scholarly journals Characterization of Cas proteins for CRISPR-Cas editing in streptomycetes

2019 ◽  
Author(s):  
Wan Lin Yeo ◽  
Elena Heng ◽  
Lee Ling Tan ◽  
Yi Wee Lim ◽  
Yee Hwee Lim ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Genome Editing ◽  
High Efficiency ◽  
Streptococcus Thermophilus ◽  
Gene Clusters ◽  
Biosynthetic Gene Clusters ◽  
Tularensis Subsp ◽  
Homology Directed Repair ◽  
Cas Proteins

Application of the well-characterized Streptococcus pyogenes CRISPR-Cas9 system in actinomycetes has enabled high efficiency multiplex genome editing and CRISPRi-mediated transcriptional regulation in these prolific bioactive metabolite producers. Nonetheless, SpCas9 has its limitations and can be ineffective depending on the strains and target sites. Here, we built and tested alternative CRISPR-Cas constructs based on the standalone pCRISPomyces-2 editing plasmid. We showed that Streptococcus thermophilus CRISPR1 (sth1Cas9), Staphylococcus aureus Cas9 (saCas9), and Francisella tularensis subsp. Novicida U112 Cpf1 (fnCpf1) are functional in multiple streptomycetes enabling efficient homology directed repair (HDR)-mediated knock-in and deletion. In strains where spCas9 was non-functional, these alternative Cas systems enabled precise genomic modifications within biosynthetic gene clusters for the discovery, production and diversification of natural products. These additional Cas proteins provide us with the versatility to overcome the limitations of individual CRISPR-Cas systems for genome editing and transcriptional regulation of these industrially important bacteria.

scholarly journals Challenges and Advances in Genome Editing Technologies in Streptomyces

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 734 ◽  
Author(s):  
Yawei Zhao ◽  
Guoquan Li ◽  
Yunliang Chen ◽  
Yinhua Lu
Keyword(s):  
Natural Product ◽  
Genome Editing ◽  
Genetic Manipulation ◽  
Chemical Compounds ◽  
Gene Clusters ◽  
Future Research ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Research Focus ◽  
Experimental Conditions

The genome of Streptomyces encodes a high number of natural product (NP) biosynthetic gene clusters (BGCs). Most of these BGCs are not expressed or are poorly expressed (commonly called silent BGCs) under traditional laboratory experimental conditions. These NP BGCs represent an unexplored rich reservoir of natural compounds, which can be used to discover novel chemical compounds. To activate silent BGCs for NP discovery, two main strategies, including the induction of BGCs expression in native hosts and heterologous expression of BGCs in surrogate Streptomyces hosts, have been adopted, which normally requires genetic manipulation. So far, various genome editing technologies have been developed, which has markedly facilitated the activation of BGCs and NP overproduction in their native hosts, as well as in heterologous Streptomyces hosts. In this review, we summarize the challenges and recent advances in genome editing tools for Streptomyces genetic manipulation with a focus on editing tools based on clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein (Cas) systems. Additionally, we discuss the future research focus, especially the development of endogenous CRISPR/Cas-based genome editing technologies in Streptomyces.

scholarly journals Genomic analysis of siderophore β-hydroxylases reveals divergent stereocontrol and expands the condensation domain family

2019 ◽  
Vol 116 (40) ◽  
pp. 19805-19814 ◽  
Author(s):  
Zachary L. Reitz ◽  
Clifford D. Hardy ◽  
Jaewon Suk ◽  
Jean Bouvet ◽  
Alison Butler
Keyword(s):  
Predictive Power ◽  
Genome Mining ◽  
Genomic Analysis ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Peptide Synthetase ◽  
Condensation Domain

Genome mining of biosynthetic pathways streamlines discovery of secondary metabolites but can leave ambiguities in the predicted structures, which must be rectified experimentally. Through coupling the reactivity predicted by biosynthetic gene clusters with verified structures, the origin of the β-hydroxyaspartic acid diastereomers in siderophores is reported herein. Two functional subtypes of nonheme Fe(II)/α-ketoglutarate–dependent aspartyl β-hydroxylases are identified in siderophore biosynthetic gene clusters, which differ in genomic organization—existing either as fused domains (IβHAsp) at the carboxyl terminus of a nonribosomal peptide synthetase (NRPS) or as stand-alone enzymes (TβHAsp)—and each directs opposite stereoselectivity of Asp β-hydroxylation. The predictive power of this subtype delineation is confirmed by the stereochemical characterization of β-OHAsp residues in pyoverdine GB-1, delftibactin, histicorrugatin, and cupriachelin. The l-threo (2S, 3S) β-OHAsp residues of alterobactin arise from hydroxylation by the β-hydroxylase domain integrated into NRPS AltH, while l-erythro (2S, 3R) β-OHAsp in delftibactin arises from the stand-alone β-hydroxylase DelD. Cupriachelin contains both l-threo and l-erythro β-OHAsp, consistent with the presence of both types of β-hydroxylases in the biosynthetic gene cluster. A third subtype of nonheme Fe(II)/α-ketoglutarate–dependent enzymes (IβHHis) hydroxylates histidyl residues with l-threo stereospecificity. A previously undescribed, noncanonical member of the NRPS condensation domain superfamily is identified, named the interface domain, which is proposed to position the β-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through mapping characterized β-OHAsp diastereomers to the phylogenetic tree of siderophore β-hydroxylases, methods to predict β-OHAsp stereochemistry in silico are realized.

scholarly journals Recent advances in the genome mining of Aspergillus secondary metabolites (covering 2012–2018)

MedChemComm ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 840-866 ◽  
Author(s):  
Jillian Romsdahl ◽  
Clay C. C. Wang
Keyword(s):  
Aspergillus Terreus ◽  
Genome Mining ◽  
Gene Clusters ◽  
Genetic Identification ◽  
Biosynthetic Pathways ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
The Past ◽  
Made In

This review covers advances made in genome mining SMs produced by Aspergillus nidulans, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus in the past six years (2012–2018). Genetic identification and molecular characterization of SM biosynthetic gene clusters, along with proposed biosynthetic pathways, is discussed in depth.

scholarly journals Phenogenomic characterization of a newly domesticated and novel species from the genus Verrucosispora

2021 ◽  
Author(s):  
Sarah Kennedy ◽  
Celine Atkinson ◽  
Brooke Tomlinson ◽  
Lauren Hammond ◽  
Prahathees J. Eswara ◽  
...  
Keyword(s):  
Amino Acids ◽  
Novel Species ◽  
Gene Clusters ◽  
Genetic Redundancy ◽  
Biosynthetic Gene Clusters ◽  
Rare Actinomycetes ◽  
Rich Media ◽  
Initial Recovery ◽  
Gene Status

The concept of bacterial dark matter stems from our inability to culture most microbes and represents a fundamental hole in our knowledge of microbial diversity. Herein we present the domestication of such an organism: a previously uncultured, novel species from the rare-Actinomycetes genus Verrucosispora. Although initial recovery took >4 months, isolation of phenotypically distinct, domesticated generations occurred within weeks. Two isolates were subjected to phenogenomic analyses, revealing domestication correlated with enhanced growth rates in nutrient-rich media, but diminished capacity to metabolize diverse amino acids. This is seemingly mediated by genomic decay through the pseudogenization of amino acids metabolism genes. Conversely, later generational strains had enhanced spore germination rates, potentially through the reversion of a sporulation-associated kinase from pseudogene to true gene status. We observed that our most wild-type isolate had the greatest potential for antibacterial activity, which correlated with extensive mutational attrition of biosynthetic gene clusters in domesticated strains. Comparative analyses revealed wholesale genomic reordering in strains, with widespread SNP, indel and pseudogene mutations observed. We hypothesize that domestication of this previously unculturable organism resulted from the shedding of genomic flexibility required for life in a dynamic marine environment, parsing out genetic redundancy to allow for a newfound cultivable amenability.

scholarly journals Recent Advances in Strategies for the Cloning of Natural Product Biosynthetic Gene Clusters

2021 ◽  
Vol 9 ◽  
Author(s):  
Wenfang Wang ◽  
Guosong Zheng ◽  
Yinhua Lu
Keyword(s):  
High Efficiency ◽  
Therapeutic Potential ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
High Molecular Weight Dna ◽  
Discovery Research ◽  
Recent Advances ◽  
Drug Discovery Research ◽  
Dna Fragment

Microbial natural products (NPs) are a major source of pharmacological agents. Most NPs are synthesized from specific biosynthetic gene clusters (BGCs). With the rapid increase of sequenced microbial genomes, large numbers of NP BGCs have been discovered, regarded as a treasure trove of novel bioactive compounds. However, many NP BGCs are silent in native hosts under laboratory conditions. In order to explore their therapeutic potential, a main route is to activate these silent NP BGCs in heterologous hosts. To this end, the first step is to accurately and efficiently capture these BGCs. In the past decades, a large number of effective technologies for cloning NP BGCs have been established, which has greatly promoted drug discovery research. Herein, we describe recent advances in strategies for BGC cloning, with a focus on the preparation of high-molecular-weight DNA fragment, selection and optimization of vectors used for carrying large-size DNA, and methods for assembling targeted DNA fragment and appropriate vector. The future direction into novel, universal, and high-efficiency methods for cloning NP BGCs is also prospected.

scholarly journals Activation and Identification of a Griseusin Cluster in Streptomyces sp. CA-256286 by Employing Transcriptional Regulators and Multi-Omics Methods

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6580
Author(s):  
Charlotte Beck ◽  
Tetiana Gren ◽  
Francisco Javier Ortiz-López ◽  
Tue Sparholt Jørgensen ◽  
Daniel Carretero-Molina ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Genome Mining ◽  
Gene Clusters ◽  
Transcriptional Regulators ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Heterologous Host ◽  
Streptomyces Sp ◽  
Identification And Characterization

Streptomyces are well-known producers of a range of different secondary metabolites, including antibiotics and other bioactive compounds. Recently, it has been demonstrated that “silent” biosynthetic gene clusters (BGCs) can be activated by heterologously expressing transcriptional regulators from other BGCs. Here, we have activated a silent BGC in Streptomyces sp. CA-256286 by overexpression of a set of SARP family transcriptional regulators. The structure of the produced compound was elucidated by NMR and found to be an N-acetyl cysteine adduct of the pyranonaphtoquinone polyketide 3′-O-α-d-forosaminyl-(+)-griseusin A. Employing a combination of multi-omics and metabolic engineering techniques, we identified the responsible BGC. These methods include genome mining, proteomics and transcriptomics analyses, in combination with CRISPR induced gene inactivations and expression of the BGC in a heterologous host strain. This work demonstrates an easy-to-implement workflow of how silent BGCs can be activated, followed by the identification and characterization of the produced compound, the responsible BGC, and hints of its biosynthetic pathway.

scholarly journals Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

2021 ◽  
Author(s):  
Ali Seleit ◽  
Alexander Aulehla ◽  
Alexandre Paix
Keyword(s):  
Fusion Proteins ◽  
High Efficiency ◽  
Single Copy ◽  
Germline Transmission ◽  
Fluorescent Reporter ◽  
Homology Directed Repair ◽  
Endogenous Protein ◽  
Initial Characterization ◽  
Protein Tagging

The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology directed repair in a variety of species. Despite its success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient and precise strategy for CRISPR/Cas9 mediated endogenous protein tagging in medaka. We use a cloning-free approach that relies on PCR amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40bp), a synthetic sgRNA and streptavidin tagged Cas9. We generate six novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole Genome Sequencing results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion-protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna knock-in has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.

scholarly journals Establishment of recombineering genome editing system in Paraburkholderia megapolitana empowers activation of silent biosynthetic gene clusters

2020 ◽  
Vol 13 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Wentao Zheng ◽  
Xue Wang ◽  
Haibo Zhou ◽  
Youming Zhang ◽  
Aiying Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters

Phylogeny of the Bacillus altitudinis Complex and Characterization of a Newly Isolated Strain with Antilisterial Activity

2021 ◽  
Author(s):  
Lauren Kathryn Hudson ◽  
Leticia A.G. Orellana ◽  
Daniel W Bryan ◽  
Andrew Moore ◽  
John P. Munafo ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Foodborne Pathogens ◽  
Food Industry ◽  
Gene Clusters ◽  
Single Species ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Bacillus Altitudinis ◽  
Biocontrol Potential

Here, a Bacillus strain, UTK D1-0055, is described that was isolated from a laboratory environment. It was of interest as it demonstrated antilisterial activity. The genome was sequenced, which identified the strain as Bacillus altitudinis, and a high-quality complete annotated genome was produced. Additionally, the taxonomy of this and related species was evaluated, including B. aerophilus, B. pumilus, B. safensis, B. stratosphericus, and B. xiamensis, which is unclear and contains errors in public databases such as NCBI. The included strains grouped into seven clusters based on average nucleotide identity (ANI). Strains designated as B. aerophilus, B. altitudinis, and B. stratosphericus grouped together in the cluster containing the B. altitudinis type strain, suggesting that they should be considered a single species, B. altitudinis. Furthermore, the antimicrobial activity of UTK D1-0055 was determined against a panel of 15 Listeria spp. strains (including nine L. monocytogenes serotypes, L. innocua, and L. marthii), other foodborne pathogens (six Salmonella enterica serotypes and Escherichia coli), and three representative fungi (Saccharomyces cerevisiae, Botrytis cinerea, and Hyperdermium pulvinatum). Antibacterial activity was observed against all Listeria spp. strains, but no antagonistic effects were observed against the other bacterial or fungal strains tested. Biosynthetic gene clusters were identified in silico that may be related to the observed antibacterial activity, which included clusters that putatively encode bacteriocins and nonribosomally synthesized peptides. The Bacillus altitudinis strain identified in the present investigation showed a broad range of antilisterial activity, suggesting that it and other related strains may potentially be evaluated for their biocontrol potential in the food industry.

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