*2.2. Activation of a Cryptic Lobophorin BGC in the Genetically Engineered Mutant*

In actuality, only a minority of potential chemicals are produced under standard laboratory culture conditions. Furthermore, the corresponding products are likely to be overlooked for multiple reasons, including low production rates, a large metabolic background, or improper culture conditions [22]. Fermented using modified-RA medium, the secondary metabolites produced by *S. olivaceus* SCSIO T05 were subsequently profiled using HPLC-DAD-UV. Multiple peaks were detected in the fermentation extract (Figure 2, trace i). We previously reported that five known NPs, rishirilides B (**2**) and C (**3**), lupinacidin A (**4**), galvaquinone B (**5**), and xiamycin A (**6**), were produced as major secondary metabolites from the wild-type strain [19,23]. In addition, an orphan dibenzoxazepinone biosynthetic pathway was mutagenically activated, leading to the production of new mycemycins [24], suggesting that *S. olivaceus* SCSIO T05 has a great potential for producing new NPs.

**Figure 2.** HPLC-based analyses of fermentation broths: (i) *S. olivaceus* SCSIO T05; (ii) *S. olivaceus* SCSIO T05R; (iii) *S. olivaceus* SCSIO T05RX; and (iv) *S. olivaceus* SCSIO T05RXL. Compound **1** is lobophorin CR4. Compounds **2**–**6** were previously identified as rishirilide B, rishirilide C, lupinacidin A, galvaquinone B, and xiamycin A, respectively.

For exploring other secondary metabolites from the strain, *S. olivaceus* SCSIO T05/Δ*rsdK*<sup>2</sup> (*S. olivaceus* SCSIO T05R) was constructed to abolish the production of the anthracenes [19]. The production of the second major secondary metabolites xiamycins was accumulated, along with a new peak around 26 min, distinct from the UV absorption characteristics of xiamycins (Figure 2, trace ii). For further background elimination of xiamycins, a "double-deletion" mutant *S. olivaceus* SCSIO T05/Δ*rsdK*2/Δ*xmcP* (*S. olivaceus* SCSIO T05RX) was constructed [23] in which the new peak (**1**) appeared to be the major product (Figure 2, trace iii). Accordingly, the *S. olivaceus* SCSIO T05RX mutant was fermented at a large scale, enabling the isolation and structure elucidation of this newly generated compound. It was identified as a known compound designated as lobophorin CR4 (Figure 3), by comparing HRESIMS, 1H, and 13C NMR data (SI, Figures S2–S4) to the reported data of an intermediate isolated from the *Streptomyces* sp. SCSIO 01127/Δ*lobG1* mutant [11]. It is reported that shifting metabolic flux of a wild-type strain by blocking the predominant product pathways may afford new secondary metabolites [5]. During our efforts to acquire new secondary metabolites by shifting the metabolic flux of marine actinomycetes [5,23,24], the production of nocardamine, olimycins, and mycemycins was turned on at the expense of major products by using gene knock-out methods. Similarly, the "double-deletion" mutant (*S. olivaceus* SCSIO T05RX) was constructed to abolish the production of two major secondary metabolites, anthracenes and xiamycins, from the wild-type strain [19,23]. With the engineered shifting of *S. olivaceus* metabolic flux, the newly produced lobophorin CR4 was activated.

**Figure 3.** Structure of the isolated lobophorin CR4.
