1. |
Skory CD,
( 2003 ) Induction of Rhizopus oryzae pyruvate decarboxylase genes. PMID : 12783195 : DOI : 10.1007/s00284-002-3933-0 Abstract >>
Two pyruvate decarboxylase genes, pdcA, and pdcB, were cloned from Rhizopus oryzae. These genes are similar to each other with approximately 85% nucleotide sequence identity within the coding region. Multiple transcriptional start sites and polyadenylation sites were found for both genes. The deduced translation product of each gene results in a 561 amino acid protein with approximate molecular weight of 61 kDa each. The amino acid identity between the two proteins was 91% as calculated by Lipmann-Pearson comparisons. Transcriptional control appears to be important in regulation of the PDC, since much of the transcript accumulation parallels enzymatic activity. There was no detectable pdc transcript from cultures grown in glycerol-containing medium. Induction of transcription for pdcA and pdcB was initiated within 1.5 h of adding glucose to the culture. Shifting the aerobically grown cultures to anoxic conditions at this time resulted in enhanced pdc transcription, PDC enzymatic activity, and ethanol production, compared to cultures with continued aerobic growth.
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2. |
Moriya T,
Murashima K,
Nakane A,
Yanai K,
Sumida N,
Koga J,
Murakami T,
Kono T,
( 2003 ) Molecular cloning of endo-beta-D-1,4-glucanase genes, rce1, rce2, and rce3, from Rhizopus oryzae. PMID : 12591897 : DOI : 10.1128/jb.185.5.1749-1756.2003 PMC : PMC148074 Abstract >>
Three endoglucanase genes, designated the rce1, rce2, and rce3 genes, were isolated from Rhizopus oryzae as the first cellulase genes from the subdivision ZYGOMYCOTA: All the amino acid sequences deduced from the rce1, rce2, and rce3 genes consisted of three distinct domains: cellulose binding domains, linker domains, and catalytic domains belonging to glycosyl hydrolase family 45. The rce3 gene had two tandem repeated sequences of cellulose binding domains, while rce1 and rce2 had only one. rce1, rce2, and rce3 had various lengths of linker sequences.
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3. |
Skory CD,
( 2002 ) Homologous recombination and double-strand break repair in the transformation of Rhizopus oryzae. PMID : 12436261 : DOI : 10.1007/s00438-002-0760-8 Abstract >>
Genetic transformation of the Mucorales fungi has been problematic, since DNA transformed into the host rarely integrates and usually is mitotically unstable in the absence of selective pressure. In this study, transformation of Rhizopus oryzae was investigated to determine if the fate of introduced DNA could be predicted based on double-strand break repair and recombination mechanisms found in other fungi. A transformation system was developed with uracil auxotrophs of Rhizopus oryzae that could be complemented with the pyrG gene isolated in this work. DNA transformed as circular plasmids was maintained extrachromosomally in high-molecular-weight (>23 kb) concatenated arrangement. Type-I crossover integration into the pyrG locus and type-III pyrG gene replacement events occurred in approximately 1-5% of transformants. Linearization of the plasmid pPyr225 with a single restriction enzyme that cleaves within the vector sequence almost always resulted in isolates with replicating concatenated plasmids that had been repaired by end-joining recombination that restored the restriction site. The addition of a 40-bp direct repeat on either side of this cleavage site led to repair by homologous recombination between the repeated sequences on the plasmid, resulting in loss of the restriction site. When plasmid pPyr225 was digested with two different enzymes that cleave within the vector sequence to release the pyrG containing fragment, only pyrG gene replacement recombination occurred in transformants. Linearization of plasmid pPyr225 within the pyrG gene itself gave the highest percentage (20%) of type-I integration at the pyrG locus. However, end-joining repair and gene replacement events were still the predominant types of recombination found in transformations with this plasmid topology.
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4. |
Ben Salah A,
Sayari A,
Verger R,
Gargouri Y,
( 2001 ) Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique. PMID : 11506890 : DOI : 10.1016/s0300-9084(01)01283-4 Abstract >>
Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus lipases. Purified ROL possesses the same N-terminal sequence as the mature Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that Rhizopus oryzae lipase is markedly stereoselective of the sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (Rhizopus delemar lipase and Rhizopus arrhizus lipase).
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5. |
Voigt K,
Wöstemeyer J,
( 2001 ) Phylogeny and origin of 82 zygomycetes from all 54 genera of the Mucorales and Mortierellales based on combined analysis of actin and translation elongation factor EF-1alpha genes. PMID : 11404008 : DOI : 10.1016/s0378-1119(01)00464-4 Abstract >>
True fungi (Eumycota) are heterotrophic eukaryotic microorganisms encompassing ascomycetes, basidiomycetes, chytridiomycetes and zygomycetes. The natural systematics of the latter group, Zygomycota, are very poorly understood due to the lack of distinguishing morphological characters. We have determined sequences for the nuclear-encoded genes actin (act) from 82 zygomycetes representing all 54 currently recognized genera from the two zygomycetous orders Mucorales and Mortierellales. We also determined sequences for translation elongation factor EF-1alpha (tef) from 16 zygomycetes (total of 96,837 bp). Phylogenetic analysis in the context of available sequence data (total 2,062 nucleotide positions per species) revealed that current classification schemes for the mucoralean fungi are highly unnatural at the family and, to a large extent, at the genus level. The data clearly indicate a deep, ancient and distinct dichotomy of the orders Mucorales and Mortierellales, which are recognized only in some zygomycete systems. Yet at the same time the data show that two genera - Umbelopsis and Micromucor - previously placed within the Mortierellales on the basis of their weakly developed columella (a morphological structure of the sporangiophore well-developed within all Mucorales) are in fact members of the Mucorales. Phylogenetic analyses of the encoded amino acid sequences in the context of homologues from eukaryotes and archaebacterial outgroups indicate that the Eumycota studied here are a natural group but provide little or no support for the monophyly of either zygomycetes, ascomycetes or basidiomycetes. The data clearly indicate that a complete revision of zygomycete natural systematics is necessary.
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6. |
Skory CD,
( 2000 ) Isolation and expression of lactate dehydrogenase genes from Rhizopus oryzae. PMID : 10831409 : DOI : 10.1128/aem.66.6.2343-2348.2000 PMC : PMC110528 Abstract >>
Rhizopus oryzae is used for industrial production of lactic acid, yet little is known about the genetics of this fungus. In this study I cloned two genes, ldhA and ldhB, which code for NAD(+)-dependent L-lactate dehydrogenases (LDH) (EC 1.1.1.27), from a lactic acid-producing strain of R. oryzae. These genes are similar to each other and exhibit more than 90% nucleotide sequence identity and they contain no introns. This is the first description of ldh genes in a fungus, and sequence comparisons revealed that these genes are distinct from previously isolated prokaryotic and eukaryotic ldh genes. Protein sequencing of the LDH isolated from R. oryzae during lactic acid production confirmed that ldhA codes for a 36-kDa protein that converts pyruvate to lactate. Production of LdhA was greatest when glucose was the carbon source, followed by xylose and trehalose; all of these sugars could be fermented to lactic acid. Transcripts from ldhB were not detected when R. oryzae was grown on any of these sugars but were present when R. oryzae was grown on glycerol, ethanol, and lactate. I hypothesize that ldhB encodes a second NAD(+)-dependent LDH that is capable of converting L-lactate to pyruvate and is produced by cultures grown on these nonfermentable substrates. Both ldhA and ldhB restored fermentative growth to Escherichia coli (ldhA pfl) mutants so that they grew anaerobically and produced lactic acid.
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7. |
Burmester A,
Richter M,
Schultze K,
Voelz K,
Schachtschabel D,
Boland W,
Wöstemeyer J,
Schimek C,
( 2007 ) Cleavage of beta-carotene as the first step in sexual hormone synthesis in zygomycetes is mediated by a trisporic acid regulated beta-carotene oxygenase. PMID : 17822929 : DOI : 10.1016/j.fgb.2007.07.008 Abstract >>
Carotene cleavage is the necessary initial step in the biosynthesis of trisporic acid, the sexual signal in zygomycete fungi. Two genes encoding putative carotene oxygenases, designated tsp3 and tsp4, were identified in the genome of the zygomycete Rhizopus oryzae. Using heterologous primers, tsp3 was cloned and sequenced also from Blakeslea trispora. tsp3 transcription correlates with sexual development in both species. Northern hybridization of B. trispora mRNA revealed strong induction of tsp3 transcription in mated cultures. A very strong and direct transient induction of transcription by trisporic acid was proven by quantitative real-time PCR analysis. In R. oryzae, transcriptional induction is also inducible by stimulation with trisporoids and depends on the developmental stage of the mycelium. The functionality of the tsp3 gene product as carotene cleavage enzyme was shown as loss of carotene in an Escherichia coli strain transformed to carotene production and tsp3 expression.
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8. |
DeMarco R,
Venancio TM,
Verjovski-Almeida S,
( 2006 ) SmTRC1, a novel Schistosoma mansoni DNA transposon, discloses new families of animal and fungi transposons belonging to the CACTA superfamily. PMID : 17090310 : DOI : 10.1186/1471-2148-6-89 PMC : PMC1636069 Abstract >>
The CACTA (also called En/Spm) superfamily of DNA-only transposons contain the core sequence CACTA in their Terminal Inverted Repeats (TIRs) and so far have only been described in plants. Large transcriptome and genome sequence data have recently become publicly available for Schistosoma mansoni, a digenetic blood fluke that is a major causative agent of schistosomiasis in humans, and have provided a comprehensive repository for the discovery of novel genes and repetitive elements. Despite the extensive description of retroelements in S. mansoni, just a single DNA-only transposon belonging to the Merlin family has so far been reported in this organism. We describe a novel S. mansoni transposon named SmTRC1, for S. mansoni Transposon Related to CACTA 1, an element that shares several characteristics with plant CACTA transposons. Southern blotting indicates approximately 30-300 copies of SmTRC1 in the S. mansoni genome. Using genomic PCR followed by cloning and sequencing, we amplified and characterized a full-length and a truncated copy of this element. RT-PCR using S. mansoni mRNA followed by cloning and sequencing revealed several alternatively spliced transcripts of this transposon, resulting in distinct ORFs coding for different proteins. Interestingly, a survey of complete genomes from animals and fungi revealed several other novel TRC elements, indicating new families of DNA transposons belonging to the CACTA superfamily that have not previously been reported in these kingdoms. The first three bases in the S. mansoni TIR are CCC and they are identical to those in the TIRs of the insects Aedes aegypti and Tribolium castaneum, suggesting that animal TRCs may display a CCC core sequence. The DNA-only transposable element SmTRC1 from S. mansoni exhibits various characteristics, such as generation of multiple alternatively-spliced transcripts, the presence of terminal inverted repeats at the extremities of the elements flanked by direct repeats and the presence of a Transposase_21 domain, that suggest a distant relationship to CACTA transposons from Magnoliophyta. Several sequences from other Metazoa and Fungi code for proteins similar to those encoded by SmTRC1, suggesting that such elements have a common ancestry, and indicating inheritance through vertical transmission before separation of the Eumetazoa, Fungi and Plants.
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9. |
Watkins RF,
Gray MW,
( 2006 ) The frequency of eubacterium-to-eukaryote lateral gene transfers shows significant cross-taxa variation within amoebozoa. PMID : 17086451 : DOI : 10.1007/s00239-006-0031-0 Abstract >>
Single-celled bacterivorous eukaryotes offer excellent test cases for evaluation of the frequency of prey-to-predator lateral gene transfer (LGT). Here we use analysis of expressed sequence tag (EST) data sets to quantify the extent of LGT from eubacteria to two amoebae, Acanthamoeba castellanii and Hartmannella vermiformis. Stringent screening for LGT proceeded in several steps intended to enrich for authentic events while at the same time minimizing the incidence of false positives due to factors such as limitations in database coverage and ancient paralogy. The results were compared with data obtained when the same methodology was applied to EST libraries from a number of other eukaryotic taxa. Significant differences in the extent of apparent eubacterium-to-eukaryote LGT were found between taxa. Our results indicate that there may be substantial inter-taxon variation in the number of LGT events that become fixed even between amoebozoan species that have similar feeding modalities.
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10. |
Mertens JA,
Skory CD,
Ibrahim AS,
( 2006 ) Plasmids for expression of heterologous proteins in Rhizopus oryzae. PMID : 16804680 : DOI : 10.1007/s00203-006-0121-9 Abstract >>
Rhizopus oryzae has long been used for enzyme production (e.g., glucoamylase and lipase), organic acid synthesis, and various fermented food applications. In this work, we describe a set of plasmid-based expression vectors that can be used for the production of heterologous proteins in R. oryzae. Three plasmid vectors have been created using either the glucoamylase A (amyA), pyruvate decarboxylase (pdcA), or phosphoglycerate kinase (pgk1) promoters to drive expression of heterologous proteins. All three plasmids use the pdcA terminator for transcription termination, the pyrG gene for restoration of uracil prototrophy, and an ampicillin resistance gene and origin of replication for maintenance in Escherichia coli. We have expressed green fluorescent protein (GFP) and compared transcription and protein accumulation for each of the expression vectors. Accumulation of GFP transcript and protein was directly correlated with the choice of promoter with pdcA > amyA > pgk1. Transcript level appears to parallel GFP protein accumulation. Plasmid copy number had little impact on transcription or protein accumulation. These vectors should be useful for overexpression of heterologous proteins and potentially, metabolic engineering of Rhizopus strains.
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11. |
Salah RB,
Mosbah H,
Fendri A,
Gargouri A,
Gargouri Y,
Mejdoub H,
( 2006 ) Biochemical and molecular characterization of a lipase produced by Rhizopus oryzae. PMID : 16842350 : DOI : 10.1111/j.1574-6968.2006.00323.x Abstract >>
A novel strain of Rhizopus oryzae WPG secretes a noninduced lipase (ROLw) in the culture medium; purified ROLw is a protein of 29 kDa, the 45 N-terminal amino acid residues were sequenced, this sequence is very homologous to Rhizopus delemar lipase (RDL), Rhizopus niveus lipase (RNL) and R. oryzae lipase (ROL29) sequences; the cloning and sequencing of the part of the gene encoding the mature ROLw, shows two nucleotides differences with RDL, RNL and ROL29 sequences corresponding to the change of the residues 134 and 200; ROLw does not present the interfacial activation phenomenon when using tripropionin or vinyl propionate as substrates; the lipase activity is maximal at pH 8 and at 37 degrees C, specific activities of 3500 or 900 U mg(-1) were measured at 37 degrees C and at pH 8, using olive oil emulsion or tributyrin as substrates, respectively; ROLw is unable to hydrolyse triacylglycerols in the presence of high concentration of bile salts; it is a serine enzyme as it is inhibited by tetrahydrolipstatin and was stable between pH 5 and pH 8.
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12. |
Niu WN,
Li ZP,
Tan T,
( 2006 ) Secretion of pro- and mature Rhizopus arrhizus lipases by Pichia pastoris and properties of the proteins. PMID : 16382184 : DOI : 10.1385/MB:32:1:073 Abstract >>
The lipases of Rhizopus spp. share a high 1,3-regiospecificity toward triacylglycerols, which makes them important enzymes in lipid modification. In the present study, the extracellularly active production of recombinant Rhizopus arrhizuslipase was carried out with genes encoding the mature region (mRAL) and the mRAL having the prosequence (ProRAL) in Pichia pastoris. Two transformed P. pastoris clones containing the multicopy of mRAL and ProRAL genes were separately selected for the production of recombinant enzymes. In a fed-batch cultivation, where methanol feeding was controlled by an on-line methanol analyzer, the supernatant contained 91 mg/L recombinant pro-form lipase (rProRAL) and 80 mg/L recombinant mature lipase (rRAL) after 92 h of cultivation. rProRAL and rRAL were purified by ultrafiltration, SP-Sepharose Fast Flow chromatography, and Butyl-Sepharose Fast Flow chromatography. Molecular weights of rProRAL and rRAL are 32 kDa and 29 kDa, respectively. The amino-terminal analysis showed that the 32-kDa protein was mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL). The specific lipase activities of mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL) and rRAL were 1543 U/mg and 2437 U/mg. The rPro28RAL was more stable than rRAL at pH 4.0-7.0, whereas rRAL was more stable at pH 7.0-10.0. The rPro28RAL had the highest lipase activity toward tributyrin (C4), whereas rRAL had the highest lipase activity toward tricaprylin (C8).
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13. |
Yoshida S,
Suzuki F,
Tsukiboshi T,
Shinohara H,
( 2004 ) Cloning and characterization of a gene rpg1 encoding polygalacturonase of Rhizopus oryzae. PMID : 15757176 : Abstract >>
The polygalacturonase (PG)-encoding gene (rpg1) of Rhizopus oryzae, the causal pathogen of rhizopus rot of mulberry, was cloned and sequenced. PGs were partially purified from incubation mixture of 2% pectin medium and their N-terminal amino acid sequences were determined by a gas-phase protein sequencer. RT-PCR was performed using degenerate primers designed from the amino acid sequences, which resulted in part of a PG-encoding gene being obtained. By 3'-RACE and TAIL-PCR analyses, the entire region of the PG-encoding gene was cloned and sequenced. The structural gene comprised 1199 bp coding for 383 amino acids with a putative signal peptide of 26 amino acids, and the open reading frame was interrupted by single intron of 47 bp. Phylogenetic analysis using the deduced amino acid sequence revealed that R. oryzae RPG1 belonged to a clade consisting of exo-PGs of ascomycete fungi.
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14. |
Sayari A,
Frikha F,
Miled N,
Mtibaa H,
Ben Ali Y,
Verger R,
Gargouri Y,
( 2005 ) N-terminal peptide of Rhizopus oryzae lipase is important for its catalytic properties. PMID : 15710378 : DOI : 10.1016/j.febslet.2004.12.068 Abstract >>
In a culture medium, the Rhizopus oryzae strain produces only one form of lipase, ROL32. When the concentrated culture medium was stored at 0 degrees C during several months or kept at 6 degrees C during a few days, we noticed the appearance of a second shorter form of ROL32 lacking its N-terminal 28 amino acid (ROL29). ROL29 was purified to homogeneity and its 21 N-terminal amino acid residues were found to be identical to the 29-49 sequence of ROL32. The cleavage of the N-terminal peptide reduced the specific activity of ROL29 by 50% using either triolein or tributyrin as substrates. In order to explain this decrease of the specific activity of ROL29, we measured its critical surface pressure of penetration into phosphatidyl choline from egg yolk films which was found to be 10 mN/m, in contrast to a value of 23 mN/m found in ROL32. A kinetic study on the surface pressure dependency, stereoselectivity and regioselectivity of ROL29 was performed using the three dicaprin isomers spread as monomolecular films at the air-water interface. Our results showed that in contrast to ROL32, ROL29 presented a preference for the distal ester groups of one diglyceride isomer (1,3-sn-dicaprin). Furthermore, ROL32 was markedly more stereoselective than ROL29 for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. A structural explanation of the enhanced penetration capacity as well as the catalytic activity of ROL32 was proposed by molecular modeling. We concluded that the N-terminal peptide of ROL32 can play an important role in the specific activity, the regioselectivity, the stereoselectivity and the binding of the enzyme to its substrate.
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15. |
Seif E,
Leigh J,
Liu Y,
Roewer I,
Forget L,
Lang BF,
( 2005 ) Comparative mitochondrial genomics in zygomycetes: bacteria-like RNase P RNAs, mobile elements and a close source of the group I intron invasion in angiosperms. PMID : 15689432 : DOI : 10.1093/nar/gki199 PMC : PMC548346 Abstract >>
To generate data for comparative analyses of zygomycete mitochondrial gene expression, we sequenced mtDNAs of three distantly related zygomycetes, Rhizopus oryzae, Mortierella verticillata and Smittium culisetae. They all contain the standard fungal mitochondrial gene set, plus rnpB, the gene encoding the RNA subunit of the mitochondrial RNase P (mtP-RNA) and rps3, encoding ribosomal protein S3 (the latter lacking in R.oryzae). The mtP-RNAs of R.oryzae and of additional zygomycete relatives have the most eubacteria-like RNA structures among fungi. Precise mapping of the 5' and 3' termini of the R.oryzae and M.verticillata mtP-RNAs confirms their expression and processing at the exact sites predicted by secondary structure modeling. The 3' RNA processing of zygomycete mitochondrial mRNAs, SSU-rRNA and mtP-RNA occurs at the C-rich sequence motifs similar to those identified in fission yeast and basidiomycete mtDNAs. The C-rich motifs are included in the mature transcripts, and are likely generated by exonucleolytic trimming of RNA 3' termini. Zygomycete mtDNAs feature a variety of insertion elements: (i) mtDNAs of R.oryzae and M.verticillata were subject to invasions by double hairpin elements; (ii) genes of all three species contain numerous mobile group I introns, including one that is closest to an intron that invaded angiosperm mtDNAs; and (iii) at least one additional case of a mobile element, characterized by a homing endonuclease insertion between partially duplicated genes [Paquin,B., Laforest,M.J., Forget,L., Roewer,I., Wang,Z., Longcore,J. and Lang,B.F. (1997) Curr. Genet., 31, 380-395]. The combined mtDNA-encoded proteins contain insufficient phylogenetic signal to demonstrate monophyly of zygomycetes.
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16. |
Wei D,
Li M,
Zhang X,
Ren Y,
Xing L,
( 2004 ) Identification and characterization of a novel delta12-fatty acid desaturase gene from Rhizopus arrhizus. PMID : 15327973 : DOI : 10.1016/j.febslet.2004.06.100 Abstract >>
Based on the sequence information of Delta12-fatty acid desaturase genes (from Mucor circinelloides, Mortierella alpina, Mucor rouxii and Aspergillus nidulans), which were involved in the conversion from C18:1 to C18:2, a cDNA sequence putatively encoding a Delta12-fatty acid desaturase was isolated from Rhizopus arrhizus using the combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. Sequence analysis indicated that it had an open reading frame (ORF) of 1170 bp, coding for 389 amino acid residues of 45 kDa, pI of the deduced protein was 7.01. The deduced amino acid sequence of this cloned cDNA showed high identity to those filamentous fungal Delta12-desaturases mentioned above, including three conserved histidine-rich motifs and two hydrophobic domains. Functional identification was done heterologously in Saccharomyces cerevisiae strain INVScl. The result demonstrated that the deduced amino acid sequence exhibited Delta12-fatty acid desaturase activity, suggesting that this gene encoded for a membrane-bound desaturase, Delta12-fatty acid desaturase.
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17. |
Saito K,
Saito A,
Ohnishi M,
Oda Y,
( 2004 ) Genetic diversity in Rhizopus oryzae strains as revealed by the sequence of lactate dehydrogenase genes. PMID : 15278242 : DOI : 10.1007/s00203-004-0691-3 Abstract >>
Twenty-seven strains of Rhizopus oryzae accumulating predominantly lactic acid were shown to possess two ldh genes, ldhA and ldhB, encoding NAD-dependent lactate dehydrogenases. Variation in nucleotide sequence was identified for each gene from different strains, and similar phylogenetic trees were obtained based on the nucleotide sequences of both genes. The other 21 strains of R. oryzae accumulating predominantly fumaric and malic acids contained a single ORF of ldhB. Compared to the strains accumulating predominantly lactic acid, a lower degree of sequence divergence was found in ldhB, resulting in a separate cluster in the phylogenetic tree. The high similarity (>90%) spanning the ORF and adjacent regions demonstrates that ldhA and ldhB are derived from the same ancestor gene. The strains accumulating predominantly fumaric and malic acids lack functional ldhA, which plays a role in lactic acid synthesis and may form a lineage separated from the strains accumulating predominantly lactic acid in the genus Rhizopus.
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18. |
Song P,
Li S,
Ding Y,
Xu Q,
Huang H,
( 2011 ) Expression and characterization of fumarase (FUMR) from Rhizopus oryzae. PMID : 21215954 : DOI : 10.1016/j.funbio.2010.10.003 Abstract >>
Fumarase catalyzes the reversible hydration of fumarate to l-malate in Rhizopus oryzae. A recombinant pET22b-fumR harboring a fumarase gene (fumR) from R. oryzae was constructed for high level expression in E. coli BL21 (DE3). The FUMR activity was optimal at 30�XC and pH 7.2. The enzyme was stable below 45�XC and at pH 3.0-9.0. No effects of Zn(2+), Fe(2+), or EDTA were observed on enzyme activity. A slight inhibition of FUMR activity was seen with Mg(2+), while Ca(2+) had a small stimulatory effect. The K(m) for l-malic acid and fumaric acid were 0.46 mM and 3.07 mM, respectively. The activity of FUMR catalyzing hydration of fumarate to l-malate was completely inhibited by 2mM fumaric acid. The unique enzymatic properties suggested that overexpression of FUMR could enhance fumaric acid accumulation in R. oryzae.
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19. |
Li S,
Zuo Z,
Niu D,
Singh S,
Permaul K,
Prior BA,
Shi G,
Wang Z,
( 2011 ) Gene cloning, heterologous expression, and characterization of a high maltose-producing �\-amylase of Rhizopus oryzae. PMID : 21243443 : DOI : 10.1007/s12010-011-9159-5 Abstract >>
A putative �\-amylase gene, designated as RoAmy, was cloned from Rhizopus oryzae. The deduced amino acid sequence showed the highest (42.8%) similarity to the �\-amylase from Trichoderma viride. The RoAmy gene was successfully expressed in Pichia pastoris GS115 under the induction of methanol. The molecular weight of the purified RoAmy determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was approximately 48 kDa. The optimal pH and temperature were 4-6 and 60 �XC, respectively. The enzyme was stable at pH ranges of 4.5-6.5 and temperatures below 50 �XC. Purified RoAmy had a K(m) and V(max) of 0.27 mg/ml and 0.068 mg/min, respectively, with a specific activity of 1,123 U/mg on soluble starch. Amylase activity was strongly inhibited by 5 mM Cu(2+) and 5 mM Fe(2+), whereas 5 mM Ca(2+) showed no significant effect. The RoAmy hydrolytic activity was the highest on wheat starch but showed only 55% activity on amylopectin relative to soluble corn starch, while the pullulanase activity was negligible. The main end products of the polysaccharides tested were glucose and maltose. Maltose reached a concentration of 74% (w/w) with potato starch as the substrate. The enzyme had an extremely high affinity (K(m) = 0.22 mM) to maltotriose. A high ratio of glucose/maltose of 1:4 was obtained when maltotriose was used at an initial concentration of 40 mM.
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20. |
Gryganskyi AP,
Lee SC,
Litvintseva AP,
Smith ME,
Bonito G,
Porter TM,
Anishchenko IM,
Heitman J,
Vilgalys R,
( 2010 ) Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex. PMID : 21151560 : DOI : 10.1371/journal.pone.0015273 PMC : PMC3000332 Abstract >>
The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (-) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species.
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21. |
Abe A,
Asano K,
Sone T,
( 2009 ) Identification and characterization of Rhizot, a novel LTR retrotransposon of Rhizopus oryzae and R. delemar. PMID : 19661716 : DOI : 10.1271/bbb.90017 Abstract >>
A novel retrotransposon Rhizot was identified in Rhizopus oryzae and R. delemar. Rhizot has a unique structure that consists of a pol ORF similar to non-LTR (long terminal repeat) retorotransposons between two LTRs. Rhizot was distributed in all Rhizopus species tested. The Rhizot pol gene was transcribed in the liquid culture, and was induced by UV and oxidative stress.
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22. |
Chen CC,
Cho YC,
Lai CC,
Hsu WH,
( 2009 ) Purification and characterization of a new Rhizopuspepsin from Rhizopus oryzae NBRC 4749. PMID : 19722576 : DOI : 10.1021/jf8040337 Abstract >>
A secretory aspartic protease (also termed as rhizopuspepsin) was purified from Rhizopus oryzae NBRC 4749 by ion exchange chromatography with a yield of 45%. The enzyme was a nonglycoprotein with a molecular mass of 37 kDa as determined by SDS-PAGE analysis. N-terminal sequence and LC-MS/MS analyses revealed that this rhizopuspepsin corresponded to the hypothetical protein RO3G_12822.1 in the R. oryzae genome database. Comparison of genomic and cDNA genes demonstrated that the rhizopuspepsin contained two introns, whereas only one intron was reported in other rhizopuspepsin genes. Phylogenetic analysis also indicated that this rhizopuspepsin was distinct from other rhizopuspepsins. The temperature and pH optima for the purified rhizopuspepsin were 50 degrees C and pH 3.0, respectively, and a half-life of about 3.5 h was observed at 40 degrees C. The enzyme preferentially cleaved the peptides with hydrophobic and basic amino acids in the P1 site but had no activity for the Glu, Pro, Trp, and aliphatic amino acids containing the beta-branch side chain.
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23. |
Tung JY,
Chang MD,
Chou WI,
Liu YY,
Yeh YH,
Chang FY,
Lin SC,
Qiu ZL,
Sun YJ,
( 2008 ) Crystal structures of the starch-binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide-binding path. PMID : 18588504 : DOI : 10.1042/BJ20080580 Abstract >>
GA (glucoamylase) hydrolyses starch and polysaccharides to beta-D-glucose. RoGA (Rhizopus oryzae GA) consists of two functional domains, an N-terminal SBD (starch-binding domain) and a C-terminal catalytic domain, which are connected by an O-glycosylated linker. In the present study, the crystal structures of the SBD from RoGA (RoGACBM21) and the complexes with beta-cyclodextrin (SBD-betaCD) and maltoheptaose (SBD-G7) were determined. Two carbohydrate binding sites, I (Trp(47)) and II (Tyr(32)), were resolved and their binding was co-operative. Besides the hydrophobic interaction, two unique polyN loops comprising consecutive asparagine residues also participate in the sugar binding. A conformational change in Tyr(32) was observed between unliganded and liganded SBDs. To elucidate the mechanism of polysaccharide binding, a number of mutants were constructed and characterized by a quantitative binding isotherm and Scatchard analysis. A possible binding path for long-chain polysaccharides in RoGACBM21 was proposed.
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24. |
Xiao Z,
Wang S,
Bergeron H,
Zhang J,
Lau PC,
( 2008 ) A flax-retting endopolygalacturonase-encoding gene from Rhizopus oryzae. PMID : 18704748 : DOI : 10.1007/s10482-008-9274-7 Abstract >>
A polygalacturonase from the filamentous fungus Rhizopus oryzae strain sb (NRRL 29086), previously shown to be effective in the retting of flax fibers, was shown by the analysis of its reaction products on polygalacturonic acid to be an endo-type. By zymogram analysis, the enzyme in the crude culture filtrate appeared as two active species of 37 and 40 kD. The endopolygalacturonase-encoding gene was cloned in Escherichia coli and its translated 383-amino acid sequence found to be identical to that of a presumed exopolygalacturonase found in R. oryzae strain YM9901 and 96% identical to a hypothetical protein (RO3G_04731.1) in the sequenced genome of R. oryzae strain 99-880. Phylogenetic analysis revealed the presence of an unique cluster of Rhizopus polygalacturonase sequences that are separate from other fungal polygalacturonases. Conservation of 12 cysteines appears to be a special feature of this family of Rhizopus polygalacturonase sequences.
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25. |
Schmitt I,
Partida-Martinez LP,
Winkler R,
Voigt K,
Einax E,
Dölz F,
Telle S,
Wöstemeyer J,
Hertweck C,
( 2008 ) Evolution of host resistance in a toxin-producing bacterial-fungal alliance. PMID : 18309361 : DOI : 10.1038/ismej.2008.19 Abstract >>
The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for the production of the virulence factor, the antimitotic agent rhizoxin. Since the toxin highly efficiently blocks mitosis in most eukaryotes, it remained elusive how self-resistance emerged in the fungal host. In this study, rhizoxin sensitivity was systematically correlated with the nature of beta-tubulin sequences in the kingdom Fungi. A total of 49 new beta-tubulin sequences were generated for representative species of Ascomycota, Basidiomycota and Zygomycota. Rhizoxin sensitivity assays revealed two further amino acids at position 100 (Ser-100 and Ala-100), in addition to the known Ile-100 and Val-100, which convey rhizoxin resistance. All sensitive strains feature Asn-100. This hot spot was verified by modeling studies, which support the finding that rhizoxin preferentially interacts with the tubulin molecule in a cavity near position 100. Ancestral character state reconstructions conducted in a Bayesian framework suggest that rhizoxin sensitivity represents the ancestral character state in fungi, and that evolution of rhizoxin resistance took place in the ancestor of extant resistant Zygomycota. These findings support a model according to which endosymbiosis became possible through a parasitism--mutualism shift in insensitive fungi.
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26. |
Nyilasi I,
Papp T,
Csernetics A,
Krizsán K,
Nagy E,
Vágvölgyi C,
( 2008 ) High-affinity iron permease (FTR1) gene sequence-based molecular identification of clinically important Zygomycetes. PMID : 18190575 : DOI : 10.1111/j.1469-0691.2007.01932.x Abstract >>
The clinical importance of zygomycosis, an emerging and frequently fatal mycotic disease, has increased during recent years. This report describes an identification method based on PCR amplification and sequencing of the high-affinity iron permease 1 gene (FTR1). Primers and amplification protocols were established and tested for the identification of Rhizopus oryzae, Rhizopus microsporus var. rhizopodiformis, R. microsporus var. oligosporus, Rhizopus schipperae, Rhizopus niveus and Rhizopus stolonifer. Rhizomucor and Syncephalastrum could be identified at the genus level. PCR-restriction fragment length polymorphism analysis of the amplified gene fragment using AluI digestion distinguished three subgroups among the R. oryzae isolates.
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27. |
Abe A,
Oda Y,
Asano K,
Sone T,
( N/A ) Rhizopus delemar is the proper name for Rhizopus oryzae fumaric-malic acid producers. PMID : 18268905 : Abstract >>
The zygomycete Rhizopus oryzae currently is identified by sporangiophore morphology and growth temperature, but heterogeneity of the species has been reported. We examined the suitability of organic acid production as an effective taxonomic character for reclassification of the species. Strains were divided into two groups, LA (lactic acid producer) and FMA (fumaric-malic acid producers) according to organic acid production. These grouping were confirmed as phylogenetically distinct because analyses of rDNA ITS, lactate dehydrogenase B, actin, translation elongation factor-1alpha and genomewide AFLP resolved the same two exclusive clusters, corresponding with the organic acid grouping. Reclassification of strains in the FMA group as R. delemar was proposed.
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28. |
Haas MJ,
Allen J,
Berka TR,
( 1991 ) Cloning, expression and characterization of a cDNA encoding a lipase from Rhizopus delemar. PMID : 1756969 : DOI : 10.1016/0378-1119(91)90594-2 Abstract >>
A lambda gt11 cDNA library was constructed in Escherichia coli using poly(A)-selected mRNA from the fungus, Rhizopus (Rp.) delemar. Lipase-producing members of the library were identified by means of a phenotypic score wherein the release of fatty acids by lipase causes a characteristic color change in the growth medium. One such isolate contained a 1287-bp insert (LIP cDNA) which hybridizes to 1.25- to 1.35-kb mRNA species from Rp. delemar. The lipase produced in E. coli containing the LIP cDNA exhibits the same substrate selectivity as the authentic fungal enzyme, hydrolyzing ester bonds at the stereospecific numbering (sn) sn-1 and sn-3, but not the sn-2, positions of triglycerides. The complete nucleotide sequence of the LIP cDNA was determined. By reference to the N-terminal sequence of authentic Rp. delemar lipase, the lipase-encoding region was identified within this fragment. The LIP cDNA encodes a putative preprolipase consisting of a 26-amino-acid(aa) signal sequence, a 97-aa propeptide, and a 269-aa mature enzyme. The predicted mature lipase has the same molecular weight and aa composition as that of Rp. delemar, is highly homologous to that produced by the fungus Rhizomucor miehei, and contains the consensus pentapeptide (Gly-Xaa-Ser-Yaa-Gly) which is conserved among lipolytic enzymes. It is concluded that the LIP cDNA is an essentially full-length analogue of the lipase-encoding gene of Rp. delemar. The lipase encoded by the LIP cDNA occupies a cytoplasmic location when synthesized in E. coli. Unprocessed forms of the lipase accumulate in E. coli.
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29. |
Yang L,
Dai X,
Hou J,
Ma C,
Wang C,
Wu Z,
Li M,
( 2008 ) Construction of a Rhizopus arrhizus glucoamylase gene suitable for expression in distinct host: introns spliced artificially by PCR. PMID : 17578683 : DOI : 10.1007/s11033-006-9045-3 Abstract >>
Glucoamylase is an industrially extremely important enzyme in the fermentative production of ethanol, used in the enzymatic conversion of starch into high glucose and fructose syrups. The aim of this study is to construct a Rhizopus arrhizus glucoamylase gene (RaGA)-introns artificially spliced by PCR-suitable for expression in S. cerevisiae host and tried expressing in Picha pastoris. In previous work, we failed in amplifying glucoamylase gene from R. arrhizus by RT-PCR, so several primers were designed to splicing the introns by PCR in vitro. Sequence analysis shown that all introns in the RaGA were deleted correctly and no mutant was induced in the extrons compared with the RaGA gene originally cloned. The RaGA gene artificially constructed was transferred into P. pastoris integrative expression vectors pPIC9 (containing small a, Cyrillic-factor). Consequently, the plasmids pPIC9-RaGA was lineared by SacI and inserted into P. pastoris GS115 (His-) genome downstream of the 5'AOX1 promoter by the method of electroporation. Induction by 0.75% methanol for 72 h led to synthesis of secreted glucoamylase. So it is demonstrated that the glucoamylase gene has been expressed in and secreted from P. pastoris.
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30. |
Xiao Z,
Grosse S,
Bergeron H,
Lau PC,
( 2014 ) Cloning and characterization of the first GH10 and GH11 xylanases from Rhizopus oryzae. PMID : 24760228 : DOI : 10.1007/s00253-014-5741-4 Abstract >>
The only available genome sequence for Rhizopus oryzae strain 99-880 was annotated to not encode any �]-1,4-endoxylanase encoding genes of the glycoside hydrolase (GH) family 10 or 11. Here, we report the identification and cloning of two such members in R. oryzae strain NRRL 29086. Strain 29086 was one of several selected fungi grown on wheat or triticale bran and screened for xylanase activity among other hydrolytic actions. Its high activity (138 U/ml) in the culture supernatant led to the identification of two activity-stained proteins, designated Xyn-1 and Xyn-2 of respective molecular masses 32,000 and 22,000. These proteins were purified to electrophoretic homogeneity and characterized. The specific activities of Xyn-1 and Xyn-2 towards birchwood xylan were 605 and 7,710 U/mg, respectively. Kinetic data showed that the lower molecular weight Xyn-2 had a higher affinity (K m=3.2 �� 0.2 g/l) towards birchwood xylan than Xyn-1 by about 4-fold. The melting temperature (T m) of the two proteins, estimated to be in the range of 49.5-53.7 �XC indicated that they are rather thermostable proteins. N-terminal and internal peptide sequences were obtained by chemical digestion of the purified xylanases to facilitate cloning, expression in Escherichia coli, and sequencing of the respective gene. The cloned Rhizopus xylanases were used to demonstrate release of xylose from flax shives-derived hemicellulose as model feedstock. Overall, this study expands the catalytic toolbox of GH10 and 11 family proteins that have applications in various industrial and bioproducts settings.
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31. |
Yuzbashev TV,
Larina AS,
Vybornaya TV,
Yuzbasheva EY,
Gvilava IT,
Sineoky SP,
( 2015 ) Repetitive genomic sequences as a substrate for homologous integration in the Rhizopus oryzae genome. PMID : 25986546 : DOI : 10.1016/j.funbio.2015.02.001 Abstract >>
The vast number of repetitive genomic elements was identified in the genome of Rhizopus oryzae. Such genomic repeats can be used as homologous regions for integration of plasmids. Here, we evaluated the use of two different repeats: the short (575 bp) rptZ, widely distributed (about 34 copies per genome) and the long (2053 bp) rptH, less prevalent (about 15 copies). The plasmid carrying rptZ integrated, but did so through a 2256-bp region of homology to the pyrG locus, a unique genomic sequence. Thus, the length of rptZ was below the minimal requirements for homologous strand exchange in this fungus. In contrast, rptH was used efficiently for homologous integration. The plasmid bearing this repeat integrated in multicopy fashion, with up to 25 copies arranged in tandem. The latter vector, pPyrG-H, could be a valuable tool for integration at homologous sequences, for such purposes as high-level expression of proteins.
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32. |
Dolatabadi S,
de Hoog GS,
Meis JF,
Walther G,
( 2014 ) Species boundaries and nomenclature of Rhizopus arrhizus (syn. R. oryzae). PMID : 25266947 : DOI : 10.1111/myc.12228 Abstract >>
Rhizopus arrhizus (Mucorales, Mucoromycotina) is the prevalent opportunist worldwide among the mucoralean species causing human infections. On the other hand the species has been used since ancient times to ferment African and Asian traditional foods and condiments based on ground soybeans. As producer of organic acids and hydrolytic enzymes it is widely applied in food industry and biotechnology. Using a set of 82 strains we studied phylogenetic and biological species boundaries within Rhizopus arrhizus s.l. to test the taxonomic status of R. delemar that was recently separated from R. arrhizus. Sequence analyses based on the internal transcribed spacer region, the gene of the largest subunit of the RNA polymerase II, a part of the actin gene, and the translation elongation factor 1-�\ as well as amplified fragment length polymorphism analysis were performed. Phenotypic characters such as enzyme profiles and growth kinetics were examined and the mating behavior was tested. Molecular analyses supported the existence of two phylogenetic species. However, the results of the mating test suggest that the mating barrier is still not complete. No physiological, ecological or epidemiological distinction could be found beside the difference in the production of organic acids. Consequently the status of varieties is proposed for the two phylogenetic species. Because the description of the first described R. arrhizus is considered to be conclusive we recommend the use of Rhizopus arrhizus var. arrhizus and var. delemar.
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33. |
Chu CH,
Li KM,
Lin SW,
Chang MD,
Jiang TY,
Sun YJ,
( 2014 ) Crystal structures of starch binding domain from Rhizopus oryzae glucoamylase in complex with isomaltooligosaccharide: insights into polysaccharide binding mechanism of CBM21 family. PMID : 24108499 : DOI : 10.1002/prot.24446 Abstract >>
Glucoamylases are responsible for hydrolysis of starch and polysaccharides to yield �]-D-glucose. Rhizopus oryzae glucoamylase (RoGA) is composed of an N-terminal starch binding domain (SBD) and a C-terminal catalytic domain connected by an O-glycosylated linker. Two carbohydrate binding sites in RoSBD have been identified, site I is created by three highly conserved aromatic residues, Trp47, Tyr83, and Tyr94, and site II is built up by Tyr32 and Phe58. Here, the two crystal structures of RoSBD in complex with only �\-(1,6)-linked isomaltotriose (RoSBD-isoG3) and isomaltotetraose (RoSBD-isoG4) have been determined at 1.2 and 1.3 ?, respectively. Interestingly, site II binding is observed in both complexes, while site I binding is only found in the RoSBD-isoG4 complex. Hence, site II acts as the recognition binding site for carbohydrate and site I accommodates site II to bind isoG4. Site I participates in sugar binding only when the number of glucosyl units of oligosaccharides is more than three. Taken together, two carbohydrate binding sites in RoSBD cooperate to reinforce binding mode of glucoamylase with polysaccharides as well as the starch.
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34. |
Schoch CL,
Seifert KA,
Huhndorf S,
Robert V,
Spouge JL,
Levesque CA,
Chen W,
N/A N/A,
N/A N/A,
( 2012 ) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. PMID : 22454494 : DOI : 10.1073/pnas.1117018109 PMC : PMC3341068 Abstract >>
Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.
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35. |
( 1996 ) Analysis of the catalytic mechanism of a fungal lipase using computer-aided design and structural mutants. PMID : 8862551 : Abstract >>
Both an active enzyme conformation and stabilization of tetrahedral transition states are essential for the catalysis of ester bond hydrolysis by lipases. X-ray structural data and results from site-directed mutagenesis experiments with proteases have been used as a basis for predictions of amino acid residues likely to have key functions in lipases. The gene encoding a lipase from Rhizopus oryzae was cloned and expressed in Escherichia coli. Site-directed mutagenesis of this gene was used to test the validity of computer-aided predictions of the functional roles of specific amino acids in this enzyme. Examination of the kinetic constants of the Rhizopus oryzae lipase variants allowed us to identify amino acid residues which are directly involved in the catalytic reaction or which stabilize the active geometry of the enzyme. The combination of these results with molecular mechanics simulations, based on a homology-derived structural model, provided new information about structure-function relationships. The interpretation of the data is consistent with results obtained with other hydrolases, such as proteases.
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36. |
( 1996 ) The folding and activity of the extracellular lipase of Rhizopus oryzae are modulated by a prosequence. PMID : 8912667 : DOI : 10.1042/bj3190351 PMC : PMC1217776 Abstract >>
The fungus Rhizopus oryzae synthesizes an extracellular lipase precursor bearing N-terminal pre- and pro-sequences. Our studies in Escherichia coli and using recombinant lipase in vitro indicate that the prosequence of 97 amino acids has at least two functions. First, it modulates the enzyme activity of the lipase so that this enzyme can initially be synthesized in a non-destructive form. Direct synthesis of the mature form of the lipase in the cell has toxic consequences, at least partly because of phospholipase activity that is suppressed in the proprotein. Secondly, it supports folding of the lipase via a pathway influenced by a single cysteine residue at position - 68. Mutational analysis of the prosequence demonstrates not only the key role of this cysteine residue but also the importance of the neighbouring amino acids. In particular, Arg-69 probably enhances the leaving group character of Cys-68. We propose a model in which Cys-68 acts as an intramolecular thiodisulphide reagent, playing a catalytic role in the folding of the enzyme. The prosequence is capable of performing the described functions both in cis and in trans.
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37. |
( 1994 ) Conformational lability of lipases observed in the absence of an oil-water interface: crystallographic studies of enzymes from the fungi Humicola lanuginosa and Rhizopus delemar. PMID : 8014587 : Abstract >>
Considerable controversy exists regarding the exact nature of the molecular mechanism of interfacial activation, a process by which most lipases achieve maximum catalytic activity upon adsorption to an oil water interface. X-ray crystallographic studies show that lipases contain buried active centers and that displacements of entire secondary structure elements, or "lids," take place when the enzymes assume active conformations [Derewenda, U., A. M. Brzozowski, D. M. Lawson, and Z. S. Derewenda. 1992. Biochemistry: 31: 1532-1541; van Tilbeurgh, H., M-P. Egloff, C. Martinez, N. Rugani, R. Verger, and C. Cambillau. 1993. Nature: 362: 814-820; Grochulski, P., L. Yunge, J. D. Schrag, F. Bouthillier, P. Smith, D. Harrison, B. Rubin, and M. Cygler. 1993. J. Biol. Chem. 268: 12843-12847]. A simple two-state model inferred from these results implies that the "closed" conformation is stable in an aqueous medium, rendering the active centers inaccessible to water soluble substrates. We now report that in crystals of the Humicola lanuginosa lipase the "lid" is significantly disordered irrespective of the ionic strength of the medium, while in a related enzyme from Rhizopus delemar, crystallized in the presence of a detergent, the two molecules that form the asymmetric unit show different "lid" conformations. These new results call into question the simplicity of the "enzyme theory" of interfacial activation.
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38. |
Friedberg D,
Peleg Y,
Monsonego A,
Maissi S,
Battat E,
Rokem JS,
Goldberg I,
( 1995 ) The fumR gene encoding fumarase in the filamentous fungus Rhizopus oryzae: cloning, structure and expression. PMID : 7557464 : DOI : 10.1016/0378-1119(95)00367-f Abstract >>
The filamentous fungus Rhizopus oryzae (Ro) is known for its ability to overproduce and accumulate high levels of fumaric acid (FA) under stress conditions. In order to study the molecular mechanisms involved in the increased biosynthesis of FA, the gene (designated fumR) encoding Ro fumarase was cloned and analysed for its structure and expression. Nucleotide (nt) sequence and comparison of the fumR product with fumarases from various sources established that fumR contains nine introns and encodes a deduced product of 494 amino acids (aa), related to class-II fumarases. A fumarase protein of 50 kDa was immuno-detected in crude Ro extracts. Primer extension experiments mapped the 5' end of the fumR RNA 159 nt upstream from the putative translation start codon. Both primer extension and Northern analysis showed the existence of one transcript of fumR. The level of fumR RNA increased in cells producing FA under stress conditions (high carbon and low nitrogen levels in the medium), suggesting that transcriptional regulation of fumR might be involved in the overproduction and accumulation of FA by Ro cells under stress conditions. The possibility that additional mechanisms are responsible for this phenomenon is discussed.
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39. |
Macedo D,
Leonardelli F,
Dudiuk C,
Theill L,
Cabeza MS,
Gamarra S,
Garcia-Effron G,
( 2018 ) Molecular Confirmation of the Linkage between the Rhizopus oryzae CYP51A Gene Coding Region and Its Intrinsic Voriconazole and Fluconazole Resistance. PMID : 29891608 : DOI : 10.1128/AAC.00224-18 PMC : PMC6105841 Abstract >>
Rhizopus oryzae is the most prevalent causative agent of mucormycosis, an increasingly reported opportunistic fungal infection. These Mucorales are intrinsically resistant to Candida- and Aspergillus-active antifungal azole drugs, such as fluconazole (FLC) and voriconazole, respectively. Despite its importance, the molecular mechanisms of its intrinsic azole resistance have not been elucidated yet. The aim of this work was to establish if the Rhizopus oryzaeCYP51 genes are uniquely responsible for intrinsic voriconazole and fluconazole resistance in these fungal pathogens. Two CYP51 genes were identified in the R. oryzae genome. We classified them as CYP51A and CYP51B based on their sequence similarity with other known fungal CYP51 genes. Later, we obtained a chimeric Aspergillus fumigatus strain harboring a functional R. oryzae CYP51A gene expressed under the regulation of the wild-type A. fumigatusCYP51A promoter and terminator. The mutant was selected after transformation by using a novel procedure taking advantage of the FLC hypersusceptibility of the A. fumigatusCYP51A deletion mutant used as the recipient strain. The azole susceptibility patterns of the A. fumigatus transformants harboring R. oryzae CYP51A mimicked exactly the azole susceptibility patterns of this mucormycete. The data presented in this work demonstrate that the R. oryzae CYP51A coding sequence is uniquely responsible for the R. oryzae azole susceptibility patterns.
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40. |
( 2013 ) Molecular cloning and functional characterization of a �G6-fatty acid desaturase gene from Rhizopus oryzae. PMID : 22961300 : DOI : 10.1002/jobm.201200189 Abstract >>
The objective was to screen for and isolate a novel enzyme with the specific activity of a �G6-fatty acid desaturase from Rhizopus oryzae. In this study, R. oryzae was identified as a novel fungal species that produces large amounts of �^-linolenic acid. A full-length cDNA, designated here as RoD6D, with high homology to fungal �G6-fatty acid desaturase genes was isolated from R. oryzae by using the rapid amplification of cDNA ends method. It had an open reading frame of 1176 bp encoding a deduced polypeptide of 391 amino acids. Bioinformatics analysis characterized the putative RoD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, a hydropathy profile, and a cytochrome b5 -like domain in the N terminus. When the coding sequence was expressed in the Saccharomyces cerevisiae strain INVScl, the encoded product of RoD6D exhibited �G6-fatty acid desaturase activity that led to the accumulation of �^-linolenic acid. The corresponding genomic sequence of RoD6D was 1565 bp in length, with five introns. This is the first report on the characterization and gene cloning of a �G6-fatty acid desaturase of R. oryzae from Douchi.
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41. |
( 2012 ) Fumaric acid production in Saccharomyces cerevisiae by in silico aided metabolic engineering. PMID : 23300594 : DOI : 10.1371/journal.pone.0052086 PMC : PMC3530589 Abstract >>
Fumaric acid (FA) is a promising biomass-derived building-block chemical. Bio-based FA production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here we report on FA production by direct fermentation using metabolically engineered Saccharomyces cerevisiae with the aid of in silico analysis of a genome-scale metabolic model. First, FUM1 was selected as the target gene on the basis of extensive literature mining. Flux balance analysis (FBA) revealed that FUM1 deletion can lead to FA production and slightly lower growth of S. cerevisiae. The engineered S. cerevisiae strain obtained by deleting FUM1 can produce FA up to a concentration of 610��31 mg L(-1) without any apparent change in growth in fed-batch culture. FT-IR and (1)H and (13)C NMR spectra confirmed that FA was synthesized by the engineered S. cerevisiae strain. FBA identified pyruvate carboxylase as one of the factors limiting higher FA production. When the RoPYC gene was introduced, S. cerevisiae produced 1134��48 mg L(-1) FA. Furthermore, the final engineered S. cerevisiae strain was able to produce 1675��52 mg L(-1) FA in batch culture when the SFC1 gene encoding a succinate-fumarate transporter was introduced. These results demonstrate that the model shows great predictive capability for metabolic engineering. Moreover, FA production in S. cerevisiae can be efficiently developed with the aid of in silico metabolic engineering.
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42. |
( 2012 ) Crystal structure of circular permuted RoCBM21 (CP90): dimerisation and proximity of binding sites. PMID : 23226294 : DOI : 10.1371/journal.pone.0050488 PMC : PMC3511584 Abstract >>
Glucoamylases, containing starch-binding domains (SBD), have a wide range of scientific and industrial applications. Random mutagenesis and DNA shuffling of the gene encoding a starch-binding domain have resulted in only minor improvements in the affinities of the corresponding protein to their ligands, whereas circular permutation of the RoCBM21 substantially improved its binding affinity and selectivity towards longer-chain carbohydrates. For the study reported herein, we used a standard soluble ligand (amylose EX-I) to characterize the functional and structural aspects of circularly permuted RoCBM21 (CP90). Site-directed mutagenesis and the analysis of crystal structure reveal the dimerisation and an altered binding path, which may be responsible for improved affinity and altered selectivity of this newly created starch-binding domain. The functional and structural characterization of CP90 suggests that it has significant potential in industrial applications.
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43. |
( 1998 ) Cloning, expression, characterization and role of the leader sequence of a lipase from Rhizopus oryzae. PMID : 9765593 : DOI : 10.1016/s0167-4781(98)00104-3 Abstract >>
A lipase from Rhizopus oryzae DSM 853 (ROL) was cloned from a chromosomal gene bank, sequenced and overexpressed in Escherichia coli. ROL and its precursors ProROL and PreProROL were purified and their pH and temperature profile was determined. In contrast to ROL, ProROL and PreProROL had considerably higher thermostability and a slightly higher pH optimum. Moreover, it could be demonstrated by in vitro experiments that the natural leader sequence of ROL is able to inhibit the folding supporting properties of the prosequence, resulting in a retardation of folding. In addition, there is strong evidence that all different lipase forms derived from Rhizopus sp. described in the literature are a result of different proteolytic processing and originate from the same gene.
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44. |
( 1998 ) The Rhizopus oryzae secreted aspartic proteinase gene family: an analysis of gene expression. PMID : 9720058 : DOI : 10.1099/00221287-144-8-2355 Abstract >>
Rhizopus oryzae was shown to possess a secreted aspartic proteinase gene family (sap) of at least four members (sap1-sap4). Within the family there was 77-87% identity at the nucleotide level and 76-92% identity at the amino acid level. Transcription of three members of this gene family (sap1-sap3) required an acidic medium (pH < 4.5) and either nitrogen or sulphur depression. Regulation was co-ordinate and hierarchical, with pH occupying the higher position in the hierarchy. Exogenous protein increased transcript levels, probably via the provision of metabolic intermediates rather than by direct induction of gene expression. sap4 was not expressed under these conditions. SAP1-SAP4 are predicted to have almost identical substrate-binding sites and therefore substrate specificity. It is proposed that sap1-sap3 exist to provide amplified expression of the secreted aspartic proteinase because protein, an important secondary nitrogen source for this fungus, requires extensive degradation to make its nitrogen available to the cell.
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