CRISPR-Cas9 has proven as a very powerful gene editing tool for
Actinomyces
, allowing scarless and precise genome editing in selected strains of these biotechnologically relevant microorganisms. ...However, its general application in actinomycetes has been limited due to its inefficacy when applying the system in an untested strain. Here, we provide evidence of how Cas9 levels are toxic for the model actinomycetes
Streptomyces coelicolor
M145 and
Streptomyces lividans
TK24, which show delayed or absence of growth. We overcame this toxicity by lowering Cas9 levels and have generated a set of plasmids in which Cas9 expression is either controlled by theophylline-inducible or constitutive promoters. We validated the targeting of these CRISPR-Cas9 system using the glycerol uptake operon and the actinorhodin biosynthesis gene cluster. Our results highlight the importance of adjusting Cas9 expression levels specifically in strains to gain optimum and efficient gene editing in
Actinomyces
.
Bacterial and fungal secondary metabolism is a rich source of novel bioactive compounds with potential pharmaceutical applications as antibiotics, anti-tumor drugs or cholesterol-lowering drugs. To ...find new drug candidates, microbiologists are increasingly relying on sequencing genomes of a wide variety of microbes. However, rapidly and reliably pinpointing all the potential gene clusters for secondary metabolites in dozens of newly sequenced genomes has been extremely challenging, due to their biochemical heterogeneity, the presence of unknown enzymes and the dispersed nature of the necessary specialized bioinformatics tools and resources. Here, we present antiSMASH (antibiotics & Secondary Metabolite Analysis Shell), the first comprehensive pipeline capable of identifying biosynthetic loci covering the whole range of known secondary metabolite compound classes (polyketides, non-ribosomal peptides, terpenes, aminoglycosides, aminocoumarins, indolocarbazoles, lantibiotics, bacteriocins, nucleosides, beta-lactams, butyrolactones, siderophores, melanins and others). It aligns the identified regions at the gene cluster level to their nearest relatives from a database containing all other known gene clusters, and integrates or cross-links all previously available secondary-metabolite specific gene analysis methods in one interactive view. antiSMASH is available at http://antismash.secondarymetabolites.org.
As the field of synthetic biology is developing, the prospects for de novo design of biosynthetic pathways are becoming more and more realistic. Hence, there is an increasing need for computational ...tools that can support these efforts. A range of algorithms has been developed that can be used to identify all possible metabolic pathways and their corresponding enzymatic parts. These can then be ranked according to various properties and modelled in an organism-specific context. Finally, design software can aid the biologist in the integration of a selected pathway into smartly regulated transcriptional units. Here, we review key existing tools and offer suggestions for how informatics can help to shape the future of synthetic microbiology.
Bacterial secondary metabolites are widely used as antibiotics, anticancer drugs, insecticides and food additives. Attempts to engineer their biosynthetic gene clusters (BGCs) to produce unnatural ...metabolites with improved properties are often frustrated by the unpredictability and complexity of the enzymes that synthesize these molecules, suggesting that genetic changes within BGCs are limited by specific constraints. Here, by performing a systematic computational analysis of BGC evolution, we derive evidence for three findings that shed light on the ways in which, despite these constraints, nature successfully invents new molecules: 1) BGCs for complex molecules often evolve through the successive merger of smaller sub-clusters, which function as independent evolutionary entities. 2) An important subset of polyketide synthases and nonribosomal peptide synthetases evolve by concerted evolution, which generates sets of sequence-homogenized domains that may hold promise for engineering efforts since they exhibit a high degree of functional interoperability, 3) Individual BGC families evolve in distinct ways, suggesting that design strategies should take into account family-specific functional constraints. These findings suggest novel strategies for using synthetic biology to rationally engineer biosynthetic pathways.
Streptomyces species produce a vast diversity of secondary metabolites of clinical and biotechnological importance, in particular antibiotics. Recent developments in metabolic engineering, synthetic ...and systems biology have opened new opportunities to exploit Streptomyces secondary metabolism, but achieving industry-level production without time-consuming optimization has remained challenging. Genome-scale metabolic modelling has been shown to be a powerful tool to guide metabolic engineering strategies for accelerated strain optimization, and several generations of models of Streptomyces metabolism have been developed for this purpose.
Here, we present the most recent update of a genome-scale stoichiometric constraint-based model of the metabolism of Streptomyces coelicolor, the major model organism for the production of antibiotics in the genus. We show that the updated model enables better metabolic flux and biomass predictions and facilitates the integrative analysis of multi-omics data such as transcriptomics, proteomics and metabolomics.
The updated model presented here provides an enhanced basis for the next generation of metabolic engineering attempts in Streptomyces.
Antibiotic production is coordinated in the Streptomyces coelicolor population through the use of diffusible signaling molecules of the γ-butyrolactone (GBL) family. The GBL regulatory system ...involves a small, and not completely defined two-gene network which governs a potentially bi-stable switch between the “on” and “off” states of antibiotic production. The use of this circuit as a tool for synthetic biology has been hampered by a lack of mechanistic understanding of its functionality. We here present the creation and analysis of a versatile and adaptable ensemble model of the Streptomyces GBL system (detailed information on all model mechanisms and parameters is documented in http://www.systemsbiology.ls.manchester.ac.uk/wiki/index.php/Main_Page). We use the model to explore a range of previously proposed mechanistic hypotheses, including transcriptional interference, antisense RNA interactions between the mRNAs of the two genes, and various alternative regulatory activities. Our results suggest that transcriptional interference alone is not sufficient to explain the system’s behavior. Instead, antisense RNA interactions seem to be the system's driving force, combined with an aggressive scbR promoter. The computational model can be used to further challenge and refine our understanding of the system’s activity and guide future experimentation.
The previously reported Streptomyces coelicolor M1146 is commonly used as a host strain for engineering of secondary metabolite production. In this study, absolute quantification of intracellular and ...extracellular metabolites of M1146 was performed in mid-log phase and stationary phase to observe major metabolites and the changes that occurred during growth. Decreased levels of central carbon metabolites (glycolysis, TCA cycle, and pentose phosphate pathway) and increased levels of amino acids were observed in stationary phase compared to mid-log phase. Furthermore, comparative metabolome analyses of M1146 upon expression of the actinorhodin biosynthetic gene cluster (M1146+ACT), a point mutation on the rpoB gene encoding RNA polymerase beta-subunit (M1152), and both expression of actinorhodin biosynthetic gene cluster and a rpoB point mutation (M1152+ACT) were performed. M1146+ACT showed higher levels of important cofactors, such as ATP, NADPH, and FMN while M1152 led to higher levels of intracellular S-adenosyl-methionine, acyl-CoAs, and extracellular nucleosides compared to M1146. M1152+ACT exhibited the highest levels of actinorhodin with elevated bases, nucleosides, and nucleotides, such as intracellular PRPP (phosphoribosyl phosphate), ATP, along with extracellular inosine, uridine, and guanine compared to the other three strains, which were considered to be combined effects of actinorhodin gene cluster expression and a rpoB point mutation. Metabolites analysis by means of absolute quantification demonstrated changes in precursors of secondary metabolites before and after phosphate depletion in M1146. Comparative metabolome analysis provided further insights into the effects of actinorhodin gene cluster expression along with a rpoB point mutation on the metabolome of S. coelicolor.
PDF
