Cytosine Base Editor-Mediated Multiplex Genome Editing to Accelerate Discovery of Novel Antibiotics in Bacillus subtilis and Paenibacillus polymyxa

Genome-based identification of new antibiotics is emerging as an alternative to traditional methods. However, uncovering hidden antibiotics under the background of known antibiotics remains a challenge. To over this problem using a quick and effective genetic approach, we developed a multiplex genome editing system using a cytosine base editor (CBE). The CBE system achieved simultaneous double, triple, quadruple, and quintuple gene editing with efficiencies of 100, 100, 83, and 75%, respectively, as well as the 100% editing efficiency of single targets in Bacillus subtilis. Whole-genome sequencing of the edited strains showed that they had an average of 8.5 off-target single-nucleotide variants at gRNA-independent positions. The CBE system was used to simultaneously knockout five known antibiotic biosynthetic gene clusters to leave only an uncharacterized polyketide biosynthetic gene cluster in Paenibacillus polymyxa E681. The polyketide showed antimicrobial activities against gram-positive bacteria, but not gram-negative bacteria and fungi. Therefore, our findings suggested that the CBE system might serve as a powerful tool for multiplex genome editing and greatly accelerating the unraveling of hidden antibiotics in Bacillus and Paenibacillus species.

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Identification of the kinanthraquinone biosynthetic gene cluster by expression of an atypical response regulator

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa082 ◽

2021 ◽

Vol 85 (3) ◽

pp. 714-721

Author(s):

Risa Takao ◽

Katsuyuki Sakai ◽

Hiroyuki Koshino ◽

Hiroyuki Osada ◽

Shunji Takahashi

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Secondary Metabolite ◽

Response Regulator ◽

Bac Library ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Gene Organization ◽

Biosynthetic Gene ◽

Streptomyces Sp

ABSTRACT Recent advances in genome sequencing have revealed a variety of secondary metabolite biosynthetic gene clusters in actinomycetes. Understanding the biosynthetic mechanism controlling secondary metabolite production is important for utilizing these gene clusters. In this study, we focused on the kinanthraquinone biosynthetic gene cluster, which has not been identified yet in Streptomyces sp. SN-593. Based on chemical structure, 5 type II polyketide synthase gene clusters were listed from the genome sequence of Streptomyces sp. SN-593. Among them, a candidate gene cluster was selected by comparing the gene organization with grincamycin, which is synthesized through an intermediate similar to kinanthraquinone. We initially utilized a BAC library for subcloning the kiq gene cluster, performed heterologous expression in Streptomyces lividans TK23, and identified the production of kinanthraquinone and kinanthraquinone B. We also found that heterologous expression of kiqA, which belongs to the DNA-binding response regulator OmpR family, dramatically enhanced the production of kinanthraquinones.

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Blocks in the pseudouridimycin pathway unlock hidden metabolites in the Streptomyces producer strain

Scientific Reports ◽

10.1038/s41598-021-84833-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marianna Iorio ◽

Sahar Davatgarbenam ◽

Stefania Serina ◽

Paolo Criscenzo ◽

Mitja M. Zdouc ◽

...

Keyword(s):

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Systematic Investigation ◽

Biosynthetic Gene ◽

Wild Type ◽

Bacterial Rna ◽

Soil Isolate ◽

Mutant Strains ◽

In The Wild ◽

Polymerase Inhibitor

AbstractWe report a metabolomic analysis of Streptomyces sp. ID38640, a soil isolate that produces the bacterial RNA polymerase inhibitor pseudouridimycin. The analysis was performed on the wild type, on three newly constructed and seven previously reported mutant strains disabled in different genes required for pseudouridimycin biosynthesis. The results indicate that Streptomyces sp. ID38640 is able to produce, in addition to lydicamycins and deferroxiamines, as previously reported, also the lassopeptide ulleungdin, the non-ribosomal peptide antipain and the osmoprotectant ectoine. The corresponding biosynthetic gene clusters were readily identified in the strain genome. We also detected the known compound pyridindolol, for which we propose a previously unreported biosynthetic gene cluster, as well as three families of unknown metabolites. Remarkably, the levels of most metabolites varied strongly in the different mutant strains, an observation that enabled detection of metabolites unnoticed in the wild type. Systematic investigation of the accumulated metabolites in the ten different pum mutants identified shed further light on pseudouridimycin biosynthesis. We also show that several Streptomyces strains, able to produce pseudouridimycin, have distinct genetic relationship and metabolic profile with ID38640.

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Identification of Putative Biosynthetic Gene Clusters for Tolyporphins in Multiple Filamentous Cyanobacteria

Life ◽

10.3390/life11080758 ◽

2021 ◽

Vol 11 (8) ◽

pp. 758

Author(s):

Xiaohe Jin ◽

Yunlong Zhang ◽

Ran Zhang ◽

Kathy-Uyen Nguyen ◽

Jonathan S. Lindsey ◽

...

Keyword(s):

Gene Cluster ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Filamentous Cyanobacterium ◽

Biosynthetic Gene ◽

Filamentous Cyanobacteria ◽

Biosynthetic Gene Clusters ◽

Common Component ◽

Homology Searching ◽

Similar Gene

Tolyporphins A–R are unusual tetrapyrrole macrocycles produced by the non-axenic filamentous cyanobacterium HT-58-2. A putative biosynthetic gene cluster for biosynthesis of tolyporphins (here termed BGC-1) was previously identified in the genome of HT-58-2. Here, homology searching of BGC-1 in HT-58-2 led to identification of similar BGCs in seven other filamentous cyanobacteria, including strains Nostoc sp. 106C, Nostoc sp. RF31YmG, Nostoc sp. FACHB-892, Brasilonema octagenarum UFV-OR1, Brasilonema octagenarum UFV-E1, Brasilonema sennae CENA114 and Oculatella sp. LEGE 06141, suggesting their potential for tolyporphins production. A similar gene cluster (BGC-2) also was identified unexpectedly in HT-58-2. Tolyporphins BGCs were not identified in unicellular cyanobacteria. Phylogenetic analysis based on 16S rRNA and a common component of the BGCs, TolD, points to a close evolutionary history between each strain and their respective tolyporphins BGC. Though identified with putative tolyporphins BGCs, examination of pigments extracted from three cyanobacteria has not revealed the presence of tolyporphins. Overall, the identification of BGCs and potential producers of tolyporphins presents a collection of candidate cyanobacteria for genetic and biochemical analysis pertaining to these unusual tetrapyrrole macrocycles.

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Clone of plipastatin biosynthetic gene cluster by transformation-associated recombination technique and high efficient expression in model organism Bacillus subtilis

Journal of Biotechnology ◽

10.1016/j.jbiotec.2018.10.006 ◽

2018 ◽

Vol 288 ◽

pp. 1-8 ◽

Cited By ~ 7

Author(s):

Yimin Hu ◽

Fang Nan ◽

Sarah Wanjiku Maina ◽

Jia Guo ◽

Shenglu Wu ◽

...

Keyword(s):

Bacillus Subtilis ◽

Gene Cluster ◽

Model Organism ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

High Efficient ◽

Efficient Expression

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Draft Genome Sequence of Streptomyces sp. TP-A0874, a Catechoserine Producer

Genome Announcements ◽

10.1128/genomea.01163-16 ◽

2016 ◽

Vol 4 (5) ◽

Author(s):

Hisayuki Komaki ◽

Akira Hosoyama ◽

Natsuko Ichikawa ◽

Yasuhiro Igarashi

Keyword(s):

Genome Sequence ◽

Cell Invasion ◽

Draft Genome ◽

Gene Clusters ◽

Bioinformatic Analysis ◽

Biosynthetic Gene Cluster ◽

Tumor Cell Invasion ◽

Draft Genome Sequence ◽

Biosynthetic Gene ◽

Streptomyces Sp

We report the draft genome sequence of Streptomyces sp. TP-A0874 isolated from compost. This strain produces catechoserine, a new catecholate-type inhibitor of tumor cell invasion. The genome harbors at least six gene clusters for polyketide and nonribosomal peptide biosyntheses. The biosynthetic gene cluster for catechoserines was identified by bioinformatic analysis.

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Phylogenetic Distribution of Secondary Metabolites in the Bacillus subtilis Species Complex

mSystems ◽

10.1128/msystems.00057-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Kat Steinke ◽

Omkar S. Mohite ◽

Tilmann Weber ◽

Ákos T. Kovács

Keyword(s):

Bacillus Subtilis ◽

Secondary Metabolites ◽

Secondary Metabolite ◽

Species Complex ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Content Type ◽

Frameshift Mutations ◽

Metabolite Production

ABSTRACT Microbes produce a plethora of secondary (or specialized) metabolites that, although not essential for primary metabolism, benefit them to survive in the environment, communicate, and influence cell differentiation. Biosynthetic gene clusters (BGCs), responsible for the production of these secondary metabolites, are readily identifiable on bacterial genome sequences. Understanding the phylogeny and distribution of BGCs helps us to predict the natural product synthesis ability of new isolates. Here, we examined 310 genomes from the Bacillus subtilis group, determined the inter- and intraspecies patterns of absence/presence for all BGCs, and assigned them to defined gene cluster families (GCFs). This allowed us to establish patterns in the distribution of both known and unknown products. Further, we analyzed variations in the BGC structures of particular families encoding natural products, such as plipastatin, fengycin, iturin, mycosubtilin, and bacillomycin. Our detailed analysis revealed multiple GCFs that are species or clade specific and a few others that are scattered within or between species, which will guide exploration of the chemodiversity within the B. subtilis group. Surprisingly, we discovered that partial deletion of BGCs and frameshift mutations in selected biosynthetic genes are conserved within phylogenetically related isolates, although isolated from around the globe. Our results highlight the importance of detailed genomic analysis of BGCs and the remarkable phylogenetically conserved erosion of secondary metabolite biosynthetic potential in the B. subtilis group. IMPORTANCE Members of the B. subtilis species complex are commonly recognized producers of secondary metabolites, among those, the production of antifungals, which makes them promising biocontrol strains. While there are studies examining the distribution of well-known secondary metabolites in Bacilli, intraspecies clade-specific distribution has not been systematically reported for the B. subtilis group. Here, we report the complete biosynthetic potential within the B. subtilis group to explore the distribution of the biosynthetic gene clusters and to reveal an exhaustive phylogenetic conservation of secondary metabolite production within Bacillus that supports the chemodiversity within this species complex. We identify that certain gene clusters acquired deletions of genes and particular frameshift mutations, rendering them inactive for secondary metabolite biosynthesis, a conserved genetic trait within phylogenetically conserved clades of certain species. The overview guides the assignment of the secondary metabolite production potential of newly isolated Bacillus strains based on genome sequence and phylogenetic relatedness.

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Refactoring the formicamycin biosynthetic gene cluster to make high-level producing strains and new molecules

10.1101/2020.09.15.275776 ◽

2020 ◽

Cited By ~ 1

Author(s):

Rebecca Devine ◽

Hannah McDonald ◽

Zhiwei Qin ◽

Corinne Arnold ◽

Katie Noble ◽

...

Keyword(s):

Gene Cluster ◽

Large Scale ◽

Liquid Culture ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Resistant Bacteria ◽

Drug Resistant ◽

Biosynthetic Gene ◽

Biosynthetic Genes

AbstractThe formicamycins are promising antibiotics with potent activity against Gram-positive pathogens including VRE and MRSA and display a high barrier to selection of resistant isolates. They were first identified in Streptomyces formicae KY5, which produces the formicamycins at low levels on solid agar but not in liquid culture, thus hindering further investigation of these promising antibacterial compounds. We hypothesised that by understanding the organisation and regulation of the for biosynthetic gene cluster, we could rationally refactor the cluster to increase production levels. Here we report that the for biosynthetic gene cluster consists of 24 genes expressed on nine transcripts. Seven of these transcripts, including those containing all the major biosynthetic genes, are repressed by the MarR-regulator ForJ which also controls the expression of the ForGF two-component system that initiates biosynthesis. A third cluster-situated regulator, ForZ, autoregulates and controls production of the putative MFS transporter ForAA. Consistent with these findings, deletion of forJ increased formicamycin biosynthesis 5-fold, while over-expression of forGF in the ΔforJ background increased production 10-fold compared to the wild-type. De-repression by deleting forJ also switched on biosynthesis in liquid-culture and induced the production of two novel formicamycin congeners. By combining mutations in regulatory and biosynthetic genes, six new biosynthetic precursors with antibacterial activity were also isolated. This work demonstrates the power of synthetic biology for the rational redesign of antibiotic biosynthetic gene clusters both to engineer strains suitable for fermentation in large scale bioreactors and to generate new molecules.ImportanceAntimicrobial resistance is a growing threat as existing antibiotics become increasingly ineffective against drug resistant pathogens. Here we determine the transcriptional organisation and regulation of the gene cluster encoding biosynthesis of the formicamycins, promising new antibiotics with activity against drug resistant bacteria. By exploiting this knowledge, we construct stable mutant strains which over-produce these molecules in both liquid and solid culture whilst also making some new compound variants. This will facilitate large scale purification of these molecules for further study including in vivo experiments and the elucidation of their mechanism of action. Our work demonstrates that understanding the regulation of natural product biosynthetic pathways can enable rational improvement of the producing strains.

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Multigenome analysis implicates miniature inverted-repeat transposable elements (MITEs) in metabolic diversification in eudicots

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1721318115 ◽

2018 ◽

Vol 115 (28) ◽

pp. E6650-E6658 ◽

Cited By ~ 12

Author(s):

Alexander M. Boutanaev ◽

Anne E. Osbourn

Keyword(s):

Transposable Elements ◽

Inverted Repeat ◽

Cluster Formation ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Terpene Synthase ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Systematic Analysis ◽

Plant Genomes

Plants produce a plethora of natural products, including many drugs. It has recently emerged that the genes encoding different natural product pathways may be organized as biosynthetic gene clusters in plant genomes, with >30 examples reported so far. Despite superficial similarities with microbes, these clusters have not arisen by horizontal gene transfer, but rather by gene duplication, neofunctionalization, and relocation via unknown mechanisms. Previously we reported that two Arabidopsis thaliana biosynthetic gene clusters are located in regions of the genome that are significantly enriched in transposable elements (TEs). Other plant biosynthetic gene clusters also harbor abundant TEs. TEs can mediate genomic rearrangement by providing homologous sequences that enable illegitimate recombination and gene relocation. Thus, TE-mediated recombination may contribute to plant biosynthetic gene cluster formation. TEs may also facilitate establishment of regulons. However, a systematic analysis of the TEs associated with plant biosynthetic gene clusters has not been carried out. Here we investigate the TEs associated with clustered terpene biosynthetic genes in multiple plant genomes and find evidence to suggest a role for miniature inverted-repeat transposable elements in cluster formation in eudicots. Through investigation of the newly sequenced Amborella trichopoda, Aquilegia coerulea, and Kalanchoe fedtschenkoi genomes, we further show that the “block” mechanism of founding of biosynthetic gene clusters through duplication and diversification of pairs of terpene synthase and cytochrome P450 genes that is prevalent in the eudicots arose around 90–130 million years ago, after the appearance of the basal eudicots and before the emergence of the superrosid clade.

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Screening of tenuazonic acid production-inducing compounds and identification of NPD938 as a regulator of fungal secondary metabolism

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab143 ◽

2021 ◽

Author(s):

Takayuki Motoyama ◽

Tomoaki Ishii ◽

Takashi Kamakura ◽

Hiroyuki Osada

Keyword(s):

Secondary Metabolism ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

Tenuazonic Acid ◽

Cellular Functions ◽

Biosynthetic Gene Clusters ◽

Chemical Library ◽

Tea Production ◽

Fungal Secondary Metabolism

Abstract The control of secondary metabolism in fungi is essential for the regulation of various cellular functions. In this study, we searched the RIKEN Natural Products Depository (NPDepo) chemical library for inducers of tenuazonic acid (TeA) production in the rice blast fungus Pyricularia oryzae and identified NPD938. NPD938 transcriptionally induced TeA production. We explored the mode of action of NPD938 and observed that this compound enhanced TeA production via LAE1, a global regulator of fungal secondary metabolism. NPD938 could also induce production of terpendoles and pyridoxatins in Tolypocladium album RK99-F33. Terpendole production was induced transcriptionally. We identified the pyridoxatin biosynthetic gene cluster among transcriptionally induced secondary metabolite biosynthetic gene clusters. Therefore, NPD938 is useful for the control of fungal secondary metabolism.

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