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Chartreusin is a potent antiproliferative agent that contains a unique aromatic pentacyclic bislactone carbon scaffold. The biosynthesis of type II polyketide aglycone has been extensively investigated and shown to proceed through a tetracyclic anthracycline intermediate. The last remaining unknown steps are the conversion of auramycinone to resomycin C. Here we have discovered three enzymes that play crucial roles in two mechanistically distinct dehydration reactions. We show that ChaX is an NAD(P)H-dependent auramycinone quinone reductase that allows the cyclase-like ChaU to catalyze the formation of 9,10-dehydroauramycinone via a carbanion intermediate. In contrast, the cyclase-like ChaJ, homologous to ChaU, is responsible for subsequent 7,8-dehydration via a canonical carbocation intermediate, yielding resomycin C. The results were confirmed via assembly of the biosynthetic pathway for production of resomycin C in Streptomyces coelicolor M1152ΔmatAB. The work expands the catalytic repertoire of the SnoaL protein family, which has previously been associated with anthracycline fourth-ring cyclization and two-component 1-hydroxylation. 

High-copy-number plasmids are indispensable tools for gene overexpression studies in prokaryotes to engineer pathways or probe phenotypes of interest. The development of genetic tools for the industrially relevant Actinobacteria is of special interest, given their utility in producing keratolytic enzymes and biologically active natural products. Within the Actinobacteria, Streptomyces–Escherichia coli shuttle vectors based on the SCP2* and pIJ101 incompatibility groups are widely employed for molecular cloning and gene expression studies. Here, the sequences of two commonly used pIJ101-based Streptomyces–E. coli shuttle vectors, pEM4 and pUWL201, were determined using next-generation sequencing. These plasmids drive the expression of heterologous genes using the constitutive ermE*p promoter. pEM4 was found to be 8.3 kbp long, containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, a ColE1 origin of replication and the Streptomyces pIJ101 origin of replication. pUWL201 was found to be 6.78 kbp long, containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, a ColE1 origin of replication and the Streptomyces pIJ101 origin of replication. Interestingly, the sequences for both pEM4 and pUWL201 exceed their previously reported size by 1.1 and 0.4 kbp, respectively. This report updates the literature with the corrected sequences for these shuttle vectors, ensuring their compatibility with modern synthetic biology cloning methodologies.