Open AccessFormation and Attachment of the Deoxysugar Moiety and Assembly of the Gene Cluster for Caprazamycin Biosynthesis
Author(s) -
Leonard Kaysser,
Emmanuel Wemakor,
Stefanie Siebenberg,
José A. Salas,
Jae Kyung Sohng,
Bernd Kammerer,
Bertolt Gust
Publication year - 2010
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.02740-09
Subject(s) - gene cluster , streptomyces coelicolor , rhamnose , biosynthesis , cosmid , streptomyces , heterologous expression , biology , gene , streptomyces albus , moiety , biochemistry , heterologous , genetics , chemistry , stereochemistry , galactose , recombinant dna , bacteria , mutant
Caprazamycins are antimycobacterials produced byStreptomyces 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 inStreptomyces 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 thel -rhamnose to the caprazamycin aglycones, generating a rare acylated deoxyhexose. Anl -rhamnose gene cluster was identified elsewhere on theStreptomyces sp. MK730-62F2 genome, and its involvement in caprazamycin formation was demonstrated by insertional inactivation ofcpzDIII . Thel -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 ofcpzDI andcpzDV in thel -rhamnose subcluster confirmed that four genes,cpzDII ,cpzDIII ,cpzDIV , andcpzDVI , 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.