifts in the late Permian period about 200-million years ago (Luo et al., 2014). Xiangxi is well-known for its characterized karstic topography and biodiversity (Chen et al., 2015). In this report, we describe the isolation, structure elucidation, biosynthesis, and biological activities of three aromatic polyketides from strain S. sp. CB09001, isolated from a karstic cave in Xiangxi (Figure 1).

Fermentation
The strain S. sp. CB09001 was grown in 250 mL Erlenmeyer flasks containing 50 mL of G1 medium and were incubated at 30 • C on rotary shakers (230 rpm) for 72 h. Then 3% macroporous resin DA201-H (Jiangsu Su Qing Water Treatment Engineering Group Co., Ltd., Jiangyin, China) or different concentration triclosan (0-20 µM) were added, and fermented for additional 4 days. S. sp. CB09001 was also fermented in G1 fermentation medium with different fermentation volume in 250-mL flatbottom Erlenmeyer flasks from 25 to 150 mL per flask. In scale-up fermentation, each of the seed cultures in G1 medium (50 mL) was aseptically transferred to 2 L Erlenmeyer flasks containing 1 L of G1 medium. All of these flasks were then incubated at 30 • C on rotary shakers (200 rpm) for 7 days.

Accession Numbers
The 16S rRNA sequence and genome sequence of S. sp. CB09001 have been submitted to GenBank with accession number MG890330 and CP026730, respectively.

Isolation and Structure Elucidation of Huanglongmycins
We collected several soil samples from Huanglong cave in Xiangxi, from which new actinomycetes strains were isolated. One of the strains, CB09001, was classified as a Streptomyces species based on its morphological characteristics and the 16S rRNA sequence (NCBI accession no. MG890330). Encouraged by its unique metabolite profiles, S. sp CB09001 was fermented under various fermentation conditions, such as the addition of elicitor triclosan, and the variation of the fermentation volume and the addition of macroporous resin (Figures S1-S3) (Bode et al., 2002;Yoon and Nodwell, 2014;Tanaka et al., 2017). For large scale fermentation, S. sp. CB9001 was fermented in six 2-L Erlenmeyer flasks, containing 1 L of G1 medium, and isolation of natural products from the fermentation culture afforded three compounds, we named huanglongmycin (HLM) A-C (7-9). Their structures were established on the basis of HR-ESI-MS and extensive 1D and 2D NMR analysis (Figure 2, Figures S4-S25, Table 1).

Identification of the Putative Biosynthetic Gene Cluster of Huanglongmycins
Inspired by the previous combinatorial biosynthesis approaches to generate the "unnatural" natural products by type II PKSs, such as 5 and 6, and the fact that only a few biosynthetic gene clusters of nonaketides have been reported, we decided to identify the huanglongmycin (hlm) gene cluster in S. sp. CB09001 to shed light on the biosynthesis of HLM A-C (Figure 3, Table 2). The genome of S. sp. CB09001 was sequenced and bioinformatic analysis using antiSMASH revealed that there are three putative type II PKS gene clusters (GenBank accession number CP026730) (Blin et al., 2017). One gene cluster is likely to be involved in the biosynthesis of HLMs, and the other two share very high sequence homology to gray pigment or angucycline biosynthesis (Tables S1, S2). The putative KS β gene hlmF is located in the KS β group for nonaketide biosynthesis, based on the phylogenetic analysis of known 42 type II PKS gene clusters whose secondary metabolite products have been structurally characterized (Figure 4) (Hillenmeyer et al., 2015). Pairwise comparison of the annotated proteins between the hlm cluster with the julichrome biosynthetic gene cluster revealed high amino acid identity, strongly supporting that the hlm gene FIGURE 3 | Predicted gene organization of huanglongmycin (hlm) cluster and julichrome (jul) cluster. Genes are color-coded according to their proposed functions. Red, purple, green, and gray represent core biosynthetic genes, additional biosynthetic genes, regulatory genes and other genes, respectively.
Frontiers in Chemistry | www.frontiersin.org cluster most likely encodes the biosynthesis of HLMs in S. sp. CB09001 (Table 2). Thus, we now propose a pathway for HLM A-C biosynthesis, featuring a nonaketide intermediate by the HLM minimal PKS, containing KS α (HlmG), KS β (HlmF), and ACP (HlmE), which catalyzes the decarboxylative condensation of nine molecules of malonyl-CoA (Figure 5). Reduction of the poly-β-keto intermediate by the C-9 specific KR (HlmD), followed by the di-domain BexL-type reducing cyclase HlmC might afford the initial C7-C12 cyclization (Caldara-Festin et al., 2015). Interestingly, the isolation of the shunt metabolites HLM B and C suggested the leakage of the HLM polyketide intermediate during its transfer to the cyclase HlmJ under specific fermentation conditions, which were often observed in the engineered mutants or in vitro enzymatic assays (Shen and Hutchinson, 1993;Kharel et al., 2010). The formation of HLM A might be from the previous SEK26 (6) polyketide through decarboxylation, the MS of which was also observed in the fermentation extract from an E. coli strain containing a fungi PKS, one KR and two cyclases (Zhang et al., 2008). It was similar to the formation of anthraquione aloesaponarin II from DMAC (McDaniel et al., 1993). The above proposal was in agreement with the previous engineered biosynthesis of aromatic polyketide RM 18 (11) and nonaSEK14 (12) from the same nonaketide intermediate, albeit from a wild-type Streptomyces strain (Figure 5) (McDaniel et al., 1993;Hopwood, 1997;Zhang et al., 2008). The similar biosynthetic pathway for huanglongmycin A might be also operational in the Streptomyces strain isolated from Egypt, which suggested that natural nonaketides might be underappreciated (Abdelfattah, 2009).

The Biological Evaluation of Huanglongmycins
HLM A-C (7-9) were first evaluated for their antibacterial activities against Staphylococcus aureus ATCC 29213, and several clinical isolates from local hospitals, including methicillinsensitive S. aureus (MSSA) strain and methicillin-resistant S. aureus (MRSA) strains, as well as strains of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, using chloramphenicol and linezolid as controls ( Table 3). HLM A FIGURE 5 | Biosynthetic Pathways for 18-Carbon Aromatic Polyketides. Biosynthesis between the known aromatic polyketides (i.e., nonaSEK4 and RM 18) and the putative HLM gene cluster were compared. The putative hlm minimal PKS contains HlmG, HlmF, and HlmE, the Fren PKS is the minimal PKS of frenolicin, while act KR is the ketoacyl reductase from actinorhodin PKS. The fungal iterative PKS is the megasynthase PKS4 from Gibberella fujikuroi. ZhuI, MtmQ, and StfQ are the cyclases of aromatic polyketide R1128, mithramycin, and steffimycin, respectively, which catalyzed the region-specific cyclization at C7-C12. showed weak antibacterial activities against the gram-negative pathogens P. aeruginosa and E. coli, with minimum inhibitory concentrations (MICs) of 64 µg/mL. HLM B showed weak antibacterial activities against the tested S. aureus strains, with a MIC of 64 µg/mL, while HLM C showed no antibacterial activities against the five indicator strains ( Table 3).

CONCLUSION
In summary, this work describes the isolation and characterization of three natural products HLM A-C, and the identification of a putative gene cluster, featuring a type II PKS specific for nonaketide biosynthesis from S. sp. CB09001. The proposal for HLMs biosynthesis agreed well with the previous engineered biosynthesis for bacteria aromatic polyketides in the past few decades (McDaniel et al., 1993(McDaniel et al., , 1995Hopwood, 1997;Kramer et al., 1997;Yu et al., 1998;Shen, 2000;Hertweck et al., 2007;Zhang et al., 2008Zhang et al., , 2017Das and Khosla, 2009;Zhou et al., 2010). Albeit previously isolated, HLM A was reported for the first time, to exhibit moderate cytotoxicity against A549 cancer cell line (IC 50 = 13.8 ± 1.5 µM) and weak antibacterial activity against P. aeruginosa (MICs = 64 µg/mL). This study showcased once again Nature is the ultimate combinatorial biosynthetic chemist, and should inspire continued efforts for natural product discovery from unexplored and underexplored ecological niches.

AUTHOR CONTRIBUTIONS
YH, YD, and BS: conceived the project; LJ and HP: performed experiments; JX and MS: carried out the cytotoxicity assays; XY, DY, and XZ: contributed to bioinformatics analysis; YH and LJ: wrote the manuscript with help from the other authors.