Differential mass spectrometry-based proteome analyses unveil major regulatory hubs in rifamycin B production in Amycolatopsis mediterranei

Rifamycin B is produced by Amycolatopsis mediterranei S699 as a secondary metabolite. Its semi-synthetic derivatives have been used for curing tuberculosis caused by Mycobacterium tuberculosis. But the emergence of rifampicin-resistant strains required analogs of rifamycin B to be developed by rifamycin biosynthetic gene cluster manipulation. In 2014 genetic engineering of the rifamycin polyketide synthase gene cluster in 5699 led to a mutant, A. mediterranei DCO#34, that produced 24-desmethylrifamycin B. Unfortunately, the productivity was strongly reduced to 20 mgL(-1) as compared to 50 mgL(-1) of rifamycin B. To understand the mechanisms leading to reduced productivity and rifamycin biosynthesis by A. mediterranei 5699 during the early and late growth phase we performed a proteome study for wild type strain 5699, mutant DCO#34, and the non-producer strain SCO2-2. Proteins identification and relative label-free quantification were performed by nLC-MS/MS. Data are available via ProteomeXchange with identifier PXD016416. Also, in-silico protein-protein interaction approach was used to determine the relationship between different structural and regulatory proteins involved in rifamycin biosynthesis. Our studies revealed RifA, RifK, RifL, Rif-Orf19 as the major regulatory hubs. Relative abundance expression values revealed that genes encoding RifC-Rifl and the transporter RifP, down-regulated in DCO#34 and genes encoding RifR, RifZ, other regulatory proteins up-regulated. Significance: The study is designed mainly to understand the underlying mechanisms of rifamycin biosynthesis in Amycolatopsis mediterranei. This resulted in the identification of regulatory hubs which play a crucial role in regulating secondary metabolism. It elucidates the complex mechanism of secondary metabolite biosynthesis and their conversion and extracellular transportation in temporal correlation with the different growth phases. The study also elucidated the mechanisms leading to reduced production of analog, 24-desmethylrifamycin B by the genetically modified strain DCO#34, derivatives of which have been found effective against rifampicin-resistant strains of Mycobacterium tuberculosis. These results can be useful while carrying out genetic manipulations to improve the strains of Amycolatopsis to produce better analogs/drugs and promote the eradication of TB. Thus, this study is contributing significantly to the growing knowledge in the field of the crucial drug, rifamycin B biosynthesis by an economically important bacterium Amycolatopsis mediterranei.

Automated summary

This study revealed the mechanisms of rifamycin biosynthesis in Amycolatopsis mediterranei through genetic engineering, identifying regulatory hubs crucial for improving strain production of analogs/drugs. This contributes significantly to the knowledge in the field and can be useful for eradicating TB caused by rifampicin-resistant strains.