*2.3. Identification of a Putative Lobophorin (lbp) BGC via Genome Mining*

The antiSMASH analysis of the complete genome of *S. olivaceus* SCSIO T05 revealed a 99.1 kb type I PKS BGC named as lobophorin BGC (*lbp*), showing highly similar traits to the reported *lob* BGCs from *Streptomyces* sp. FXJ7.023 [16] and *Streptomyces* sp. SCSIO 01127 [11]. The complete *lbp* contains 38 open reading frames (ORFs). The genetic organization of *lbp* is shown in Figure 4A, with genes color-coded on the basis of their proposed functions summarized in Table 3. The nucleotide sequences were deposited in GenBank (MN396889). The *lbp* BGC contains six inconsecutive genes *lbpA1*–*A6*, similar to *lobA1*–*A5* in *lob* from *S.* sp. SCSIO 01127. Differently, the LobA4 homologue is separated into two polyketide synthases (PKSs), LbpA4 and LbpA5, in *lbp*. The high similarity between the PKS modules in *lbp* and in *lob* enables us to propose that the assembly of the linear polyketide chain catalyzed by LbpA1–A6 utilizes six malonyl CoAs, six methylmalonyl-CoAs, and a 3-carbon glycerol unit (Figure 5) [11]. The *lbp* harbors four putative regulator genes (*lbpR1–R4*) (Figure 4 and Table 3) that are highly similar to *lobR1*, *lobR3*, *lobR4*, and *lobR5* in *lob*, respectively. These four regulators are assumed to be involved in the regulation network of lobophorin CR4 biosynthesis, which seems to be less complex than *lob* but more complex than *kij* [7] and *tca* [8]. In contrast, five regulator genes *lobR1–R5* are identified in *lob*; three regulator genes, *kijA8*, *kijC5*, and *kijD12,* are included in *kij*; *tcaR1* and *tcaR2* both encode regulators in *tca*. There is only one gene, *lbpU2* in *lbp*, with no apparent homologue in *lob* (Figure 4 and Table 3). The other genes included in *lbp* are putatively associated with the biosynthesis of kijanose and l-digitoxose units by virtue of high similarities to corresponding counterparts in *lob* (Figure 4 and Table 3).

**Figure 4.** Genetic organizations: (**A**) the *lbp* BGC from *S. olivaceus* SCSIO T05; (**B**) the *lob* BGC from *S.* sp. SCSIO 01127.

**Figure 5.** Proposed biosynthetic pathway of lobophorin CR4.


**Table 3.** Deduced function of open reading frames (ORFs) in the *lbp* BGC.

<sup>a</sup> Amino acids. <sup>b</sup> Identity/similarity.

To demonstrate the validity of the putative *lbp* BGC, *lbpC4* coding for ketosynthase-III-like protein, which incorporates a 3-carbon glycerol unit into the biosynthetic precursor LOB aglycon [11], was disrupted by using PCR-targeting methods. As expected, the production of lobophorin CR4 was completely blocked in *S. olivaceus* SCSIO T05/Δ*rsdK*2/Δ*xmcP*/Δ*lbpC4* (*S. olivaceus* SCSIO T05RXL) (Figure 2, trace iv), demonstrating that the *lbp* BGC is indeed responsible for lobophorin biosynthesis. With high similarity to the *lob* BGC, the *lbp* BGC accounts for lobophorin CR4 without the attachment of kijanose to C17-OH, rather than lobophorins A and B in *lob*. Based on bioinformatics analysis, a series of enzymes are proposed to be involved in kijanose biosynthesis (Figure 5) [7]. Among them, the amino acid sequence of the putative FAD-dependent oxidoreductase LbpP2 is far shorter than its homologues LobP2 [11] and KijB3 [7]. KijB3 is proposed to oxidize the methyl group to a carboxylate group, essential for the generation of the kijanose moiety [7]. Multiple protein sequence alignments of LbpP2, LobP2, and KijB3 revealed that the conserved FAD binding domain is missing in LbpP2 (Figure S5). Thus, we speculate that LbpP2 is nonfunctional, failing to catalyze the carboxylation and hinder the generation of kijanose.

Given the high similarity of LbpG3 and LobG3, we envision that LbpG3 has a similar function as LobG3, a glycosyltransferase from *S.* sp. SCSIO 01127, tandemly attaching the first two l-digitoxose at C-9 in lobophorins [11]. LbpG2 has 99% similarity to LobG2, another glycosyltransferase from the same strain, which was established to transfer the terminal l-digitoxose [11]. Both LbpG2 and LbpG3 are likely to be involved in the transfers of three sugar units, sugars A, B, and C, in lobophorin CR4 (Figure 5), consistent with the metabolite profile of Δ*lobG1* in *S.* sp. SCSIO 01127 [11].
