Affiliation:
1. First Affiliated Hospital and Institute of Pharmaceutical Biotechnology, Zhejiang University School of Medicine , Hangzhou, China
2. Zhejiang Provincial Key Laboratory for Microbial Biochemistry and Metabolic Engineering, Institute of Pharmaceutical Biotechnology , Hangzhou, China
Abstract
ABSTRACT
Fidaxomicin is an 18-membered glycosidic macrolide polyketide that was clinically used in the treatment of
Clostridium difficile
infection in 2011. Although the biosynthetic mechanism and pathway-specific regulator of fidaxomicin were well elaborated, little is known about the pleiotropic regulators outside the fidaxomicin gene cluster. To further reveal the transcriptional regulatory mechanism of fidaxomicin biosynthesis in
Actinoplanes deccanensis
YP-1, we used the 5′-biotin-labeled
fadR1
promoter (
fadS5R1p
) as a probe to affinity isolate the
fadS5R1p
-interactive protein MtrA, a response regulator of the two-component system MtrAB, from the proteome of mycelia. MtrA is bound directly to
fadS5R1p
to positively regulate gene cluster expression and fidaxomicin production. Fidaxomicin production was improved by 37% in the
mtrA
overexpressed strain. Furthermore, we revealed that MtrA tends to bind GTKAYS motifs and influence genes involved in secondary metabolites, phosphate transport, methionine transport, nitrogen metabolism, drug transport, membrane properties, etc. Overall, for the first time at the transcriptional level, we comprehensively revealed a pleiotropic regulator, MtrA, that not only directly mediates the FadR1-specific pathway to regulate the proper production of fidaxomicin but also participates in bacterial growth and development.
IMPORTANCE
Cascade regulation networks are almost present in various kinds of microorganisms, but locating and systematically elucidating specific pleiotropic regulators related to a certain gene cluster can be a tricky problem. Here, based on the promoter of the fidaxomicin pathway-specific regulator FadR1, we utilized a “DNA to Proteins” affinity purification method and captured a global regulator MtrA, which positively regulates fidaxomicin biosynthesis. In the
mtrA
overexpressed strain, the production of fidaxomicin was improved by 37% compared to the native strain. Then, we combined the “Protein to DNAs” affinity purification method (DAP-seq) with the results of RNA-seq and systematically elucidated the primary and secondary metabolic processes in which MtrA directly or indirectly participates. Thus, our work brought up a new way to improve fidaxomicin production from the perspective of global regulation and analyzed the regulatory mechanism of MtrA. Meanwhile, we provided a novel methodology for the research of cascade regulation networks and vital secondary metabolites.
Funder
MOST | National Key Research and Development Program of China
MOST | National Natural Science Foundation of China
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Cell Biology,Microbiology (medical),Genetics,General Immunology and Microbiology,Ecology,Physiology