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

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.

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Characterization of squalene synthase gene from Gymnema sylvestre R. Br.

Beni-Suef University Journal of Basic and Applied Sciences ◽

10.1186/s43088-020-00094-4 ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Kuldeepsingh A. Kalariya ◽

Ram Prasnna Meena ◽

Lipi Poojara ◽

Deepa Shahi ◽

Sandip Patel

Keyword(s):

Dna Sequences ◽

Genomic Dna ◽

Competitive Inhibition ◽

Sequence Data ◽

Homology Model ◽

Squalene Synthase ◽

Gymnema Sylvestre ◽

Gardenia Jasminoides ◽

Ramachandran Plots ◽

Flanking Regions

Abstract Background Squalene synthase (SQS) is a rate-limiting enzyme necessary to produce pentacyclic triterpenes in plants. It is an important enzyme producing squalene molecules required to run steroidal and triterpenoid biosynthesis pathways working in competitive inhibition mode. Reports are available on information pertaining to SQS gene in several plants, but detailed information on SQS gene in Gymnema sylvestre R. Br. is not available. G. sylvestre is a priceless rare vine of central eco-region known for its medicinally important triterpenoids. Our work aims to characterize the GS-SQS gene in this high-value medicinal plant. Results Coding DNA sequences (CDS) with 1245 bp length representing GS-SQS gene predicted from transcriptome data in G. sylvestre was used for further characterization. The SWISS protein structure modeled for the GS-SQS amino acid sequence data had MolProbity Score of 1.44 and the Clash Score 3.86. The quality estimates and statistical score of Ramachandran plots analysis indicated that the homology model was reliable. For full-length amplification of the gene, primers designed from flanking regions of CDS encoding GS-SQS were used to get amplification against genomic DNA as template which resulted in approximately 6.2-kb sized single-band product. The sequencing of this product through NGS was carried out generating 2.32 Gb data and 3347 number of scaffolds with N50 value of 457 bp. These scaffolds were compared to identify similarity with other SQS genes as well as the GS-SQSs of the transcriptome. Scaffold_3347 representing the GS-SQS gene harbored two introns of 101 and 164 bp size. Both these intronic regions were validated by primers designed from adjoining outside regions of the introns on the scaffold representing GS-SQS gene. The amplification took place when the template was genomic DNA and failed when the template was cDNA confirmed the presence of two introns in GS-SQS gene in Gymnema sylvestre R. Br. Conclusion This study shows GS-SQS gene was very closely related to Coffea arabica and Gardenia jasminoides and this gene harbored two introns of 101 and 164 bp size.

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Rapid Amplification of Uncharacterized Transposon-tagged DNA Sequences from Genomic DNA

Yeast ◽

10.1002/(sici)1097-0061(19970315)13:3<233::aid-yea88>3.0.co;2-e ◽

1997 ◽

Vol 13 (3) ◽

pp. 233-240 ◽

Cited By ~ 70

Author(s):

KRISTIN T. CHUN ◽

HOWARD J. EDENBERG ◽

MARK R. KELLEY ◽

MARK G. GOEBL

Keyword(s):

Dna Sequences ◽

Genomic Dna ◽

Rapid Amplification

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Meiotic Recombination Between Paralogous RBCSB Genes on Sister Chromatids of Arabidopsis thaliana

Genetics ◽

10.1093/genetics/166.2.947 ◽

2004 ◽

Vol 166 (2) ◽

pp. 947-957 ◽

Cited By ~ 1

Author(s):

John G Jelesko ◽

Kristy Carter ◽

Whitney Thompson ◽

Yuki Kinoshita ◽

Wilhelm Gruissem

Keyword(s):

Gene Cluster ◽

Dna Sequences ◽

Meiotic Recombination ◽

Sequence Similarity ◽

Specific Gene ◽

High Sequence Similarity ◽

Paralogous Genes ◽

Chimeric Genes ◽

Unequal Recombination ◽

Sister Chromatids

Abstract Paralogous genes organized as a gene cluster can rapidly evolve by recombination between misaligned paralogs during meiosis, leading to duplications, deletions, and novel chimeric genes. To model unequal recombination within a specific gene cluster, we utilized a synthetic RBCSB gene cluster to isolate recombinant chimeric genes resulting from meiotic recombination between paralogous genes on sister chromatids. Several F1 populations hemizygous for the synthRBCSB1 gene cluster gave rise to Luc+ F2 plants at frequencies ranging from 1 to 3 × 10-6. A nonuniform distribution of recombination resolution sites resulted in the biased formation of recombinant RBCS3B/1B::LUC genes with nonchimeric exons. The positioning of approximately half of the mapped resolution sites was effectively modeled by the fractional length of identical DNA sequences. In contrast, the other mapped resolution sites fit an alternative model in which recombination resolution was stimulated by an abrupt transition from a region of relatively high sequence similarity to a region of low sequence similarity. Thus, unequal recombination between paralogous RBCSB genes on sister chromatids created an allelic series of novel chimeric genes that effectively resulted in the diversification rather than the homogenization of the synthRBCSB1 gene cluster.

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Distribution of sequence-dependent curvature in genomic DNA sequences

FEBS Letters ◽

10.1016/s0014-5793(97)00236-6 ◽

1997 ◽

Vol 406 (1-2) ◽

pp. 69-74 ◽

Cited By ~ 24

Author(s):

Andrei Gabrielian ◽

Kristian Vlahovicek ◽

Sándor Pongor

Keyword(s):

Dna Sequences ◽

Genomic Dna ◽

Sequence Dependent

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FIE2: a program for the extraction of genomic DNA sequences around the start and translation initiation site of human genes

Nucleic Acids Research ◽

10.1093/nar/gkg604 ◽

2003 ◽

Vol 31 (13) ◽

pp. 3546-3553 ◽

Cited By ~ 8

Author(s):

A. Chong

Keyword(s):

Translation Initiation ◽

Dna Sequences ◽

Genomic Dna ◽

Initiation Site ◽

Translation Initiation Site ◽

Human Genes

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Thermodynamic Model for B-Z Transition of DNA Induced by Z-DNA Binding Proteins

Molecules ◽

10.3390/molecules23112748 ◽

2018 ◽

Vol 23 (11) ◽

pp. 2748 ◽

Cited By ~ 5

Author(s):

Ae-Ree Lee ◽

Na-Hyun Kim ◽

Yeo-Jin Seo ◽

Seo-Ree Choi ◽

Joon-Hwa Lee

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Genomic Dna ◽

Binding Proteins ◽

Dna Binding Proteins ◽

Multiple Sequence ◽

Left Handed ◽

Transition Mechanism ◽

Dna Strands ◽

Z Dna

Z-DNA is stabilized by various Z-DNA binding proteins (ZBPs) that play important roles in RNA editing, innate immune response, and viral infection. In this review, the structural and dynamics of various ZBPs complexed with Z-DNA are summarized to better understand the mechanisms by which ZBPs selectively recognize d(CG)-repeat DNA sequences in genomic DNA and efficiently convert them to left-handed Z-DNA to achieve their biological function. The intermolecular interaction of ZBPs with Z-DNA strands is mediated through a single continuous recognition surface which consists of an α3 helix and a β-hairpin. In the ZBP-Z-DNA complexes, three identical, conserved residues (N173, Y177, and W195 in the Zα domain of human ADAR1) play central roles in the interaction with Z-DNA. ZBPs convert a 6-base DNA pair to a Z-form helix via the B-Z transition mechanism in which the ZBP first binds to B-DNA and then shifts the equilibrium from B-DNA to Z-DNA, a conformation that is then selectively stabilized by the additional binding of a second ZBP molecule. During B-Z transition, ZBPs selectively recognize the alternating d(CG)n sequence and convert it to a Z-form helix in long genomic DNA through multiple sequence discrimination steps. In addition, the intermediate complex formed by ZBPs and B-DNA, which is modulated by varying conditions, determines the degree of B-Z transition.

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