RedEx: a method for seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters

Abstract Biosynthesis reprograming is an important way to diversify chemical structures. The large repetitive DNA sequences existing in polyketide synthase genes make seamless DNA manipulation of the polyketide biosynthetic gene clusters extremely challenging. In this study, to replace the ethyl group attached to the C-21 of the macrolide insecticide spinosad with a butenyl group by refactoring the 79-kb gene cluster, we developed a RedEx method by combining Redαβ mediated linear-circular homologous recombination, ccdB counterselection and exonuclease mediated in vitro annealing to insert an exogenous extension module in the polyketide synthase gene without any extra sequence. RedEx was also applied for seamless deletion of the rhamnose 3′-O-methyltransferase gene in the spinosad gene cluster to produce rhamnosyl-3′-desmethyl derivatives. The advantages of RedEx in seamless mutagenesis will facilitate rational design of complex DNA sequences for diverse purposes.

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The Fusarium verticillioides FUM Gene Cluster Encodes a Zn(II)2Cys6 Protein That Affects FUM Gene Expression and Fumonisin Production

Eukaryotic Cell ◽

10.1128/ec.00400-06 ◽

2007 ◽

Vol 6 (7) ◽

pp. 1210-1218 ◽

Cited By ~ 137

Author(s):

Daren W. Brown ◽

Robert A. E. Butchko ◽

Mark Busman ◽

Robert H. Proctor

Keyword(s):

Gene Cluster ◽

Polyketide Synthase ◽

Fusarium Verticillioides ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Regulatory Proteins ◽

Wild Type ◽

Polyketide Synthase Gene ◽

Splice Forms ◽

Self Protection

ABSTRACT Fumonisins are mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in the synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. Such genes can encode structural enzymes, regulatory proteins, and/or proteins that provide self-protection. The fumonisin biosynthetic gene cluster includes 16 genes, none of which appear to play a role in regulation. In this study, we identified a previously undescribed gene (FUM21) located adjacent to the fumonisin polyketide synthase gene, FUM1. The presence of a Zn(II)2Cys6 DNA-binding domain in the predicted protein suggested that FUM21 was involved in transcriptional regulation. FUM21 deletion (Δfum21) mutants produce little to no fumonisin in cracked maize cultures but some FUM1 and FUM8 transcripts in a liquid GYAM medium. Complementation of a Δfum21 mutant with a wild-type copy of the gene restored fumonisin production. Analysis of FUM21 cDNAs identified four alternative splice forms (ASFs), and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how FUM21 ASFs may regulate fumonisin biosynthesis.

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Characterization of the Nodularin Synthetase Gene Cluster and Proposed Theory of the Evolution of Cyanobacterial Hepatotoxins

Applied and Environmental Microbiology ◽

10.1128/aem.70.11.6353-6362.2004 ◽

2004 ◽

Vol 70 (11) ◽

pp. 6353-6362 ◽

Cited By ~ 169

Author(s):

Michelle C. Moffitt ◽

Brett A. Neilan

Keyword(s):

Gene Cluster ◽

Rational Design ◽

Polyketide Synthase ◽

Alternative Hypothesis ◽

Phylogenetic Analyses ◽

Cyanobacterial Bloom ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

Microcystin Synthetase

ABSTRACT Nodularia spumigena is a bloom-forming cyanobacterium which produces the hepatotoxin nodularin. The complete gene cluster encoding the enzymatic machinery required for the biosynthesis of nodularin in N. spumigena strain NSOR10 was sequenced and characterized. The 48-kb gene cluster consists of nine open reading frames (ORFs), ndaA to ndaI, which are transcribed from a bidirectional regulatory promoter region and encode nonribosomal peptide synthetase modules, polyketide synthase modules, and tailoring enzymes. The ORFs flanking the nda gene cluster in the genome of N. spumigena strain NSOR10 were identified, and one of them was found to encode a protein with homology to previously characterized transposases. Putative transposases are also associated with the structurally related microcystin synthetase (mcy) gene clusters derived from three cyanobacterial strains, indicating a possible mechanism for the distribution of these biosynthetic gene clusters between various cyanobacterial genera. We propose an alternative hypothesis for hepatotoxin evolution in cyanobacteria based on the results of comparative and phylogenetic analyses of the nda and mcy gene clusters. These analyses suggested that nodularin synthetase evolved from a microcystin synthetase progenitor. The identification of the nodularin biosynthetic gene cluster and evolution of hepatotoxicity in cyanobacteria reported in this study may be valuable for future studies on toxic cyanobacterial bloom formation. In addition, an appreciation of the natural evolution of nonribosomal biosynthetic pathways will be vital for future combinatorial engineering and rational design of novel metabolites and pharmaceuticals.

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Transcriptome Analysis Identifies a Gene Cluster for the Biosynthesis of Biruloquinone, a Rare Phenanthraquinone, in a Lichen-Forming Fungus Cladonia macilenta

Journal of Fungi ◽

10.3390/jof7050398 ◽

2021 ◽

Vol 7 (5) ◽

pp. 398

Author(s):

Won-Yong Kim ◽

Min-Hye Jeong ◽

Sung-Hwan Yun ◽

Jae-Seoun Hur

Keyword(s):

Gene Cluster ◽

Transcriptome Analysis ◽

Polyketide Synthase ◽

Expression Patterns ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Whole Transcriptome Analysis ◽

High Degree ◽

Whole Transcriptome

Lichens are prolific producers of natural products of polyketide origin. We previously described a culture of lichen-forming fungus (LFF) Cladonia macilenta that produces biruloquinone, a purple pigment that is a phenanthraquinone rarely found in nature. However, there was no genetic information on the biosynthesis of biruloquinone. To identify a biosynthetic gene cluster for biruloquinone, we mined polyketide synthase (PKS) genes from the genome sequence of a LFF isolated from thalli of C. macilenta. The 38 PKS in C. macilenta are highly diverse, many of which form phylogenetic clades with PKS previously characterized in non-lichenized fungi. We compared transcriptional profiles of the 38 PKS genes in two chemotypic variants, one producing biruloquinone and the other producing no appreciable metabolite in vitro. We identified a PKS gene (hereafter PKS21) that was highly upregulated in the LFF that produces biruloquinone. The boundaries of a putative biruloquinone gene cluster were demarcated by co-expression patterns of six clustered genes, including the PKS21. Biruloquinone gene clusters exhibited a high degree of synteny between related species. In this study we identified a novel PKS family responsible for the biosynthesis of biruloquinone through whole-transcriptome analysis.

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Microfluidic automated plasmid library enrichment for biosynthetic gene cluster discovery

Nucleic Acids Research ◽

10.1093/nar/gkaa131 ◽

2020 ◽

Vol 48 (8) ◽

pp. e48-e48 ◽

Cited By ~ 1

Author(s):

Peng Xu ◽

Cyrus Modavi ◽

Benjamin Demaree ◽

Frederick Twigg ◽

Benjamin Liang ◽

...

Keyword(s):

Gene Cluster ◽

Polyketide Synthase ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Bioactive Molecules ◽

Type I ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Plasmid Library ◽

Polyketide Synthase Gene

Abstract Microbial biosynthetic gene clusters are a valuable source of bioactive molecules. However, because they typically represent a small fraction of genomic material in most metagenomic samples, it remains challenging to deeply sequence them. We present an approach to isolate and sequence gene clusters in metagenomic samples using microfluidic automated plasmid library enrichment. Our approach provides deep coverage of the target gene cluster, facilitating reassembly. We demonstrate the approach by isolating and sequencing type I polyketide synthase gene clusters from an Antarctic soil metagenome. Our method promotes the discovery of functional-related genes and biosynthetic pathways.

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In Silico Analysis of PKS and NRPS Gene Clusters in Arisostatin- and Kosinostatin-Producers and Description of Micromonospora okii sp. nov.

Antibiotics ◽

10.3390/antibiotics10121447 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1447

Author(s):

Hisayuki Komaki ◽

Natsuko Ichikawa ◽

Akira Hosoyama ◽

Moriyuki Hamada ◽

Yasuhiro Igarashi

Keyword(s):

Gene Cluster ◽

Polyketide Synthase ◽

Sequence Similarity ◽

In Silico Analysis ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Rrna Gene ◽

Type I ◽

Phenotypic Differences ◽

Polyketide Synthase Gene

Micromonospora sp. TP-A0316 and Micromonospora sp. TP-A0468 are producers of arisostatin and kosinostatin, respectively. Micromonospora sp. TP-A0316 showed a 16S rRNA gene sequence similarity of 100% to Micromonosporaoryzae CP2R9-1T whereas Micromonospora sp. TP-A0468 showed a 99.3% similarity to Micromonospora haikouensis 232617T. A phylogenetic analysis based on gyrB sequences suggested that Micromonospora sp. TP-A0316 is closely related to Micromonospora oryzae whereas Micromonospora TP-A0468 is an independent genomospecies. As Micromonospora sp. TP-A0468 showed some phenotypic differences to its closely related species, it was classified as a novel species, for which the name Micromonospora okii sp. nov. is proposed. The type strain is TP-A0468T (= NBRC 110461T). Micromonospora sp. TP-A0316 and M. okii TP-A0468T were both found to harbor 15 gene clusters for secondary metabolites such as polyketides and nonribosomal peptides in their genomes. Arisostatin-biosynthetic gene cluster (BGC) of Micromonospora sp. TP-A0316 closely resembled tetrocarcin A-BGC of Micromonospora chalcea NRRL 11289. A large type-I polyketide synthase gene cluster was present in each genome of Micromonospora sp. TP-A0316 and M. okii TP-A0468T. It was an ortholog of quinolidomicin-BGC of M. chalcea AK-AN57 and widely distributed in the genus Micromonospora.

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Total synthesis of long DNA sequences: Synthesis of a contiguous 32-kb polyketide synthase gene cluster

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0406911101 ◽

2004 ◽

Vol 101 (44) ◽

pp. 15573-15578 ◽

Cited By ~ 172

Author(s):

S. J. Kodumal ◽

K. G. Patel ◽

R. Reid ◽

H. G. Menzella ◽

M. Welch ◽

...

Keyword(s):

Total Synthesis ◽

Gene Cluster ◽

Dna Sequences ◽

Polyketide Synthase ◽

Polyketide Synthase Gene

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Structural comparison of Acinetobacter baumannii β-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis

Scientific Reports ◽

10.1038/s41598-021-86997-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Woo Cheol Lee ◽

Sungjae Choi ◽

Ahjin Jang ◽

Kkabi Son ◽

Yangmee Kim

Keyword(s):

Fatty Acid ◽

Acinetobacter Baumannii ◽

Gene Cluster ◽

Fatty Acid Synthase ◽

Acyl Carrier Protein ◽

Gene Clusters ◽

Carrier Protein ◽

Aryl Group ◽

Visible Absorption

AbstractSome Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Here, we determined the crystal structures of the β-ketoacyl-acyl carrier protein (ACP) reductase ApeQ produced by an APE gene cluster from clinically isolated virulent Acinetobacter baumannii in two states (bound and unbound to NADPH). An in vitro visible absorption spectrum assay of the APE polyene moiety revealed that the β-ketoacyl-ACP reductase FabG from the A. baumannii FAS gene cluster cannot be substituted for ApeQ in APE biosynthesis. Comparison with the FabG structure exhibited distinct surface electrostatic potential profiles for ApeQ, suggesting a positively charged arginine patch as the cognate ACP-binding site. Binding modeling for the aryl group predicted that Leu185 (Phe183 in FabG) in ApeQ is responsible for 4-benzoyl moiety recognition. Isothermal titration and arginine patch mutagenesis experiments corroborated these results. These structure–function insights of a unique reductase in the APE BGC in comparison with FAS provide new directions for elucidating host–pathogen interaction mechanisms and novel antibiotics discovery.

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Engineering DNA aptamers and DNA enzymes with fluorescence-signaling properties

Pure and Applied Chemistry ◽

10.1351/pac200476071547 ◽

2004 ◽

Vol 76 (7-8) ◽

pp. 1547-1561 ◽

Cited By ~ 18

Author(s):

R. Nutiu ◽

Shirley Mei ◽

Zhongjie Liu ◽

Y. Li

Keyword(s):

Dna Sequences ◽

Rational Design ◽

In Vitro Selection ◽

Random Sequence ◽

Pair Potential ◽

Dna Aptamers ◽

Dna Molecules ◽

Dna Enzymes ◽

Fluorescence Signaling

Single-stranded DNA molecules with ligand-binding ability and catalytic function, referred to as DNA aptamers and DNA enzymes, respectively, are special DNA sequences isolated from random-sequence DNA libraries by “in vitro selection”. These two new classes of artificial DNA molecules have the potential of being used as molecular tools in a variety of innovative applications ranging from biosensing to gene regulation. Our laboratory is interested in engineering fluorescence-signaling DNA aptamers and DNA enzymes that can be widely exploited for detection-directed applications. In this article, we will first discuss our recent efforts on the rational design of a new class of signaling aptamers denoted “structure- switching signaling aptamers”, which report target binding by switching structures from DNA/DNA duplex to DNA/target complex. We will then describe the in vitro selection of fluorescence-signaling DNA enzymes that exhibit a synchronized catalysis-signaling capability by cleaving a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by closely spaced fluorophore-quencher pair. Potential utilities of these signaling DNA molecules will also be discussed.

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Engineering the Erythromycin-Producing Strain Saccharopolyspora erythraea HOE107 for the Heterologous Production of Polyketide Antibiotics

Frontiers in Microbiology ◽

10.3389/fmicb.2020.593217 ◽

2020 ◽

Vol 11 ◽

Author(s):

Jin Lü ◽

Qingshan Long ◽

Zhilong Zhao ◽

Lu Chen ◽

Weijun He ◽

...

Keyword(s):

Gene Cluster ◽

Polyketide Synthase ◽

Streptomyces Coelicolor ◽

Genome Mining ◽

Bac Library ◽

Gene Clusters ◽

Artificial Chromosome ◽

Saccharopolyspora Erythraea ◽

Heterologous Production ◽

Integration Sites

Bacteria of the genus Saccharopolyspora produce important polyketide antibiotics, including erythromycin A (Sac. erythraea) and spinosad (Sac. spinosa). We herein report the development of an industrial erythromycin-producing strain, Sac. erythraea HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other Saccharopolyspora species and related actinomycetes. To facilitate the integration of natural product BGCs and auxiliary genes beneficial for the production of natural products, the erythromycin polyketide synthase (ery) genes were replaced with two bacterial attB genomic integration sites associated with bacteriophages ϕC31 and ϕBT1. We also established a highly efficient conjugation protocol for the introduction of large bacterial artificial chromosome (BAC) clones into Sac. erythraea strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into Sac. erythraea. The large spinosad gene cluster from Sac. spinosa and the actinorhodin gene cluster from Streptomyces coelicolor were successfully expressed in the ery deletion mutant. Deletion of the endogenous giant polyketide synthase genes pkeA1-pkeA4, the product of which is not known, and the flaviolin gene cluster (rpp) from the bacterium increased the heterologous production of spinosad and actinorhodin. Furthermore, integration of pJTU6728 carrying additional beneficial genes dramatically improved the yield of actinorhodin in the engineered Sac. erythraea strains. Our study demonstrated that the engineered Sac. erythraea strains SLQ185, LJ161, and LJ162 are good hosts for the expression of heterologous antibiotics and should aid in expression-based genome-mining approaches for the discovery of new and cryptic antibiotics from Streptomyces and rare actinomycetes.

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The Gene Cluster forpara-Nitrophenol Catabolism Is Responsible for 2-Chloro-4-Nitrophenol Degradation in Burkholderia sp. Strain SJ98

Applied and Environmental Microbiology ◽

10.1128/aem.02093-14 ◽

2014 ◽

Vol 80 (19) ◽

pp. 6212-6222 ◽

Cited By ~ 31

Author(s):

Jun Min ◽

Jun-Jie Zhang ◽

Ning-Yi Zhou

Keyword(s):

Gene Cluster ◽

Gene Knockout ◽

Gene Clusters ◽

Sole Source ◽

Pcr Analysis ◽

Content Type ◽

Reverse Transcription Pcr ◽

Biochemical Analyses ◽

Nitrophenol Degradation

ABSTRACTBurkholderiasp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) orpara-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of thepnpgenes in thepnpABA1CDEFcluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activityin vitroin the conversion of 2C4NP to CBQ. Genetic analyses indicated thatpnpAplays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels.

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