Genome mining and biosynthesis of kitacinnamycins as a STING activator† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc00815b

Genome mining targeting unique type II PKS and NRPS led to the identification of a novel class of glycopeptides named kitacinnamycins.

Compounds 7 (12 mg) and 8 (15 mg) were purified through HPLC from the extract of a large scale fermentation (4 L) of ΔKcn27 mutant strain using the same fermentation condition mentioned above.

Physicochemical data of kitacinnamycins
Kitacinnamycin A (1): light yellow amorphous powder; NMR data see

6
In vitro assay of Kcn28.
The Kcn28 reaction solution (100 μL) was performed in 100 mM phosphate buffer (pH 6.8) containing 5 µM Kcn28, 100 μM substrate, 1 mM UDP-glucose/UDP-GlcNAc. Reaction was incubated at 30 °C for 1 h, and terminated by adding 100 μL methanol. The mixture was centrifuged at 15,000 g for 10 min, and the supernatant was analyzed by LC-MS. LC-MS analysis was performed using a 18 min solvent gradient from 10% to 90% (0-15 min) and 100% (15-18 min) MeCN in water supplied with 0.1 TFA at a flow rate of 0.5 mL/min. UDPglucose and UDP-GlcNAc were obtained from Sigma-Aldrich.
Sequence similarity network (SSN) analysis. 4 The KS protein sequences for the SSN analysis include representative sequences from polyene type II PKSs (Cal30, ALG65306. For SSN analysis, an initial E value of 10 -10 was used from the local blast analysis (all vs all). The E values were converted into intergers using -log(E value), and the E value of 0 was manually assigned as 200. Cytoscape 3.7.0 were used for network visualization. Both self loops and duplicate loops were deleted. Finally, the E values of 10 -70 , 10 -55 , and 10 -15 were chosen for KS, KR, and DH figure generation, respectively.

Genome neighbouring network (GNN) analysis for putative CCNP gene clusters
For GNN analysis, all genes within the putative CCNP gene clusters were collected and translated. The total 3196 proteins from 53 BGCs (51 new identified BGCs plus two known BGCs (sky and cal)) were used for all-vs-all BLAST analysis with an initial E value of 10 -5 . The obtained E value from all-vs-all BLAST analysis were converted to intergers using -log(E value), and the E value of 0 was manually assigned as 200. Cytoscape 3.7.0 were used for newwork visualization. The self loops and duplicate loops were deleted. The E value of 10 -30 was chosen for figure generation.
Protein crystallization, structural elucidation and docking study.
The purified Kcn28 was incubated with thrombin to remove the N-terminal His tag. Crystals were grown at 4 °C with the sitting-drop vapor-diffusion method. Drops consisted of 1:1 ratio of proteins (10 mg/ml, 50 mM NaCl, 20 mM Tris, pH8.0) and crystallization buffer (1.6 M Ammonium sulfate, 0.1 M MES monohydrate pH 6.5, 10% v/v 1,4-Dioxane). Meanwhile, Kcn28-substrates complex was also achieved by crystallizing Kcn28 with different substrates at up to 1:10 ratio. Crystals of Kcn28 and Kcn28-9 complex were directly flash frozen in liquid nitrogen.
An single wavelength anomalous diffraction data of the Kcn28-9 complex was collected at BL18U1 beamline at the Shanghai Synchrotron Radiation Facility (SSRF) at wavelengths of 0.97930 Å, while the data of Kcn28 was collected at BL18U1 beamline at SSRF at wavelengths of 0.97894 Å. All diffraction datasets were processed and scaled using imosflm. 5 The phase problem of the Kcn28 and complex was solved by the molecular replacement method using the structure of the OleD protein (PDB ID: 2IYF) as the search model with PHASER, 6 and further autobuilded and refined by PHENIX, 7 COOT was used for manually model rebuilding and adjustments. 8 Finally, additional TLS refinement was performed in PHENIX. The final refinement statistics are listed in Table S17. Structural diagrams were prepared using the program PyMOL (http:// www.pymol.org/). The UDP was docked into the UDP binding pocket by using Autodock Vina. 9

Site-directed mutagenesis of Kcn28
Mutated fragments were amplified with primers listed in Table S2 by using plasmid pHG5007 as template. The purified PCR products were incubated with DpnI, T4 polynucleotide kinase and T4 DNA ligase, according to the standard procedure of Q5® Site-Directed Mutagenesis Kit purchased from NEB (USA). Each mutation was confirmed by sequencing. The recombined plasmids were expressed in E. coli BL21(DE) and purified as described above for native protein.

Cell culture
Human monocytic THP-1 cell line was purchased from Shanghai Institute of Cell Biology (Shanghai, China) and cultured at 37 °C in a 5% (v/v) CO2 atmosphere. Before further stimulation, THP-1 cells were treated with PMA (500 nM) for 3 h.

Immunoblot assay
Immunoblot assay was performed as described previously. 10 Briefly, proteins were extracted in lysis buffer. The proteins were then separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred onto polyvinylidene difluoride membranes. The membranes were probed with antibodies overnight at 4 °C, and then incubated with a horseradish peroxidase-coupled secondary antibody. Detection was performed using a LumiGLO chemiluminescent substrate system.

Name
Sequence The prediction of the substrate specificity was based on NRPS Predictor2. 13 13