Oxytetracycline hyper-production through targeted genome reduction of Streptomyces rimosus

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

Reference-grade genome and large linear plasmid of Streptomyces rimosus: pushing the limits of Nanopore sequencing

[EN] Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtainedSIThis work was supported as part of the European project “Thoroughly Optimised Production Chassis for Advanced Pharmaceutical Ingredients” (grant ID 720793, European Union’s Horizon 2020 Research and Innovation Program) and by the Slovenian Research Agency (P4-0116, P4-0077, and P1-0034). L.S. is supported by a Slovenian Research Agency young researcher grant (35220200570), and M.T. is supported by grant C3330-19-952047 funded by Republic of Slovenia Ministry of Education, Science, and Sport and the European Union European Regional Development Fun

Reference-Grade Genome and Large Linear Plasmid of Streptomyces rimosus : Pushing the Limits of Nanopore Sequencing

Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained. IMPORTANCE The genomes of Streptomyces species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids [GLPs]), rearrangements, and high GC content. To improve the quality of the S. rimosus ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters

Analysis of oxytetracycline biosynthesis and other secondary metabolites in Streptomyces rimosus

Bakterija Streptomyces rimosus je poznana predvsem zaradi produkcije široko spektralnega antibiotika oksitetraciklina (OTC). Z namenom boljšega razumevanja biosinteze OTC in drugih naravnih tetraciklinov (TC) smo naredili bioinformacijsko primerjavo gruč biosinteznih genov (GBG), ki kodirajo biosintezo: OTC, klorotetraciklina, kelokardina, daktilociklina in SF2575. Nato smo se osredotočili na izbrane gene udeležene v zgodnje in pozne stopnje biosinteze OTC, katerih vloga še ni poznana. Izbili smo izbrane gene iz otc GBG (oxyI, oxyH, oxyG, oxyM, oxyO) v visoko donosnem sevu bakterije S. rimosus. Sevom z izbitimi geni smo ovrednotili donos OTC ter z analizo netarčnega metaboloma poskušali identificirati nove intermediate/stranske produkte. Pokazali smo, da geni oxyI, oxyG, oxyM in oxyO niso nujno potrebni v biosintezi OTC. Vendar ob izbitju genov oxyI, oxyG in oxyM donos OTC upade, zaradi česar domnevamo, da imajo pomožno vlogo v biosintezi. V sevu z izbitim genom oxyH je prišlo do prekinitve produkcije OTC in ob tem smo detektirali nov metabolit. V drugem sklopu smo se osredotočili na preostali sekundarni metabolizem bakterije S. rimosus, tako da smo s pomočjo bioinformacijskih orodij identificirali in analizirali GBG na kromosomu in plazmidu. Po kultivaciji bakterije S. rimosus v različnih gojiščih in LC-MS analizi smo detektirali produkcijo devetih sekundarnih metabolitov. Poleg tega smo poskušali identificirati GBG v genomu bakterije S. rimosus, ki kodira biosintezo rdeče obarvane spojine in smo jo detektirali le ob kokultivaciji z bakterijo Bacillus subtilis. Ob izbitju izbranih GBG je bakterija S. rimosus ob kokultivaciji z bakterijo B. subtilis še vedno proizvajala rdeče obarvano spojino.The bacterium Streptomyces rimosus is mainly known for the production of broad-spectrum antibiotic oxytetracycline (OTC). To gain deeper understanding of the biosynthesis of OTC and other natural tetracyclines (TC) we initialy performed a bioinformatic comparison of the biosynthetic gene clusters (BGCs) encoding the biosynthesis of OTC, chlortetracycline, chelocardin, dactylocycline and SF2575. In the second step, we focused on selected genes involved in the early and late stages of OTC biosynthesis whose roles is not yet understood. We deleted selected genes from otc BGC (oxyI, oxyH, oxyG, oxyM, oxyO) in the S. rimosus high-producer strain. We then evaluated the titer of OTC produced by engineered strains and we attempted to identify potential new intermediates or side products by analyzing the non-targeted metabolome. We showed that the oxyI, oxyG, oxyM and oxyO genes are not essential for the OTC biosynthesis. However, when the oxyI, oxyG, and oxyM genes were deleted the OTC titer decreased suggesting that these genes play an auxiliary role. OTC production was entirely disrupted in the strain with deletion of oxyH gene, instead a new metabolite was detected. In the second part of the thesis, we focused on the analysis of all BGCs encoded in the S. rimosus genom by applying bioinformatics tools. After cultivation of S. rimosus in selected media and followed by LC-MS analysis, we detected the production of nine secondary metabolites. Finally, we aimed to identify BGCs in the S. rimosus genome encoding the biosynthesis of a red-colored metabolite, which only appears in co-cultivation with Bacillus subtilis. After deletion of selected BGCs, engineered S. rimosus strains in co-cultivation with B. subtilis still produced red-colored metabolite

Optimisation of biosynthesis of recmobinant phytase in bacteria Streptomyces rimosus

Bakterije iz rodu Streptomyces so poznane predvsem zaradi produkcije antibiotikov. Vse več pa je zanimanja za seve rodu Streptomyces za produkcijo heterolognih proteinov. Cilj magistrske naloge je bil optimizacija produkcijskega gojišča za produkcijo rekombinantne fitaze s pomočjo S. rimosus. Produkcijsko gojišče, ki se trenutno uporablja za kultivacijo S. rimosus je prilagojeno predvsem produkciji tetraciklinov. Pripravili smo različne vrste gojišč in merili fitazno aktivnost. V obsegu magistrske naloge smo izmed dveh sevov S. rimosus izbrali donosnejši sev z oznako e24 za nadaljnja testiranja. Pokazali smo, da ima razmerje med sojino moko in koruznim škrobom vpliv na biosintezo fitaze. Ko je koncentracija sojine moke v gojišču prenizka ali previsoka, se vrednosti fitazne aktivnosti znižajo v primerjavi z osnovnim (nespremenjenim) produkcijskim gojiščem. Vpliv glukoze je odvisen od koncentracije sojine moke in koruznega škroba in časa gojenja, vendar v večini primerov nima pozitivnega vpliva na produkcijo fitaze. Izmed vseh testiranih dodanih virov ogljika in dušika, imajo pozitiven vpliv na produkcijo fitaze: laktoza, dekstrin, sečnina, amonijev sulfat, kvas in kazein. Določili smo optimalno koncentracijo CaCO3, NaCl, cinka, mangana in železa. Dodatek fosforja v produkcijskem gojišču negativno vpliva na produkcijo fitaze. V okviru magistrske naloge smo določili optimalne koncentracije dodatkov in posameznih komponent gojišča za rekombinantno produkcijo fitaze v S. rimosus.Members of genus Streptomyces are well known antibiotics producers. Streptomyces sp. are also attractive host cells for heterologous protein production. The aim of our thesis was optimization of production medium for recombinant phytase production in S. rimosus. Currently used production medium for cultivation of S. rimosus is adjusted for antibiotics production. We prepared various mediums and measured phytase activity. Within master thesis we selected from two strains S. rimosu more productive strain e24 for further analysis. We have shown that ratio between soybean flour and starch effect on phytase activity. Low or high concentrations of soybean flour in medium decreased phytase activity regard unaltered production medium. Effect of added glucose depends on concentrations of soybean meal and corn starch and time of cultivation: but in most cases glucose repress phytase production. From all tested added carbon and nitrogen sources positive effect on phytase production have: lactose, dextrin, urea, ammonium sulfate, yeast and casein. We determined optimal concentration of CaCO3, NaCl, zinc, manganese and iron. Phosphate supplement has negative effect on phytase production. In context of the master\u27s thesis we determined the optimal concentration of the individual components of the medium for the recombinant production of phytase

Simple and reliable in situ CRISPR-Cas9 nuclease visualization tool is ensuring efficient editing in Streptomyces species

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

Oxytetracycline hyper-production through targeted genome reduction of Streptomyces rimosus

Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressedtherefore, 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 BGCssome 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

Multiple copies of the oxytetracycline gene cluster in selected Streptomyces rimosus strains can provide significantly increased titers.

Background: Natural products are a valuable source of biologically active compounds that have applications in medicine and agriculture. One disadvantage with natural products is the slow, time-consuming strain improvement regimes that are necessary to ensure sufficient quantities of target compounds for commercial production. Although great efforts have been invested in strain selection methods, many of these technologies have not been improved in decades, which might pose a serious threat to the economic and industrial viability of such important bioprocesses. Results: In recent years, introduction of extra copies of an entire biosynthetic pathway that encodes a target product in a single microbial host has become a technically feasible approach. However, this often results in minor to moderate increases in target titers. Strain stability and process reproducibility are the other critical factors in the industrial setting. Industrial Streptomyces rimosus strains for production of oxytetracycline are one of the most economically efficient strains ever developed, and thus these represent a very good industrial case. To evaluate the applicability of amplification of an entire gene cluster in a single host strain, we developed and evaluated various gene tools to introduce multiple copies of the entire oxytetracycline gene cluster into three different Streptomyces rimosus strains: wild-type, and medium and high oxytetracycline-producing strains. We evaluated the production levels of these engineered S. rimosus strains with extra copies of the oxytetracycline gene cluster and their stability, and the oxytetracycline gene cluster expression profiles; we also identified the chromosomal integration sites. Conclusions: This study shows that stable and reproducible increases in target secondary metabolite titers can be achieved in wild-type and in high oxytetracycline-producing strains, which always reflects the metabolic background of each independent S. rimosus strain. Although this approach is technically very demanding and requires systematic effort, when combined with modern strain selection methods, it might constitute a very valuable approach in industrial process development

Synthetic biology approaches to actinomycete strain improvement

Their biochemical versatility and biotechnological importance make actinomycete bacteria attractive targets for ambitious genetic engineering using the toolkit of synthetic biology. But their complex biology also poses unique challenges. This mini review discusses some of the recent advances in synthetic biology approaches from an actinomycete perspective and presents examples of their application to the rational improvement of industrially relevant strains