/n IMPACT FACTOR Indexed in: CITESCORE PubMed 6.4 microorganisms 4.5 Article Activation of Secondary Metabolism in Red Soil-Derived Streptomycetes via Co-Culture with Mycolic Acid-Containing Bacteria Kairui Wang, Ning Liu, Fei Shang, Jiao Huang, Bingfa Yan, Minghao Liu and Ying Huang Special Issue Secondary Metabolism of Microorganisms Edited by Dr. Carlos García-Estrada and Dr. Carlos Barreiro MDPI https://doi.org/10.3390/microorganisms9112187 MDPI wwww Article microorganisms Activation of Secondary Metabolism in Red Soil-Derived Streptomycetes via Co-Culture with Mycolic Acid-Containing Bacteria Kairui Wang 1,2, Ning Liu ¹, Fei Shang ³, Jiao Huang 1,2, Bingfa Yan 1,2, Minghao Liu ¹,* and Ying Huang 1 3 1,2,* State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; wangkairui1994@163.com (K.W.); fussliu@126.com (N.L.); huangjiao515665@163.com (J.H.); yanbingfa2014@126.com (B.Y.) 2 College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China 3 Analytical and Testing Center, Beijing University of Chemical Technology, Beijing 100029, China; shangfei@mail.buct.edu.cn * Correspondence: lysf1987313@163.com (M.L.); huangy@im.ac.cn (Y.H.) check for updates Citation: Wang, K.; Liu, N.; Shang, F.; Huang, J.; Yan, B.; Liu, M.; Huang, Y. Activation of Secondary Metabolism in Red Soil-Derived Streptomycetes via Co-Culture with Mycolic Acid-Containing Bacteria. Microorganisms 2021, 9, 2187. https://doi.org/10.3390/ microorganisms9112187 Academic Editors: Carlos García-Estrada and Carlos Barreiro Received: 28 September 2021 Accepted: 15 October 2021 Published: 20 October 2021 Abstract: Our previous research has demonstrated a promising capacity of streptomycetes isolated from red soils to produce novel secondary metabolites, most of which, however, remain to be explored. Co-culturing with mycolic acid-containing bacteria (MACB) has been used successfully in activating the secondary metabolism in Streptomyces. Here, we co-cultured 44 strains of red soil- derived streptomycetes with four MACB of different species in a pairwise manner and analyzed the secondary metabolites. The results revealed that each of the MACB strains induced changes in the metabolite profiles of 35-40 streptomycetes tested, of which 12–14 streptomycetes produced "new" metabolites that were not detected in the pure cultures. Moreover, some of the co-cultures showed additional or enhanced antimicrobial activity compared to the pure cultures, indicating that co-culture may activate the production of bioactive compounds. From the co-culture-induced metabolites, we identified 49 putative new compounds. Taking the co-culture of Streptomyces sp. FXJ1.264 and Mycobacterium sp. HX09-1 as a case, we further explored the underlying mechanism of co-culture activation and found that it most likely relied on direct physical contact between the two living bacteria. Overall, our results verify co-culture with MACB as an effective approach to discover novel natural products from red soil-derived streptomycetes. Keywords: Streptomyces; co-culture; mycolic acid-containing bacteria (MACB); secondary metabolites (SMs); activation of natural products Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. CC BY Copyright: 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1. Introduction Natural products (NPs), or their semi-synthetic derivatives, are important sources of lead compounds in drug discovery [1,2]. More than 50% of clinically used antibiotics were derived from filamentous Gram-positive bacteria of the genus Streptomyces [3,4]. Analysis of the genome sequences of Streptomyces and related genera revealed that they may contain a great variety of secondary metabolite (SM) biosynthetic gene clusters (BGCs) encoding novel NPs [5,6]. However, most of these BGCs remain silent under laboratory conditions, thus triggering research in the sense of developing activating strategies for the genome mining of microbial NPs [7–9]. Several previous studies reported that microbial cryptic BGCs could be activated by co-culturing the host strains with other species [10–13], partially due to mimicking the in situ microbial interactions in the original environment where microorganisms coexist [14]. However, traditional co-culture methods require large-scale screening to find ideal micro- bial combinations, and thus, is laborious and difficult to apply at scale. In recent years, it has been shown that co-cultures with mycolic acid-containing bacteria (MACB) widely Microorganisms 2021, 9, 2187. https://doi.org/10.3390/microorganisms9112187 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 2187 2 of 15 activate cryptic SM-BGCs in streptomycetes [15]. Hitherto, around 40 novel NPs have been discovered from Streptomyces, and rare actinobacteria by co-culture with MACB strains. These compounds comprise a variety of chemical scaffolds and bioactivities, validating this SM-BGC activation method of co-culture [16–18]. Red soils are widely distributed in tropic and subtropical areas of southern China. These soils are acidic, oligotrophic, and rich in iron and aluminum oxides, and thus provide ideal habitats for acidophilic actinobacteria [19]. Our recent studies have shown that red soil-derived streptomycetes are prolific NP producers [19], and have identified several NPs with novel scaffolds or modifications from these strains, as exemplified by azolemycins [20], NC-1 [21], and mycemycins [22]. Meanwhile, the in silico genome- mining of red soil-derived streptomycetes also reveals that these strains contain numerous unidentified SM-BGCs, the products of which remain to be unraveled. To activate the silent SM-BGCs of red soil-derived streptomycetes for NPs discovery, we selected 44 bioactive Streptomyces isolates and co-cultured them with four MACB of different species in a pairwise manner. Metabolites of the co-cultures were subjected to multi-spectroscopic analyses and bioactivity assay, which showed that the MACB strains effectively activated secondary metabolism in most of the streptomycetes. We also tried to explore the underlying mechanism of co-culture activation. Results of the study gain a deep insight into the NP biosynthetic potential of red soil-derived streptomycetes. 2. Materials and Methods 2.1. Strains and Media The strains used in this work are listed in Table S1. Eight MACB were isolated from soils collected in Haixi Mongolian and Tibetan Autonomous Prefecture, China [23], and two MACB and all Streptomyces strains were from red soils collected in Jiangxi Province, China [19,24]. All these strains were preliminarily identified by 16S rRNA gene sequencing in our previous studies [19,23,24]. Three indicator strains from different phyla were used for antimicrobial activity assay: Micrococcus luteus CGMCC 1.2567 (Gram-positive bacterium) and Trichoderma viride CGMCC 3.1913 (fungus) were obtained from the China General Microbiological Culture Collection Center (CGMCC), and extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli 4-1 (Gram-negative bacterium) was obtained from the Weifang Medical University, Shandong Province, China. A YGGS medium (glucose 5.0 g, soluble starch 20.0 g, glycerin 20.0 g, yeast extract 3.0 g in 1 L dd-H₂O, pH 7.2) [25] was used for the co-culture and pure culture of strains. A TSB medium (pancreatic digest of casein 17.0 g, papaic digest of soybean 3.0 g, dextrose 2.5 g, NaCl 5.0 g, K₂HPO4 2.5 g in 1 L dd-H₂O, pH 7.1-7.5) was used for seed culture. A GYM agar (yeast extract 4.0 g, malt extract 10.0 g, glucose 4.0 g, CaCO3 2.0 g, agar 15.0 g in 1 L dd-H2O) was used for recovering strains from glycerol stocks. An LB agar (tryptone 10.0 g, yeast extract 5.0 g, NaCl 10.0 g, agar 15.0 g in 1 L dd-H₂O) and PDA (glucose 20.0 g, potato powder 6.0 g, agar 15.0 g in 1 L dd-H2O) were used to culture the indicator bacteria and fungus, respectively. 2.2. Co-Culture and Pure Culture of Strains After the incubation of the strains on a GYM plate for 2-3 days, an agar block of about 1 cm² with bacterial lawn was cut out and transferred into a 250 mL shake flask containing 50 mL of TSB medium for seed culture. The seeds of Streptomyces and MACB were cultured at 28 °C on a rotary shaker at 160 rpm for 3 and 2 days, respectively. Then, 3 mL of Streptomyces and 1 mL of MACB seed cultures were co-transferred into a 250 mL flask containing 100 mL of YGGS medium and fermented at 28 °C, 220 rpm for 7 days. Pure culture controls were performed similarly but with single strains. Each experiment was repeated in triplicate in this study. Microorganisms 2021, 9, 2187 3 of 15 2.3. SM Extraction, Isolation, and Analysis The resulting cultures were collected and extracted three times with an equal volume of ethyl acetate. The extracts were combined and concentrated in vacuo to evaporate the solvent, and the residue was re-dissolved in 1 mL of methanol. An HPLC analysis was carried out with a Shimadzu SPD-M20A HPLC system, using a Waters Xbridge ODS column (4.6 × 150 mm, 5 μm) with a linear gradient of MeOH/H2O (see Table S2). The injection volume of the sample was 20 µL. The Dionex 3000 RS system was used to set the temperature at 30 °C and the flow rate at 1.0 mL/min; the elution curves of metabolites were monitored at 220, 254, and 300 nm, respectively. Differences in the secondary metabolism between the co-cultures and pure cultures were determined by comparing their HPLC profiles based on the retention time and UV absorption spectra of peaks. Metabolites corresponding to the differential HPLC peaks were then collected and subjected to UHPLC-HRMS (Waters Xevo G2 quadrupole time of flight-ultra performance liquid chromatography, and mass spectra scanning from 100 to 2000 atomic mass units) to obtain their accurate molecular weights (MWs). The resultant mass spectrum data were analyzed by the Mass Lynx v 4.1 software system. Compounds were identified by the comparison of MWs, UV spectra, and retention times with published chemical data from standard databases (Dictionary of Natural Prod- ucts [DNP] on DVD, version 22.2 and on web, version 30.1 [http://dnp.chemnetbase.com/, accessed on 1 September 2021]; ChemSpider [http://www.chemspider.com/, accessed on 2 September 2021]) and references. The activated metabolites with characteristic information unmatched with that in the databases were inferred as putative new products. Some of these compounds were subjected to nuclear magnetic resonance (NMR) spectroscopic analysis (Bruker AVIII 500 MHz NMR spectrometer, Bruker, Karlsruhe, Germany) for further structure elucidation. 2.4. Bioactivity Assay Bioactivities of the fermentation extracts were tested against M. luteus, ESBL-producing E. coli, and T. viride using agar-well diffusion assay. Twenty μL of each extract were added into a punched hole (7 mm in diameter) in LB/PDA plates containing indicator strains. The plates were then cultured at 37 °C for 12 h for bacterial indicators or at 28 °C for 48 h for the fungus. Antimicrobial activity was estimated by measuring the diameter of the inhibition zones: positive (7 mm < diameter ≤ 9 mm) and strongly positive (diameter > 9 mm). 2.5. Non-Contact Co-Culture of Streptomyces sp. FXJ1.264 and Mycobacterium sp. HX09-1 The co-culture was carried out in a device of two connected culture compartments separated by a 0.22-µm polyether sulfone (PES) membrane (Figure S1). Each of the com- partments contained 50 mL YGSS medium and were inoculated with 3 mL seed culture of S. sp. FXJ1.264 or 1 mL seed culture of M. sp. HX09-1. The device only allowed substance exchange between the compartments, but the cells of the two strains could not contact each other. For the control groups, only one compartment in the device was inoculated, with either a single strain or two strains mixed. The device was fixed on a shaker and the strains were fermented at 28 °C, 220 rpm for 7 days. The fermentation metabolites from each compartment were analyzed using the method described in Section 2.3. 2.6. Co-Culture of S. sp. FXJ1.264 and Heat-killed M. sp. HX09-1 M. sp. HX09-1 was cultured in 250 mL flasks each containing 50 mL of the YGGS medium for 2 or 7 days. The culture broths were then heated at 121 °C for 20 min to kill the cells. After cooling down to room temperature, the flask was added with 50 mL fresh YGGS medium and 3 mL seed culture of S. sp. FXJ1.264, and the resulting culture was incubated at 28 °C, 220 rpm for 7 days. The fermentation metabolites were analyzed as above. Microorganisms 2021, 9, 2187 4 of 15 3. Results 3.1. Preliminary Evaluation of the Activation Ability of MACB and Selection of Red Soil-Derived Streptomycetes Ten MACB candidates (listed in Table S1) and two known NPs producer strains that were isolated from red soil, Streptomyces spp. FXJ1.172 and FXJ1.264 [19], were used for preliminary co-culture evaluation. The HPLC profiles of metabolites showed that three of the MACB (Mycobacterium sp. HX10-42, Nocardia sp. HX14-21, and Rhodococcus sp. HX10-55) obviously activated S. sp. FXJ1.172 to produce new peaks compared to their individual pure cultures (Figure 1a). Meanwhile, a series of unique peaks were detected in the combined culture of S. sp. FXJ1.264 and Mycobacterium sp. HX09-1 (Figure 1b). The other six MACB did not exhibit activation ability when co-cultured with the two Streptomyces strains. Therefore, the above four strains of MACB were chosen for subsequent co-culture. In addition, based on the antimicrobial activity and 16S rRNA gene similarity of the red soil-derived Streptomyces strains [19,24], 44 bioactive streptomycetes with abundant diversity were preferentially selected for this study (Table S1). The selected MACB and streptomycetes thus formed 176 (4 × 44) co-culture pairs. a. Absorbance units A=316 nm b. 1.5 (vii) (vi) 1.5 1.0 (V) 1.172+HX10-42 HX10-42 1.172+HX14-21 (iv) HX14-21 (iii) 1.172+HX10-55 0.5 (ii) (i) HX10-55 Absorbance units 1.0 (iii) 0.5 (ii) 1.172 (i) Ми A=254 nm 1.264+HX09-1 HX09-1 1.264 0.01 0.01 15.0 17.5 20.0 Retention time (min) 22.5 25.0 15.0 17.5 20.0 Retention time (min) 22.5 25.0 Figure 1. Preliminary evaluation of the activation ability of mycolic acid-containing bacteria (MACB). (a) HPLC analysis of the fermentation extracts of S. sp. FXJ1.172 co-cultured with different MACB and the extracts of their pure cultures; (b) HPLC analysis of the fermentation extracts of S. sp. FXJ1.264, M. sp. HX09-1, and their combined culture. 3.2. Co-Culture with MACB Changed the SM Profiles of Streptomycetes M. HPLC analysis showed changes in SM profiles of most (82.9%, 146/176) of the co- cultures compared to the pure culture counterparts (Figure 2 and Table 1). The differences of metabolites were characterized by four patterns of HPLC peaks: the increase/decrease in metabolite production (the integral area of peaks changed by more than 30%), appearance of new metabolite peaks, loss of original peaks, and no change. The comparison results were mostly a combination of the above patterns due to the complex secondary metabolism in Streptomyces (Figure 2). For example, compared to the pure cultures, co-culture with sp. HX09-1 changed the secondary metabolism of 40 Streptomyces strains. Among them, 30 and 23 strains enhanced and decreased the production of some original metabolites, respectively, 14 strains produced new metabolites that were not found in the pure cultures, and seven strains lost some metabolites (Figure 2a). M. sp. HX10-42, R. sp. HX10-55, and N. sp. HX14-21 exhibited similar activation ability (Figure 2b-d) (Table 1). In summary, 29.5% (52/176) of the co-cultures activated the production of "new" SMs and 60.8% (107/176) of the co-cultures increased the production of original metabolites (Table 1). For all four MACB strains, the proportion of positive impact (increase in production and/or appearance of "new" metabolites) on the secondary metabolism of Streptomyces is significantly higher (p<0.01) than that of negative impact (decrease in production and/or loss of metabolites), as shown in Table 1. Only four of the co-cultured streptomycetes were not activated by any