Genomes database integration
We collected the genomes from a previously compiled database with 7,762 genomes retrieved from the GenBank. These sequences were processed with ad-hoc annotation and formatting tools18. To create a high-quality Streptomyces genomes database we first selected sequences assigned to the Actinomycetota phylum (formerly Actinobacteria). For this step, we parsed the taxonomic labels in our database against the genera listed in the entry for the Actinobacteria phylum at the NCBI taxonomy database (ID: 201174). This selection step resulted in >5000 genomes. Then, we selected for high quality genomes by filtering-out assemblies with more than 10 contigs. We reasoned that this cut-off would leave high quality genomes that may have one or more plasmids or chromosomes. Finally, we assessed the completeness of the selected genomes with BUSCO using the actinobacteria_class_odb10 reference lineage dataset36. We kept genomes with a minimum completeness of 85%. The final dataset included 720 genomes (Supplementary Table 1).
Taxonomic classification of Streptomyces strains
We calculated the core genome for the 720 selected strains. For this, we used the predicted proteomes of the 720 genomes to identify sets of conserved orthologs at different sequence identity cutoffs using BPGA37. We identified a set of 18 Actinomycetota-wide conserved protein sequences at a minimum sequence identity cutoff of 20% (Supplementary Figure 1). The set of 18 orthologs was then collected from each genome, the amino acid sequences were aligned, and concatenated to generate a super matrix with 6517 characters for phylogenetic analysis using IQtree 238. A substitution model was calculated for each partition in the super matrix (Supplementary table PCM2) and then a phylogenetic tree was calculated. The tree showed excellent support for most nodes, and it is consistent with the known general taxonomy of the phylum (Supplementary Figure 1). The tree features a clade with 201 taxa that we could safely assign to the Streptomycetaceae family (NCBI taxID: 2062). This clade includes strains assigned to the genera Streptomyces (190 strains), Kitasatospora (8 strains) and Streptacidiphilus (3 stranis). Within this group only a few genomes were misclassified: one Streptomyces strain classified as Kitasatospora and 6 Kitasatospora strains that were classified as Streptomyces; finally, one strain at the base of this clade (classified as Streptomyces scabrisporus NF3) did not group with the other genera and may belong to a different genus within the Streptomycetaceae family. We used the 201 genomes to construct a species tree for the Streptomyces genus using the same approach descried above for the Actinomycetota species tree. For the Streptomyces tree, we identified a set of 619 conserved orthologs across the 201 genomes at a sequence identity cutoff of 0.4. This set of orthologs was processed leading to a super matrix with 604 partitions (Supplementary Table 2) that was used to calculate a species tree that was rooted using the 17 non-Streptomyces strains as outgroup. This improved version is available at https://github.com/WeMakeMolecules/myCORASON. The Actinomycetota and Streptomyces species tree were constructed using code available at https://github.com/WeMakeMolecules/Core-to-Tree.
Functional analysis of conserved traits in in Streptomyces
The number and type of biosynthetic gene clusters for each strain was obtained using AntiSMASH39. To calculate the conserved set of orthologs of each of the 14 clades we used BPGA37. To identify the clade-specific conserved genes, we compared the clade-level cores with BPGA and extracted the genes that are uniquely conserved in clade 12 and their annotation. For the identification of PQQ biosynthetic gene clusters we used PqqB (Uniprot id: A0A0C6FBE1) as query for a search using Corason3 which has been modified from 10.1038/s41589-019-0400-9.
Strains and growth conditions
Strains and plasmids used in this study were listed in Table S12. E. coli DH5α was used for plasmids construction, and ET12567 (pUZ8002) was used for conjugation. All E.coli strains was cultivated in Luria-Bertani (LB) medium with appropriate antibiotics at 37 °C.
For cultivation and harvesting spores, S. coelicolor M145, S. albus J1074, S. cinnamonensis ATCC 15413 (recently reclassified as S. virginiae ATCC1541340), S. sp. NP10 and S. venezulae ISP5230 (ATCC 10712) were cultivated on SFM medium (20 g/L soybean powder, 20 g/L mannitol, 2% agar) plates. S. avermitilis NRRL 8165 (MA-4680), S. clavuligerus ATCC 27064, S. noursei ATCC 11455, S. cattleya NRRL 8057 were cultivated on YMG medium (4 g/L yeast extract, 10 g/L malt extract, 4 g/L glucose, 2% agar); S. rapamycinicus A-001 (a lab-stock strain close to NRRL 5491) was cultivated on oatmeal medium (20 g/L oatmeal, 2% agar); and S. griseus DSM 40236 was cultivated on ISP-4 medium (37 g/L ISP-4 medium powder (BD DIFCO™), 2% agar).
For conjugation, S. coelicolor, S. albus, S. cinnamonensis, S. sp. NP10, S. venezulae, S. avermitilis and S. rapamycinicus were performed on SFM medium supplemented with 10 mM MgCl2. S. clavuligerus, S. noursei, and S. cattleya were performed on YMG medium supplement of 10 mM MgCl2. S. griseus was performed on ISP-4 medium supplement of 10 mM MgCl2.
Unless stated, cultivation of streptomyces strains were performed according to the publications, with only a few modifications to accommodate to the present media component stock in our lab. For fermentation, strains were firstly inoculated on corresponding plates at 30 °C for 2 days, then 1 cm2 medium was inoculated into seed medium for growth of mycelium, and then 10% seed medium was transferred into fermentation medium for another round of cultivation, accumulation for secondary metabolites. Both seed cultivation and production cultures were performed in 250 mL flask filled with spring steels circle. All medium was adjusted to pH 7.0-7.5 before sterilization.
For S. coelicolor, the strain was cultivated in TSBY medium (30 g/L tryptone soya broth, 5 g/L yeast extract, 103 g/L sucrose) at 30 °C for 2 days, then transferred into YMG medium (4 g/L yeast extract, 10 g/L malt extract, 4 g/L glucose) on 30 °C for another 5 days41.
For S. albus, the strain was cultivated in TSB medium (30 g/L tryptone soya broth) at 30 °C for 24 hrs, then transferred into MFE medium (10 g/L glucose, 5 g/L soy bean flour, 21 g/L MOPS, 0.2 g/L Yeast extract, 0.6 g/L MgSO4·7H2O, 1.75 g/L K2HPO4, 5 mg/L CaCl2, 1 mg/L MnCl2, 1 mg/L ZnSO4, 5 mg/L FeSO4) on 30 °C for another 4 days42.
For S. cinnamonensis, strain was cultivated in seed medium (20 g/L dextrin, 15 g/L soybean powder, 2.5 g/L yeast extract, 5 g/L glucose, 1 g/L CaCO3) on 30 °C for 48 hrs, then transferred into fermentation medium (20 g/L soybean oil, 45 g/L glucose, 40 g/L soybean powder, 2.2 g/L NaNO3, 2.2 g/L Na2SO4, 0.07 g/L Al2(SO4)3, 0.1 g/L FeSO4, 0.33 g/L MnCl2, 0.075 g/L K2HPO4, 2.5 g/L CaCO3) at 30 °C for another 10 days43.
For S. sp. NP10, strain was cultivated in TSB medium at 30 °C for 24 hrs, then transferred into YMG medium on 30 °C for another 7 days.
For S. venezulae, strain was cultivated in TSB medium at 30 °C for 16 hrs, then transferred into MYM + MOPS medium (10 g/L malt extract, 4 g/L yeast extract, 4 g/L maltose, 2.1 g/L MOPS) on 30 °C with 10% inoculation amount, after 8 hrs (OD600 = 0.5-0.6), 6% (v/v) absolute ethanol was added, and kept on fermentation for 48 hrs44.
For S. avermitilis, strain was cultivated in seed medium (30 g/L corn starch, 2 g/L yeast extract, 20 g/L soybean powder, 0.0005 g/L CoCl2·6H2O) at 30 °C for 48 hrs, then transferred into production medium (50 g/L soluble starch, 10 g/L corn starch, 10 g/L yeast extract, 10 g/L soybean powder and 2 g/L CaCO3) and grown at 30 °C for another 10 days.
For S. rapamycinicus, strain was cultivated in seed medium (40 g/L corn starch, 5 g/L tryptone, 10 g/L yeast extract, 10 g/L glycerol and 2 g/L CaCO3) at 30 °C for 48 hrs, then transferred into production medium (13 g/L corn starch, 10 g/L soybean powder, 6 g/L tryptone, 35 g/L glucose, 3 g/L K2HPO4, 3 g/L KH2PO4, 1 g/L NaCl and 3 g/L CaCO3) and grown at 30 °C for another 7 days45.
For S. clavuligerus, strain was cultivated in the modified SNK-t medium (20 g/L Soybean powder, 5 g/L NaCl, 2.5 g/L K2HPO4, 24 g/L glycerol and 5 g/L soybean oil) at 30 °C for 48 hrs, then was transferred into the production medium (10 g/L glycerol, 20 g/L soybean powder, 23 g/L soybean oil, 1.2 g/L K2HPO4, 1 ml/L trace elements and 21 g/L MOPS) and grown at 30 °C for 4 days46.
For S. noursei, strain was cultivated in TSB medium at 30 °C for 48 hrs, then it was transferred into SAO-27 medium (12 g/L corn flour, 2 g/L tryptone soya broth, 2 g/L yeast extract) for 5 days47.
For S. cattleya, strain was cultivated in TSB medium at 30 °C for 48 hrs, then it was transferred into fermentation medium (1.5 g/L CaCO3, 2.5 g/L K2HPO4, 2.5 g/L KH2PO4, 0.125 g/L MgSO4·7H2O, 2.5 g/L NaCl, 1.25 g/L (NH4)2SO4, 7.5 g/L soybean powder, 7.5 g/L starch) for 6 days48.
For S. griseus, strain was cultivated in TSBY medium at 30 °C for 48 hrs, then it was transferred into SGG medium (5 g/L soluble starch, 5 g/L glucose, 5 g/L glycerol, 1.25 g/L corn steep liquor, 2.5 g/L soybean powder, 1 g/L yeast extract, 1.5 g/L CaCO3 and 1 ml/L trace elements) for 7 days49.
Construction of plasmids and strains
The primers used in this study are listed in Table S10. The fragment including pqqA-E was amplified from genome sequence of Streptomyces hygroscopicus XM201 and was introduced between the NdeI/EcoRI restriction sites of pLQ646 by Gibson assembly. The plasmid pLQ646 was constructed previously by introduction of kasOp* promoter in the multiple cloning site of pSET15250. The resulting plasmid pXR-1 was then introduced into E. coli ET12567 (pUZ8002) for conjugation with different Streptomyces. After conjugation, the exo-conjugants were confirmed both by apramycin resistance and PCR verification.
Metabolomic analysis
After fermentation, the culture from three biological replicates was diluted by mixing with two volumes of methanol and sonicated for 30 min. The samples were centrifuged at 13, 800 g for 5 min to remove insoluble particles and then filtered using 0.22 μm filter membrane. The filtrate was injected to Agilent 1290 series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent 6546 Accurate-Mass Q-TOF LC-MS (Agilent Technologies, Santa Clara, CA, USA) via an electrospray ionization source (ESI). LC conditions were as follows: (A, water + 0.1% formic acid; B, acetonitrile + 0.1% formic acid; 0.4 ml min-1). The data were collected using Agilent Data aquacition software (version 10.1) (Agilent Technologies, Santa Clara, CA, USA). Then the collected raw data were further processed by Agilent Profinder (version 10) (Agilent Technologies, Santa Clara, CA, USA) for peak alignment, and Agilent Mass Profiler Professional (version 15.0) (Agilent Technologies, Santa Clara, CA, USA) for grouped differential analysis and compound annotation.
For compound annotation, the compounds were annotated with dictionary of streptomeDB database covering up to now most natural products identified in Streptomyces in Agilent Mass Profiler Professional (version 15.0) according to the molecular weight and predicted addiction. The annotated compound was embedded into Agilent Qualitative analysis (version 10.0) (Agilent Technologies, Santa Clara, CA, USA) for peak confirmation to make sure the difference with the target molecular is < 5 ppm, also the shapeless peaks were removed. The detailed information for known natural products was shown in Figure S8.
Proteomic analysis
Protein extracts were prepared as previously described with minor modifications 51. To prepare protein extracts, strains were grown in YMG medium for 24 hours and cells were harvested. Cells were then resuspended in a solution containing 50 mM NH4HCO3 and 8 M urea, along with 0.5 mm glass beads (Biospec, US), and mechanically disrupted using a tissue grinding machine (10 times for 30 seconds each). The resulting cell debris was pelleted by centrifugation at 12,000×g for 10 minutes at 4°C. Protein concentration was determined using a BCA protein assay kit (Solarbio, China), and 100 μg of protein was used for digestion. The samples were first treated with 5 mM reducing dithiothreitol (DTT) for 30 minutes at 56°C, followed by 25 mM iodoacetamide (IAM) for alkylation in the dark at room temperature for 1 hour. The samples were then diluted with 50 mM NH4HCO3 to a final urea concentration of 2 M and digested overnight with trypsin (Promega, US) at an enzyme-to-protein ratio of 1:50 (w/w) at 37°C. The resulting tryptic peptides were desalted using C18 Sep-Pak cartridges (Welch, China), vacuum dried, and stored at -80°C prior to LC-MS/MS analysis.
To prepare for LC-MS/MS analysis, the vacuum dried samples were reconstituted in an aqueous solution containing 0.1% FA. Peptides were then loaded onto a 3 μm x 0.2 cm pre-column (P/N 164535, Thermo Scientific, US) and separated on a 2 μm x 15 cm capillary column (P/N 164534, Thermo Scientific, US) using the following HPLC gradient: 3-8% B in 5 minutes, 8-20% B in 65 minutes, 20-30% B in 20 minutes, 30-40% B in 10 minutes, 40-99% B in 5 minutes, held at 99% B for 5 minutes, and then held at 3% B for 10 minutes (A = 0.1% formic acid in water, B = 0.1% formic acid in 80% acetonitrile) at a flow rate of 300 nL/min. The peptide samples were analyzed using an Ultimate™ 3000 RSLC nano system (Thermo Scientific, US) coupled to a Q-Exactive™ HF Orbitrap mass spectrometer (Thermo Fisher Scientific, US). The mass spectrometer was operated in data-dependent mode with a full MS scan (350–2000 m/z) at a resolution of 60,000 followed by HCD (Higher Collisional Dissociation) fragmentation at a normalized collision energy of 28%. The MS2 automatic gain control (AGC) target was set to 45 s.
The raw MS files were processed by MaxQuant (http://maxquant.org/, version 1.6.10.43). MS/MS spectra were searched against the Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) protein database (downloaded from UniProt, version 2002) using the Andromeda search engine embedded in MaxQuant. Both peptide and protein assignments were filtered to achieve a false discovery rate (FDR) < 1%. After searching, the reverse hits, contaminants, and proteins only identified by one site were removed.
Transcriptome analysis
To perform transcriptome analysis, strains of S. rapamycinicus and S. griesus introduced PQQ gene cluster as well as the empty vectors were cultured under normal fermentation condition for 12 h. The total RNA of each sample was extracted using Total RNA Extractor kit (B511311, Sangon, China) according to the manufacturer’s protocol. The extracted RNA was then sent to the Sangon Biotech (Shanghai) Co., Ltd. for library construction and sequencing. Gene expression level was calculated by DESeq2 R package (v.1.20.0), displaying as average values of three clones from each group.
Analytical methods
Cell growth was monitored by determination of cell dry weight. Intracellular levels of ATP (ab113849), NADH/NAD+ (ab65348) and NADPH/NADP+ (ab65349) were analyzed by commercialized kits (Abcam) following the instructions.