*3.1. General Experimental Procedures*

The plasmids and bacteria used are listed in Table S1. *Streptomyces olivaceus* SCSIO T05 and its mutants were incubated on modified ISP-4 medium [25] with 3% sea salt and fermented in modified RA medium [19]. All cultures for *Streptomyces* were incubated at 28 ◦C. Luria-Bertani (LB) medium was used for *E. coli*, with appropriate antibiotics added at a final concentration of 100 μg/mL of ampicillin (Amp), 50 μg/mL of kanamycin (Kan), 50 μg/mL of apramycin (Apr), 25 μg/mL of chloroamphenicol (Cml), and 50 μg/mL of trimethoprim (TMP).

A 1260 infinity system (Agilent, Santa Clara, CA, USA), which uses a Phenomenex Prodigy ODS (2) column (150 × 4.6 mm, 5 μm, USA), was used for HPLC-based analyses. Silica gel with the size of 100–200 mesh (Jiangpeng Silica gel development, Inc., Shandong, China) was used for column chromatography (CC). A Primaide 1110 solvent delivery module, which is equipped with a 1430 photodiode array detector (Hitachi, Tokyo, Japan) and uses a YMC-Pack ODS-A column (250 mm × 10 mm, 5 μm), was used for semi-preparative HPLC. A MaXis Q-TOF mass spectrometer (Bruker, Billerica, MA, USA) was used to acquire high-resolution mass spectral data. An MCP-500 polarimeter (Anton Paar, Graz, Austria) was used to record optical rotations. A Bruker Avance 500 was used to record NMR spectra. Carbon signals and the residual proton signals of DMSO-*d*<sup>6</sup> were used for calibration (δ<sup>C</sup> 39.52 and δ<sup>H</sup> 2.50).

#### *3.2. Genome Sequencing and Bioinformatic Analysis*

Whole genome scanning and annotation of *S. olivaceus* SCSIO T05 were acquired by the single-molecule real-time (SMRT) sequencing technology (PacBio) at Shanghai Majorbio Bio-Pharm Technology Co., Ltd (Shanghai, China). AntiSMASH (AntiSMASH 5.0, available at http://antismash. secondarymetabolites.org/) was used to analyze and assess the potential BGCs. FramePlot (FramePlot 4.0 beta, available at http://nocardia.nih.go.jp/fp4/) was used to analyze ORFs whose functions were predicted based on an online BLAST program (http://blast.ncbi.nlm.nih.gov/).

## *3.3. Construction of a "Triple-Deletion" Mutant Strain*

Gene *lbpC4* from the *lbp* BGC was inactivated by the REDIRECT protocol [26]. All primers used in this study are listed in Table S2. *LbpC4* was replaced by the apramycin resistance gene *oriT*/*aac(3)IV* fragment in the target cosmids 01-07D or 21-02E. The target mutant clones, *S. olivaceus* SCSIO T05RXL, were accomplished as previously described [19,23,24].

#### *3.4. Fermentation and HPLC-based Analyses of S. olivaceus SCSIO T05 and Its Mutants*

The *Streptomyces* used in this study were incubated in modified ISP-4 medium plates for 2–3 d. For fermentation, a portion of mycelium and spores was seeded into 50 mL of modified RA medium in a 250 mL flask and then shaken at 200 rpm and 28 ◦C for 8 d. The cultures were extracted with an equal volume of butanone. Organic phases were then dissolved in CH3OH (1 mL) after having been evaporated to dryness, and 40 μL of each relevant sample was injected for HPLC-based analysis. The UV detection was at 254 nm. Solvent A is composed of 85% ddH2O and 15% CH3CN, supplemented with 0.1% HOAc. Solvent B is composed of 85% CH3CN and 15% ddH2O, supplemented with 0.1% HOAc. Samples were analyzed via the following method: a linear gradient from 0% to 80% solvent B in 20 min, and then, from 80% to 100% solvent B for 1.5 min, finally eluted with 100% solvent B in 6.5 min. The flow rate was 1.0 mL/min.

#### *3.5. Production, Isolation, and Structure Elucidation of Lobophorin CR4*

The mycelium of *S. olivaceus* SCSIO T05RX were inoculated into 50 mL of modified-RA medium and then shaken at 200 rpm and 28 ◦C for 2 d, to gain the seed cultures. After that, the seed cultures were transferred into 150 mL of modified-RA medium and shaken at 200 rpm and 28 ◦C for 8 d. After the large-scale fermentation was accomplished, a total of 12 L of the growth culture was centrifuged at 4000 g for 10 min to separate the supernatant and mycelium and further extracted by butanone and acetone, respectively. The two organic phases were concentrated (via solvent removal under vacuum), and the residues were combined. The combined sample was subjected to normal phase silica gel CC eluted with CHCl3-CH3OH (100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 85:15, 80:20, 70:30, 50:50, v/v, each solvent combination in 250 mL volume) to give ten fractions (AFr.1–AFr.10). Fractions A1-A3 were purified to afford the accumulation of compound **1** (98 mg), by preparative HPLC, eluting with 90% solvent B (A: H2O; B: CH3CN) over the course of 30 min. The flowrate was 2.5 mL/min and the UV detection was at 254 nm. The purified compound was subjected to MS, 1H, and 13C NMR spectra measurements and elucidated as a known intermediate **3** during lobophorins A and B biosynthesis [11], and we named it lobophorin CR4 (**1**).

### **4. Conclusions**

In this study, we acquired the complete genome sequence of *S. olivaceus* SCSIO T05. The biosynthetically talented strain harbors 37 putative BGCs analyzed by antiSMASH. To explore the biosynthetic potential of this strain, metabolic engineering and genome mining were performed. The major anthracenes and indolosesquiterpenes biosynthetic pathways were blocked, and an orphan spirotetronate antibiotics BGC (*lbp*) was activated in *S. olivaceus* SCSIO T05, leading to the isolation and identification of one known compound, lobophorin CR4. We have identified the *lbp* BGC accounting for lobophorin biosynthesis by gene-disruption experiments and bioinformatics analysis. The production of lobophorin CR4 without the attachment of d-kijanose to C17-OH was on account that the nonfunctional FAD-dependent oxidoreductase LbpP2 failed to generate d-kijanose. This work highlights that metabolic engineering and genome mining are the effective ways to turn on putative orphan or silent BGCs to acquire new NPs for drugs discovery.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/17/10/593/s1. This section includes HRESIMS, 1D NMR spectra for compound **1**, construction of Δ*lbpC4*.

**Author Contributions:** C.Z. performed the experiments and wrote the draft manuscript. W.D. performed the isolation of compound **1**. X.Q. helped to perform the sequence alignments. J.J. supervised the whole work and edited the manuscript. All authors read and approved the final manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (81425022, U1706206, and U1501223), and Natural Science Foundation of Guangdong Province (2016A030312014).

**Acknowledgments:** We are grateful to Aijun Sun, Xiaohong Zheng, Yun Zhang, Xuan Ma, and Zhihui Xiao, in the analytical facility center of the SCSIO for recording MS and NMR data.

**Conflicts of Interest:** The authors declare no conflicts of interest.
