Overexpression of Global Regulator SCrp Leads to the Discovery of New Angucyclines in Streptomyces sp. XS-16

Six angucyclines including three unreported compounds (1–3) were isolated from Streptomyces sp. XS-16 by overexpressing the native global regulator of SCrp (cyclic AMP receptor). The structures were characterized based on nuclear magnetic resonance (NMR) and spectrometry analysis and assisted by electronic circular dichroism (ECD) calculations. All compounds were tested for their antitumor and antimicrobial activities, and compound 1 showed different inhibitory activities against various tumor cell lines with IC50 values ranging from 0.32 to 5.33 μM.


Introduction
Streptomyces have been demonstrated to be the producers of many clinically important antibiotics, such as tetracyclines, glycopeptides, macrolides, and polypeptides [1,2]; however, genome sequencing in recent years has shown that the metabolic potentials of Streptomyces are far from being fully exploited with a large group of gene clusters remaining cryptic or poorly expressed when strains are cultivated in laboratory [3][4][5][6]. Therefore, various methods such as OSMAC (one strain many compounds) [7], ribosome engineering [8], heterologous expression [9,10], coculture [11], promoter engineering [12], and metabolic regulation [13][14][15] have been developed to awaken or upregulate their expression as well as for the identification of molecules encoded by these gene clusters.
The cyclic AMP-receptor protein (Crp) is a global transcription regulator broadly distributed in a variety of bacteria and regulates multiple biological activities such as glucose starvation, cell differentiation, and primary metabolism [16]. Crp plays a key role in spore germination and colony development of Streptomyces, and it can regulate primary metabolism and enhance the precursor flux to secondary metabolite biosynthesis [17][18][19][20]. Thus, Crp was identified as the overall regulator of primary metabolism that stimulates the production of secondary metabolites. Studies have shown that overexpression of Crp in a group of Streptomyces leads to enhanced antibiotic biosynthesis and the production of new metabolites such as high production of actinorhodin (an aromatic polyketide antibiotic), undecylprodigiosin (a tripyrrolic pigment such as prodigiosin with antimalarial, antiulcer, and apoptotic activities), and a calcium-dependent antibiotic in S. coelicolor, indicating that the regulation of Crp on secondary metabolism is widely conserved in Streptomyces [20]. In addition, overexpression of Crp has been shown to successfully stimulate the production of monensin (a polyether ionophore antibiotic widely used as a coccidiostat and a growth-promoting agent in agricultural industry) in S. cinnamonensis [21] and daptomycin 2 of 12 (a novel cyclic lipopeptide antibiotic that is effective against Gram-positive bacteria) in S. roseosporus [22].
During our ongoing work in searching for bioactive compounds from marine-derived microbes, an actinomycete strain Streptomyces sp. XS-16 was obtained from marine sediment samples, collected from the sea around Naozhou Island in Zhanjiang, Guangdong Province. Genomic sequencing and analysis revealed a 7.54 Mb genome for the strain XS-16; however, we only obtained two previously described compounds named flazaine [23] and 1-(2-hydroxyphenyl)-2-phenylethanone [24] under laboratory cultivation. Thus, in order to tap into the metabolic potential and obtain diversified secondary metabolites, we overexpressed SCrp, the native global regulatory factor of Crp discovered by genome mining from XS-16. As a result, the metabolic profile was diversified and six typical angucycline derivatives, including three undescribed ones, were isolated from the mutant strain Streptomyces sp. XS-16-SCrp. Among the isolated compounds, 1, contains an oxygen-containing ternary ring system, and showed different inhibitory activities against five cancer cell lines with IC 50 values ranging from 0.32 to 5.33 µM. Here, we report the details of metabolic profile modulation by overexpression of SCrp, the isolation and characterization of new agucyclines, as well as bioactivity evaluation of the isolated compounds.

Results and Discussion
The Crp gene analogue, SCrp, was identified via Localblast, using a Crp gene (Streptomyces lividans TK24, AIJ15031.1) as a query. The total size of the gene is 675 bp, which encodes a polypeptide of 224 amino acids. The BLAST analysis by NCBI indicated that the SCrp protein had 99% sequence identity to the protein of SGCrp (S. griseus WP_257623897.1), SGZCrp (S. griseus WP 257623897), STCrp (Streptomyces sp. WP 202418726). Phylogenetic analysis revealed that SCrp is mostly related to STCrp ( Figure S2). Sequence analysis via InterProScan showed that the SCrp protein possessed two domains: cyclic nucleotide monophosphate binding domain and Crp-type HTH domain profile, which are consistent with the putative mechanism of Crp genes [25,26]. The SCrp in Streptomyces sp. XS-16 was amplified by specific primers (Table S1) and ligated into the lined vector pSET152 (cuted by the restriction site BamHI) using the ClonExpress Ultra One Step Cloning Kit C115 (Vazyme). The recombinant vector was transferred to Streptomyces sp. XS-16 to generate the OE::SCrp mutants (the mutant of Streptomyces sp. XS-16 harboring vacant pSET152 was also generated as a control). Following a fermentation in M1 media with shaking at 28 • C for 8 days, high-performance liquid chromatography (HPLC) analysis also showed a series of new peaks present in the extract of the OE::SCrp mutant compared with that of the control strain ( Figure 1), indicating changes in secondary metabolite production.
All new compounds were investigated for their cytotoxicity against L-02 (human normal liver cells), MDA-MB-231 (breast cancer), K562 (human myeloleukemia cells), ASPC-1 (human pancreatic cancer cells), H69AR (multidrug resistant human small cell lung cancer cells), and H69 (human small cell lung cancer cells) cell lines in vitro. Adriamycin was used as positive control. As a result, compound 1 showed cytotoxicity toward five cancer cell lines and especially strongly inhibited H69AR and H69 cell lines with an IC 50 values of 0.59 and 0.32 µM, respectively (Table 2). In addition, all new compounds were tested for their antimicrobial activity against seven bacteria, including Bacillus subtilis, Proteus vulgaris, B. cereus, Escherichia coli, Mycobacterium phlei, Acinetobacter baumannii, and MRSA (methicillin-resistant Staphylococcus aureus), in addition to one yeast strain Candida albicans using previously described minimum inhibitory concentration (MIC) assay method [35]. Ciprofloxacin and nystatin were used as positive controls for pathogenic bacteria and yeast, respectively. All compounds did not show significant inhibitory effect against the tested strains (MIC > 50 µg/mL).

General Experimental Procedures
NMR spectra were recorded on a JEOLJN M-ECP 600 spectrometer (JEOL, Tokyo, Japan) or Bruker Avance Neo 400 MHz (Bruker, Beijing, China) using tetramethylsilane as an internal standard. Specific rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were recorded on HITACHI 5430. IR spectra were measured on a Bruker Tensor-27 spectrophotometer in KBr discs. HRESIMS were obtained on a Thermo Scientific LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) or Micromass Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer (Waters Corporation, Shanghai, China). A JASCO J-715 spectropolarimeter (JASCO, Tokyo, Japan) was used to obtain ECD spectra. Semipreparative HPLC was performed on an ODS column (YMC-Pack ODS-A, 10 × 250 mm, 5 µm, 3 mL/min).

Materials and Culture Conditions
The actinomycete strain, Streptomyces sp. XS-16, was isolated from marine sediment samples collected from the sea around Naozhou Island in Zhanjiang, Guangdong Province. The strain was identified by 16S ribosomal RNA sequences (GenBank No. OQ449572). The strain was incubated in 2216E agar (5 g peptone, 1 g yeast extract, 1 g glucose, 0.1 g FePO 4 , 20 g agar, per liter seawater) at 28 • C for 5 days for cultivation. For compound isolation, the strain was cultured in the fermentation medium (20 g glucose, 4 g yeast extract, 1 g beef extract, 20 g soluble starch, 5 g soybean, 15 g K 2 HPO 4 , 20 g CaCO 3 per liter water) at 28 • C, 180 rpm for 8 days. The strain was deposited at the Marine Medicinal Bioresources Center, Ocean University of China, Qingdao, China.

Sequence Analysis of the SCrp Gene
The Crp gene was analyzed by Localblast with the reported Crp obtained in National Center for Biotechnology Information (NCBI). For the multiple sequence alignment analysis, the amino acid sequences of SCrp and other Crp homologues from different species retrieved from NCBI were aligned using the ClustalX software [36]. The phylogenetic analysis was conducted with the MEGA7 software [37]. The conserved domain of the SCrp protein was scanned by the InterProScan program [38].

Construction of the SCrp Expression Vector
The overexpression vector pSET152 which mainly contains a constitutive promoter ermE*, site-specific recombinant elements phage ϕC31 integrase and the attachment site attP gene, resistant acc3(IV) apramycin gene as selection markers was digested with restriction endonuclease BamHI. The SCrp gene was PCR-amplified (from genomic DNA of the wild-type strain Streptomyces sp. XS-16) using specific primers SCrp-F/R (Table S1). The PCR products SCrp gene was introduced into pSET152 vector to generate pSET152-SCrp using the ClonExpress Ultra One Step Cloning Kit C115 (Vazyme) ( Figure S3). The recombinant vector was transformed into competent E. coli strain DH5α to extract plasmids for transformation.

Transformants Screening
After conjugation, the transformants were passaged to 2216E plates with 100 µg/mL apramycin, respectively. Apramycin-resistant mutants were transferred onto new 2216E media containing 100 µg/mL apramycin for the second screening. The strains that were able to grow were subjected to further PCR analysis validation. The putative OE::SCrp mutants and the wild-type strain were cultured on 2216E for 5 days at 28 • C.

Fermentation
For small-scale analysis, the transconjugants and the control strain were inoculated into 100 mL fermentation medium in 500 mL Erlenmeyer flasks and incubated at 28 • C for 8 days, after which the cultures were extracted with the volume of ethyl acetate for 3 times. The organic phase was evaporated, and the residue was dissolved in MeOH, which was analyzed by HPLC, which indicated that the mutants showed an apparent change in metabolite production ( Figure 1).
For compound isolation, the selected strain was initially handled as above. Then a large-scale fermentation was performed in 500 mL Erlenmeyer flasks (total 30 L). The broth was extracted 3 times with ethyl acetate to give a total of 90 L of extract solution. The organic phase was evaporated under reduced pressure to afford a crude residue (3.2 g).

Extraction and Purification
Metabolites were monitored by LC-MS. The crude extract from 30 L fermentation of Streptomyces sp. XS-16 was subjected to a C 18  3.9. Computational Section 3.9.1. NMR Calculations Conformation searches based on molecular mechanics with MMFF force fields were performed for stereoisomers to get stable conformers within 20 kJ/mol. All conformers were further optimized by the density functional theory method at the B3LYP/6-31G(d) level by the Gaussian 16 program package. Gauge Independent Atomic Orbital (GIAO) calculations of their 1 H and 13 C NMR chemical shifts using density functional theory (DFT) at the mPW1PW91/6-311+G(d,p) level with the PCM model in DMSO. The calculated NMR data of these conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy. The 1 H and 13 C NMR chemical shifts for TMS were also calculated by the same procedures and used as the reference. After calculation, the experimental and calculated data were evaluated by linear correlation coefficients (R 2 ) and the improved probability DP4+ method [40].

ECD Calculations
All stable conformers were further optimized by the density functional theory method at the B3LYP/6-31G(d) level by the Gaussian 16 program package. The ECD were calculated using density functional theory (TDDFT) at the B3LYP/6-31+G(d,p) level in methanol with IEFPCM model. The calculated ECD curves were all generated using SpecDis 1.71 program package (version number, manufacturer's name, city, and country Version 1.71, SpecDis, Berlin, Germany, https://specdis-software.jimdo.com, accessed on 25 February 2023) and the calculated ECD data of all conformers were Boltzmann averaged by Gibbs free energy.

Cytotoxicity Assay
Cytotoxic activities of new compounds were evaluated against K562 (using the MTT method), MDA-MB-231, L-02, H69AR, and ASPC-1 (using the SRB method) cell lines. Adriamycin (ADM) was used as a positive control. The detailed methodologies for biological testing have been described in previous reports [41,42].

Antimicrobial Activity
Antimicrobial activities of new compounds against seven bacteria, including B. subtilis, P. vulgaris, B. cereus, E. coli, M. phlei, A. baumannii, and MRSA, in addition to one fungal strain C. albicans were carried out by using minimum inhibitory concentration (MIC) assay method as previously reported [35]. Ciprofloxacin was used as a positive control.

Conclusions and Outlook
Crp is a global transcription regulator that controls transcription initiation and widely affects the primary and secondary metabolism of bacteria. By overexpression of the native Crp homologue SCrp in the marine-derived strain of Streptomyces sp. XS-16, we discovered three undescribed angucycline derivatives 1-3. Structurally, compounds 1 and 2 are highly oxidized angucyclines, and they are isomerized at C-6a, C-12a, and C-12b. The angucycline structures have been reported to be derived from type II polyketide biosynthetic pathway [43]. One type II PKS gene cluster (cluster 1, Figure S1) has been discovered from the genome of Streptomyces sp. XS-16. Biosynthetic study of above angucycline derivatives is currently in progress.
The isolated compounds 1-3 were evaluated for their cytotoxic and antimicrobial activities in vitro. In addition, compound 1 showed inhibitory activities against five tumor cell lines with IC 50 values ranging from 0.32 to 5.33 µM. So far, this is the first report on the application of the global regulator Crp in marine-derived Streptomyces species. Specifically, Crp could upregulate the expression of genes involved in angucyclines biosynthesis. In addition, Crp is also a multifunctional regulator that modulates primary metabolism and enhances precursor flux to secondary metabolite biosynthesis. The above results support Crp as a useful tool exploiting the metabolic potential of marine-derived Streptomyces and developing the structural diversity of secondary metabolites.

Conflicts of Interest:
The authors declare no conflict of interest.