# Naphthoquinone-Based Meroterpenoids from Marine-Derived _Streptomyces_ sp. B9173
## 3 Results
### Isolated New Natural Products Revealing New Insight into Flaviogeranin Biosynthesis  
Even though the molecular architecture of these isolated naphthoquinone-containing compounds is different, the common structural feature indicates that they may use the same early biosynthetic intermediates and biosynthetic pathway. Formation of the aromatic backbone of naphthoquinone  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Compound** & \multicolumn{2}{c}{**MIC (\(\upmu\)g/mL)**} & \multicolumn{2}{c}{**IC\({}_{50}\) (\(\upmu\)M)**} \\ \hline  & _S. aureus_ & _M. smegmatis_ & **A549** & **Hela** \\ \hline
1 & 14.6 \(\pm\) 0.3 & 12.4 \(\pm\) 0.3 & 25.7 \(\pm\) 0.4 & 34.7 \(\pm\) 0.6 \\
2 & 28.1 \(\pm\) 0.4 & 35.1 \(\pm\) 0.6 & 46.6 \(\pm\) 0.4 & 50.2 \(\pm\) 0.5 \\
3 & 9.2 \(\pm\) 0.2 & 5.2 \(\pm\) 0.2 & 0.6 \(\pm\) 0.2 utilizes a common precursor which is synthesized by type III PKS [12; 13]. To mine the genome for the biosynthetic genes of these flaviogeranin congeners, we aimed at identifying type III PKS genes that are essential for the biosynthesis of the THN skeleton and genes encoding preryltransferases in the adjacent region [22]. A \(\sim\)48 kb candidate gene cluster has been found and sequence analysis revealed type III PKS genes putatively involved in the construction of the naphthoquinone polyketide core, associated with the biosynthesis and connection of the terpenoid units [22]. Putative regulators, resistance genes, and genes with unknown function are involved in the cluster too.  
THN formed by such type III PKS appears to be an intermediate in the biosynthetic pathways for various naphthoquinone-containing secondary metabolites [27]. Type III PKS from _Streptomyces_ sp. B9173 showed 91% identity in amino acid sequence to the chalcone synthase (CHS)-like protein RppA from _Streptomyces griseus_[12]. Sequence alignment of the type III PKS with its counterparts from the gene cluster of naphthoquinone-containing compounds revealed the conserved catalytic triad residues (Cys138, His270, and Asn303) (Figure S2). The terpenoid origin of the C\({}_{10}\) moiety indicated that the mevalonic acid (MVA) pathway genes in the cluster were responsible for the formation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate building blocks.  
Based on the chemical structures of these flaviogeranin congeners isolated from _Streptomyces_ sp. B9173, we proposed a biosynthetic pathway, as shown in Figure 4. Type III PKS catalyzes naphthoquinone ring formation: it selects malonyl-coenzyme A as the start unit, carries out four successive extensions to form the pentaketide, releases the resulting nonreducing ketide, and cyclizes to form THN. The symmetric intermediate THN was subsequently oxidized to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin), which then modified to form various compounds through oxidation, methylation, and preparations to produce 3-7. Two putative preryltrasnferases likely catalyze the prenylation at two different positions: the prenylation at hydroxyl group on the C7 to generate flaviogeranin A (7) and the prenylation at the C2 to give flaviogeranin D (3). Isolation of flaviogeranin C1 (4) and flaviogeranin C2 (5) revealed the oxidation at the C8, followed by successive methylations. An aminotransferase introduced an amino group on C8 to generate 8-amino-flaviolin (8-amino-2,5,7-trihydroxynaphthalene-1,4-dione) and dedicated modification enzymes further tailored the scaffold to form flaviogeranin B1 (1) and flaviogeranin B (2). The divergent pathway represents the formation of diverse architecturally flaviogeranin family compounds via the common THN precursor.  
Figure 4: Proposed biosynthetic pathway of flaviogeranins supported by the isolation of isolated flaviogeranin intermediates and congeners. Three common intermediates of the divergent biosynthetic pathway are highlighted in gray.