Alen Pšeničnik’s research while affiliated with University of Ljubljana and other places

Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, to ensure a strong pipeline of novel antibiotics, there is a need to develop rapid and efficient strain development approaches. This study uses comparative genome analysis to instruct rational strain improvement, using Streptomyces rimosus, the producer of the important antibiotic oxytetracycline (OTC) as a model system. 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 chromosome end. We evaluated the effect of these genome deletions on the parental S. rimosus Type Strain (ATCC 10970) genome where introduction of a 145 kb deletion close to the OTC BGC in the Type Strain resulted in massive OTC overproduction, achieving titers that were equivalent to M4018 and R6-500. Transcriptome data supported the hypothesis that the reason for such an increase in OTC biosynthesis was due to enhanced transcription of the OTC BGC and not due to enhanced substrate supply. We also observed changes in the expression of other cryptic BGCs; some metabolites, undetectable in ATCC 10970, were now produced at high titers. This study demonstrated for the first time that the main force behind BGC overexpression is genome rearrangement. This new approach demonstrates great potential to activate cryptic gene clusters of yet unexplored natural products of medical and industrial value. IMPORTANCE There is a critical need to develop novel antibiotics to combat antimicrobial resistance. Streptomyces species are very rich source of antibiotics, typically encoding 20–60 biosynthetic gene clusters (BGCs). However, under laboratory conditions, most are either silent or poorly expressed so that their products are only detectable at nanogram quantities, which hampers drug development efforts. To address this subject, we used comparative genome analysis of industrial Streptomyces rimosus strains producing high titers of a broad spectrum antibiotic oxytetracycline (OTC), developed during decades of industrial strain improvement. Interestingly, large-scale chromosomal deletions were observed. Based on this information, we carried out targeted genome deletions in the native strain S. rimosus ATCC 10970, and we show that a targeted deletion in the vicinity of the OTC BGC significantly induced expression of the OTC BGC, as well as some other silent BGCs, thus suggesting that this approach may be a useful way to identify new natural products.

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.

Mediante la aplicación de la técnica de CRISPR-Cas9 se ha conseguido la deleción de fragmentos de ADN de 245 kpb a 450 kpb del cromosoma de la cepa productora de oxitetraciclina, Streptomyces rimosus ATCC 10970. Estas deleciones afectaron a la misma región de un extremo del cromosoma que, según el análisis bioinformático, contiene varios agrupamientos genéticos que codifican para enzimas implicadas en la biosíntesis de metabolitos secundarios. La generación de grandes deleciones en esta parte del cromosoma de S. rimosus probablemente hará que las cepas modificadas sean más robustas, reduciendo de esta forma la inestabilidad morfológica y genética. Para ello, mediante diferentes procedimientos de clonación se ensamblaron 3 construcciones en Escherichia coli de plásmidos que contenían la maquinaria CRISPR-Cas9, para posteriormente ser introducidas independientemente en S. rimosus usando técnicas de conjugación, para conseguir las deleciones correspondientes. A pesar de las grandes deleciones generadas, los clones modificados de S. rimosus seguían manteniendo la morfología de la cepa original de referencia, pudiendo representar atractivas factorías de células microbianas para la producción de diferentes metabolitos.

CRISPR-Cas9 technology has emerged as a promising tool for genetic engineering of Streptomyces strains. However, in practice, numerous technical hurdles have yet to be overcome when developing robust editing procedures. Here, we developed an extension of the CRISPR-Cas toolbox, a simple and reliable cas9 monitoring tool with transcriptional fusion of cas9 nuclease to a beta glucuronidase (gusA) visual reporter gene. The Cas9-SD-GusA tool enables in situ identification of cells expressing Cas9 nuclease following the introduction of the plasmid carrying the CRISPR-Cas9 machinery. Remarkably, when the Cas9-SD-GusA system was applied under optimal conditions, 100% of the colonies displaying GusA activity carried the target genotype. In contrast, it was shown that the cas9 sequence had undergone major recombination events in the colonies that did not exhibit GusA activity, giving rise to “escaper colonies” carrying unedited genotype. Our approach allows a simple detection of “escaper” phenotype and serves as an efficient CRISPR-Cas9 optimisation tool.