scholarly journals Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production of Basidiomycete Terpenes by Using Genomic DNA Sequences

2019 ◽  
Vol 85 (15) ◽  
Author(s):  
Shota Nagamine ◽  
Chengwei Liu ◽  
Jumpei Nishishita ◽  
Takuto Kozaki ◽  
Kaho Sogahata ◽  
...  
Keyword(s):  
Natural Products ◽  
Aspergillus Oryzae ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Genome Mining ◽  
Biologically Active ◽  
Biosynthetic Genes ◽  
Content Type ◽  
Content System ◽  
Basidiomycete Fungi

ABSTRACT Basidiomycete fungi are an attractive resource for biologically active natural products for use in pharmaceutically relevant compounds. Recently, genome projects on mushroom fungi have provided a great deal of biosynthetic gene cluster information. However, functional analyses of the gene clusters for natural products were largely unexplored because of the difficulty of cDNA preparation and lack of gene manipulation tools for basidiomycete fungi. To develop a versatile host for basidiomycete genes, we examined gene expression using genomic DNA sequences in the robust ascomycete host Aspergillus oryzae, which is frequently used for the production of metabolites from filamentous fungi. Exhaustive expression of 30 terpene synthase genes from the basidiomycetes Clitopilus pseudo-pinsitus and Stereum hirsutum showed two splicing patterns, i.e., completely spliced cDNAs giving terpenes (15 cases) and mostly spliced cDNAs, indicating that A. oryzae correctly spliced most introns at the predicted positions and lengths. The mostly spliced cDNAs were expressed after PCR-based removal of introns, resulting in the successful production of terpenes (14 cases). During this study, we observed relatively frequent mispredictions in the automated program. Hence, the complementary use of A. oryzae expression and automated prediction will be a powerful tool for genome mining. IMPORTANCE The recent large influx of genome sequences from basidiomycetes, which are prolific producers of bioactive natural products, may provide opportunities to develop novel drug candidates. The development of a reliable expression system is essential for the genome mining of natural products because of the lack of a tractable host for heterologous expression of basidiomycete genes. For this purpose, we applied the ascomycete Aspergillus oryzae system for the direct expression of fungal natural product biosynthetic genes from genomic DNA. Using this system, 29 sesquiterpene synthase genes and diterpene biosynthetic genes for bioactive pleuromutilin were successfully expressed. Together with the use of computational tools for intron prediction, this Aspergillus oryzae system represents a practical method for the production of basidiomycete natural products.

Efficient Reconstitution of Basidiomycota Diterpene Erinacine Gene Cluster in Ascomycota Host Aspergillus oryzae Based on Genomic DNA Sequences

2019 ◽  
Vol 141 (39) ◽  
pp. 15519-15523 ◽  
Author(s):  
Chengwei Liu ◽  
Atsushi Minami ◽  
Taro Ozaki ◽  
Jing Wu ◽  
Hirokazu Kawagishi ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Aspergillus Oryzae ◽  
Dna Sequences ◽  
Genomic Dna

scholarly journals A Pseudoalteromonas clade with remarkable biosynthetic potential

2021 ◽  
Author(s):  
Rocky Chau ◽  
Leanne A. Pearson ◽  
Jesse Cain ◽  
John A. Kalaitzis ◽  
Brett A. Neilan
Keyword(s):  
Natural Products ◽  
Gene Cluster ◽  
Genome Mining ◽  
Gene Clusters ◽  
Average Density ◽  
Biologically Active ◽  
Biosynthesis Gene Cluster ◽  
Diverse Range ◽  
Siderophore Biosynthesis ◽  
Biosynthesis Gene

Pseudoalteromonas species produce a diverse range of biologically active compounds, including those biosynthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here we report the biochemical and genomic analysis of Pseudoalteromonas sp. HM-SA03, isolated from the blue-ringed octopus, Hapalochalaena sp. Genome mining for secondary metabolite pathways revealed seven putative NRPS/PKS biosynthesis gene clusters, including those for the biosynthesis of alterochromides, pseudoalterobactins, alteramides and four hitherto novel compounds. Among these was a novel siderophore biosynthesis gene cluster with unprecedented architecture (NRPS-PKS-NRPS-PKS-NRPS-PKS-NRPS). Alterochromide production in HM-SA03 was also confirmed by liquid chromatography-mass spectrometry. An investigation of the biosynthetic potential of 42 publicly available Pseudoalteromonas genomes indicated that some of these gene clusters are distributed throughout the genus. Through phylogenetic analysis, a particular subset of strains formed a clade with extraordinary biosynthetic potential, with an average density of ten biosynthesis gene clusters per genome. In contrast, the majority of Pseudoalteromonas strains outside this clade contained an average of three clusters encoding complex biosynthesis. These results highlight the under-explored potential of Pseudoalteromonas as a source of new natural products. Importance This study demonstrates that the Pseudoalteromonas strain, HM-SA03, isolated from the venomous blue-ringed octopus, Hapalochalaena sp., is a biosynthetically talented organism, capable of producing alterochromides and potentially six other specialized metabolites. We have identified a pseudoalterobactin biosynthesis gene cluster and proposed a pathway for the production of the associated siderophore. A novel siderophore biosynthesis gene cluster with unprecedented architecture was also identified in the HM-SA03 genome. Finally, we have demonstrated that HM-SA03 belongs to a phylogenetic clade of strains with extraordinary biosynthetic potential. While our results do not support a role of HM-SA03 in Hapalochalaena sp. venom (tetrodotoxin) production, they emphasize the untapped potential of Pseudoalteromonas as a source of novel natural products.

scholarly journals Discovery of 16-Demethylrifamycins by Removing the Predominant Polyketide Biosynthesis Pathway in Micromonospora sp. Strain TP-A0468

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Qiang Zhou ◽  
Guang-Cai Luo ◽  
Huizhan Zhang ◽  
Gong-Li Tang
Keyword(s):  
Natural Products ◽  
Transcriptional Activation ◽  
Metabolic Flux ◽  
Genome Mining ◽  
Gene Clusters ◽  
Bioinformatic Analysis ◽  
Transcriptional Analysis ◽  
Content Type ◽  
In The Wild ◽  
New Compounds

ABSTRACT A number of strategies have been developed to mine novel natural products based on biosynthetic gene clusters and there have been dozens of successful cases facilitated by the development of genomic sequencing. During our study on biosynthesis of the antitumor polyketide kosinostatin (KST), we found that the genome of Micromonospora sp. strain TP-A0468, the producer of KST, contains other potential polyketide gene clusters, with no encoded products detected. Deletion of kst cluster led to abolishment of KST and the enrichment of several new compounds, which were isolated and characterized as 16-demethylrifamycins (referred to here as compounds 3 to 6). Transcriptional analysis demonstrated that the expression of the essential genes related to the biosynthesis of compounds 3 to 6 was comparable to the level in the wild-type and in the kst cluster deletion strain. This indicates that the accumulation of these compounds was due to the redirection of metabolic flux rather than transcriptional activation. Genetic disruption, chemical complementation, and bioinformatic analysis revealed that the production of compounds 3 to 6 was accomplished by cross talk between the two distantly placed polyketide gene clusters pks3 and M-rif. This finding not only enriches the analogue pool and the biosynthetic diversity of rifamycins but also provides an auxiliary strategy for natural product discovery through genome mining in polyketide-producing microorganisms. IMPORTANCE Natural products are essential in the development of novel clinically used drugs. Discovering new natural products and modifying known compounds are still the two main ways to generate new candidates. Here, we have discovered several rifamycins with varied skeleton structures by redirecting the metabolic flux from the predominant polyketide biosynthetic pathway to the rifamycin pathway in the marine actinomycetes species Micromonospora sp. strain TP-A0468. Rifamycins are indispensable chemotherapeutics in the treatment of various diseases such as tuberculosis, leprosy, and AIDS-related mycobacterial infections. This study exemplifies a useful method for the discovery of cryptic natural products in genome-sequenced microbes. Moreover, the 16-demethylrifamycins and their genetically manipulable producer provide a new opportunity in the construction of novel rifamycin derivates to aid in the defense against the ever-growing drug resistance of Mycobacterium tuberculosis.

Unique marine derived cyanobacterial biosynthetic genes for chemical diversity

2016 ◽  
Vol 33 (2) ◽  
pp. 348-364 ◽  
Author(s):  
Karin Kleigrewe ◽  
Lena Gerwick ◽  
David H. Sherman ◽  
William H. Gerwick
Keyword(s):  
Natural Products ◽  
Chemical Diversity ◽  
Biologically Active ◽  
Biosynthetic Genes ◽  
Biochemical Pathways ◽  
Active Natural

Cyanobacteria are a prolific source of structurally unique and biologically active natural products that derive from intriguing biochemical pathways.

scholarly journals CRISPR-Cpf1-Assisted Multiplex Genome Editing and Transcriptional Repression in Streptomyces

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Lei Li ◽  
Keke Wei ◽  
Guosong Zheng ◽  
Xiaocao Liu ◽  
Shaoxin Chen ◽  
...  
Keyword(s):  
Natural Products ◽  
Genetic Engineering ◽  
Genome Editing ◽  
Transcriptional Repression ◽  
High Efficiency ◽  
Streptomyces Coelicolor ◽  
Genome Mining ◽  
Strain Improvement ◽  
Strand Break ◽  
Content Type

ABSTRACT Streptomyces has a strong capability for producing a large number of bioactive natural products and remains invaluable as a source for the discovery of novel drug leads. Although the Streptococcus pyogenes CRISPR-Cas9-assisted genome editing tool has been developed for rapid genetic engineering in Streptomyces, it has a number of limitations, including the toxicity of SpCas9 expression in some important industrial Streptomyces strains and the need for complex expression constructs when targeting multiple genomic loci. To address these problems, in this study, we developed a high-efficiency CRISPR-Cpf1 system (from Francisella novicida) for multiplex genome editing and transcriptional repression in Streptomyces. Using an all-in-one editing plasmid with homology-directed repair (HDR), our CRISPR-Cpf1 system precisely deletes single or double genes at efficiencies of 75 to 95% in Streptomyces coelicolor. When no templates for HDR are present, random-sized DNA deletions are achieved by FnCpf1-induced double-strand break (DSB) repair by a reconstituted nonhomologous end joining (NHEJ) pathway. Furthermore, a DNase-deactivated Cpf1 (ddCpf1)-based integrative CRISPRi system is developed for robust, multiplex gene repression using a single customized crRNA array. Finally, we demonstrate that FnCpf1 and SpCas9 exhibit different suitability in tested industrial Streptomyces species and show that FnCpf1 can efficiently promote HDR-mediated gene deletion in the 5-oxomilbemycin-producing strain Streptomyces hygroscopicus SIPI-KF, in which SpCas9 does not work well. Collectively, FnCpf1 is a powerful and indispensable addition to the Streptomyces CRISPR toolbox. IMPORTANCE Rapid, efficient genetic engineering of Streptomyces strains is critical for genome mining of novel natural products (NPs) as well as strain improvement. Here, a novel and high-efficiency Streptomyces genome editing tool is established based on the FnCRISPR-Cpf1 system, which is an attractive and powerful alternative to the S. pyogenes CRISPR-Cas9 system due to its unique features. When combined with HDR or NHEJ, FnCpf1 enables the creation of gene(s) deletion with high efficiency. Furthermore, a ddCpf1-based integrative CRISPRi platform is established for simple, multiplex transcriptional repression. Of importance, FnCpf1-based genome editing proves to be a highly efficient tool for genetic modification of some important industrial Streptomyces strains (e.g., S. hygroscopicus SIPI-KF) that cannot utilize the SpCRISPR-Cas9 system. We expect the CRISPR-Cpf1-assisted genome editing tool to accelerate discovery and development of pharmaceutically active NPs in Streptomyces as well as other actinomycetes.

scholarly journals A Novel Rickettsia Species Detected in Vole Ticks (Ixodes angustus) from Western Canada

2013 ◽  
Vol 79 (24) ◽  
pp. 7583-7589 ◽  
Author(s):  
Clare A. Anstead ◽  
Neil B. Chilton
Keyword(s):  
Dna Sequences ◽  
Genomic Dna ◽  
Sequence Data ◽  
Phylogenetic Analyses ◽  
Spotted Fever ◽  
Ixodid Ticks ◽  
Western Canada ◽  
Rickettsia Species ◽  
Content Type ◽  
Putative Species

ABSTRACTThe genomic DNA of ixodid ticks from western Canada was tested by PCR for the presence ofRickettsia. No rickettsiae were detected inIxodes sculptus, whereas 18% of theI. angustusand 42% of theDermacentor andersoniorganisms examined were PCR positive forRickettsia. The rickettsiae from each tick species were characterized genetically using multiple genes. Rickettsiae within theD. andersoniorganisms had sequences at four genes that matched those ofR. peacockii. In contrast, theRickettsiapresent within the larvae, nymphs, and adults ofI. angustushad novel DNA sequences at four of the genes characterized compared to the sequences available from GenBank for all recognized species ofRickettsiaand all other putative species within the genus. Phylogenetic analyses of the sequence data revealed that the rickettsiae inI. angustusdo not belong to the spotted fever, transitional, or typhus groups of rickettsiae but are most closely related to “CandidatusRickettsia kingi” and belong to a clade that also includesR. canadensis, “CandidatusRickettsia tarasevichiae,” and “CandidatusRickettsia monteiroi.”

scholarly journals Regeneration ofEscherichia colifrom Minicells through Lateral Gene Transfer

2018 ◽  
Vol 200 (9) ◽  
Author(s):  
Hideki Kobayashi
Keyword(s):  
Genomic Dna ◽  
Living System ◽  
Biologically Active ◽  
Pulse Field ◽  
Natural Behavior ◽  
Content Type ◽  
E Coli ◽  
Pulse Field Gel Electrophoresis ◽  
The Difference ◽  
Genome Donors

ABSTRACTRecently, artificial life has been created with artificial materials and methods. Life can be created when genomic DNA molecules are integrated in liposomes containing biochemical reactions for biogenic needs. However, it is not yet known whether the integration of these parts will be able to occur in nature and constitute a living system. I planned to regenerate bacteria from biologically active liposomes by inserting genomic DNA using only natural materials and methods. Minicells ofEscherichia coli, containing plasmids and activated SOS proteins, act as protocells. Four newE. colistrains were regenerated from minicells by inserting the genomes by using the system for conjugation between F−and Hfr strains. Cells of the four regenerated strains showed the same genetic markers as the two genome donors. Pulse-field gel electrophoresis of their genomes showed admixing of those of both donors. In addition, the genomes of the four regenerated strains had chimeric genome of the two donors. These results show that synthesis of life can occur in nature without artificial arrangement.IMPORTANCEWhat is the difference between inanimate objects and organisms? Organisms always have genomic DNA. When organisms lose their genomes, they can neither grow nor reproduce. As the result, organisms turn into inanimate objects without their genomes. In this study, I regenerated microbes from cells that had lost their genomes (cell corpses) by inserting another genome. All steps of regeneration used the natural behavior of microbes. The same regeneration of microbes could happen in nature. These primitive lives have plasticity, which accelerates evolution and provides various kinds of life in the world.

Biologically active natural products from Cassine viburnifolia

Planta Medica ◽  
2015 ◽  
Vol 81 (11) ◽  
Author(s):  
T Grkovic ◽  
R Akee ◽  
J Evans ◽  
JM Collins ◽  
B O'Keefe
Keyword(s):  
Natural Products ◽  
Biologically Active ◽  
Active Natural

Expanding the Natural Products Heterologous Expression Repertoire in the Model Cyanobacterium Anabaena sp. Strain PCC 7120: Production of Pendolmycin and Teleocidin B-4

2019 ◽  
Author(s):  
Patrick Videau ◽  
Kaitlyn Wells ◽  
Arun Singh ◽  
Jessie Eiting ◽  
Philip Proteau ◽  
...  
Keyword(s):  
Natural Products ◽  
Genome Mining ◽  
Gene Clusters ◽  
Combinatorial Biosynthesis ◽  
Test Case ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

Cyanobacteria are prolific producers of natural products and genome mining has shown that many orphan biosynthetic gene clusters can be found in sequenced cyanobacterial genomes. New tools and methodologies are required to investigate these biosynthetic gene clusters and here we present the use of <i>Anabaena </i>sp. strain PCC 7120 as a host for combinatorial biosynthesis of natural products using the indolactam natural products (lyngbyatoxin A, pendolmycin, and teleocidin B-4) as a test case. We were able to successfully produce all three compounds using codon optimized genes from Actinobacteria. We also introduce a new plasmid backbone based on the native <i>Anabaena</i>7120 plasmid pCC7120ζ and show that production of teleocidin B-4 can be accomplished using a two-plasmid system, which can be introduced by co-conjugation.

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