Formation and Attachment of the Deoxysugar Moiety and Assembly of the Gene Cluster for Caprazamycin Biosynthesis

Abstract

Caprazamycins are antimycobacterials produced by Streptomyces sp. MK730-62F2. Previously, cosmid cpzLK09 was shown to direct the biosynthesis of caprazamycin aglycones, but not of intact caprazamycins. Sequence analysis of cpzLK09 identified 23 genes involved in the formation of the caprazamycin aglycones and the transfer and methylation of the sugar moiety, together with genes for resistance, transport, and regulation. In this study, coexpression of cpzLK09 in Streptomyces coelicolor M512 with pRHAM, containing all the required genes for dTDP-L-rhamnose biosynthesis, led to the production of intact caprazamycins. In vitro studies showed that Cpz31 is responsible for the attachment of the L-rhamnose to the caprazamycin aglycones, generating a rare acylated deoxyhexose. An L-rhamnose gene cluster was identified elsewhere on the Streptomyces sp. MK730-62F2 genome, and its involvement in caprazamycin formation was demonstrated by insertional inactivation of cpzDIII. The L-rhamnose subcluster was assembled with cpzLK09 using Red/ET-mediated recombination. Heterologous expression of the resulting cosmid, cpzEW07, led to the production of caprazamycins, demonstrating that both sets of genes are required for caprazamycin biosynthesis. Knockouts of cpzDI and cpzDV in the L-rhamnose subcluster confirmed that four genes, cpzDII, cpzDIII, cpzDIV, and cpzDVI, are sufficient for the biosynthesis of the deoxysugar moiety. The presented recombineering strategy may provide a useful tool for the assembly of biosynthetic building blocks for heterologous production of microbial compounds.Caprazamycins are potent antimycobacterials isolated from Streptomyces sp. MK730F-62F2 (23). In a pulmonary tuberculosis mouse model, they showed a therapeutic effect but no significant toxicity (24). The caprazamycins are assigned to the translocase I inhibitors (27) due to their structural similarity to the liposidomycins (34, 43), which have been studied in more detail. Translocase I catalyzes the first step in the membranelinked reaction cycle of bacterial cell wall formation (52): the transfer of phospho-N-acetylmuramic acid-L-Ala-␥-D-Glu-mdiaminopimelic acid-D-Ala-D-Ala from UMP to the lipid carrier undecaprenyl phosphate. Structurally unique in nature, the caprazamycins and liposidomycins share a 5Ј-␤-O-aminoribosyl-glycyluridine and a rare N-methylated diazepanone as their characteristic feature (Fig. 1) (25, 53). Attached at the 3Љ position are ␤-hydroxylated fatty acid groups of different chain lengths, carrying a 3-methylglutarate. While the liposidomycins are sulfated, the caprazamycins lack this group. Instead, they are glycosylated with a 2,3,4-O-methyl-L-rhamnose and therefore belong to the large number of bioactive compounds containing 6-deoxyhexoses. Usually, these moieties contribute significantly to the compounds' properties, influencing, e.g., molecule-target interactions, cell import and export, pharmacokinetics, and solubility (56). The biosynthesis of deoxysugars has been studied in detail and generally starts from NDPactiva...

Heterologous expression of the biosynthetic gene clusters of coumermycin A1, clorobiocin and caprazamycins in genetically modified Streptomyces coelicolor strains

“…MK730‐62F2, all genes required for the biosynthesis of the caprazamycin aglycones are localized in a contiguous gene cluster comprising 23 open reading frames that span ∼26 kb. In contrast, the genes for the biosynthesis of the L ‐rhamnose moiety are located elsewhere in the genome 34. Integration of cosmid cpzLK09, comprising the caprazamycin gene cluster, into the ΦC31 attachment site of S. coelicolor M512 therefore led to the formation of caprazamycin aglycones in the resulting strains.…”

Identification of a Napsamycin Biosynthesis Gene Cluster by Genome Mining

“…[13] Structureactivity studies with synthesised peptidyl nucleoside analogues revealed that the terminal amino group, the N-methyl-Daba and probably the uridine moiety are most important for the inhibition of translocase I. [14,15] Recently, the identification and analysis of the gene clusters for caprazamycins, [16,17] liposidomycins [18][19][20] and capuramycintype A-500359 [21] has given the first insights into the biosynthesis of the structurally unusual nucleoside antibiotics that target translocase I. However, little is known about the formation of the uridylpeptide-type translocase I inhibitors.…”

Biosynthetic reconstitution of deoxysugar phosphoramidate metalloprotease inhibitors using an N–P-bond-forming kinase

“…MK730-62F2 for heterologous expression. This strain is known to provide activated l -rhamnose for the endogenous production of caprazamycins and the respective deoxysugar biosynthesis genes have recently been identified 18,19. To our satisfaction, LC-MS analysis of culture extracts from the heterologous mutant showed the production of a single new peak with a mass, retention time and UV-spectrum identical to a commercial standard of 1 (Fig.…”