Oxytetracycline hyper-production through targeted genome reduction of Streptomyces rimosus

Abstract

Abstract Most of the biosynthetic gene clusters (BGC) encoding the biosynthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, robust technologies are required to secure the production of natural products for both drug discovery and any subsequent commercial fermentation processes. Industrial strain improvement has resulted almost exclusively from expensive and time-consuming approaches to strain improvement. Therefore, to ensure a strong pipeline of truly novel antibiotics there is an urgent need to develop rapid and efficient strain improvement approaches. This study uses comparative genome analysis to instruct rational strain improvement, using Streptomyces rimosus for the industrial production of the medically-important antibiotic oxytetracycline. Sequencing of the genomes of two industrial strains M4018 and R6-500, developed independently from a common ancestor, identified large DNA rearrangements located at the terminal parts of the chromosomes that occurred in approximately at the same location in both strains. We evaluated the effect of these DNA deletions at similar locations of the parental S. rimosus Type Strain (ATCC 10970) genome. Surprisingly a single engineering step in the Type Strain (introduction of a 145kb deletion close to the otc BGC) resulted in significant OTC overproduction, achieving titers that were equivalent to the M4018 and R6-500 strains used for the industrial production of OTC. Transcriptome data fully support the hypothesis that the main reason for such an increase in OTC biosynthesis was due to massively enhanced transcription of the otc BGC and not to enhanced substrate supply. Surprisingly, we also observed changes in the expression of other cryptic BGCs. Similarly, some metabolites, previously undetectable in ATCC 10970 were now produced at relatively high titers. This entirely new approach to strain improvement demonstrates great potential as a rapid and versatile technology to increase titer of the target secondary metabolite in a one-step procedure, and to activate cryptic gene clusters, which are an enormous source of yet unexplored natural products of medical and industrial value.