Serine-Type Carboxypeptidase KexA of Aspergillus oryzae Has Broader Substrate Specificity than Saccharomyces cerevisiae Kex1 and Is Required for Normal Hyphal Growth and Conidiation

Morita, Hiroto; Tomita, Sayako; Maéda, Hiroshi; Okamoto, Ayako; Yamagata, Yuriko; Kusumoto, Ken-Ichi; Amano, Hitoshi; Ishida, Hiroki; Takeuchi, Michio

doi:10.1128/aem.01601-12

Cited by 11 publications

(4 citation statements)

References 17 publications

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“…These genes are mainly involved in amino acid metabolism, microbial metabolism in diverse environments, carbohydrate metabolism, cell growth and death, energy metabolism, lipid metabolism, xenobiotics biodegradation and metabolism, metabolism of terpenoids and polyketides, nucleotide metabolism, replication and repair, signal transduction, translation, transcription, biosynthesis of secondary metabolites, glycan biosynthesis and metabolism, transport and catabolism, and metabolism of cofactors and vitamins pathways (Additional file 4 ). Many genes whose expression was up-regulated during microcycle conidiation encode proteins that function in cell division, cell proliferation, cell wall formation, and cytoskeletal rearrangement, including a tyrosine-protein phosphatase [ 29 ], a transcriptional coactivator [ 30 ], a zinc knuckle domain protein [ 31 ], a serine-type carboxypeptidase [ 32 ], sedoheptulose-1, 7-bisphosphatase [ 33 ], a catalase [ 34 , 35 ], cytochrome P450 [ 36 ], a mannan endo-1, 6-α-mannosidase-like protein [ 37 , 38 ], an actin-associated protein [ 39 ], and a HLH transcription factor [ 40 ], suggesting that these up-regulated genes play a role in microcycle conidiation. Interestingly, members of the normal conidiation FluG pathway, including snaD , GNAT , fluG , pkaA [ 41 ], fadA [ 42 ], and gasA [ 43 ], were up-regulated during microcycle conidiation (Additional file 5 ).…”

Section: Resultsmentioning

confidence: 99%

Transcriptional analysis of the conidiation pattern shift of the entomopathogenic fungus Metarhizium acridum in response to different nutrients

2016

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BackgroundMost fungi, including entomopathogenic fungi, have two different conidiation patterns, normal and microcycle conidiation, under different culture conditions, eg, in media containing different nutrients. However, the mechanisms underlying the conidiation pattern shift are poorly understood.ResultsIn this study, Metarhizium acridum undergoing microcycle conidiation on sucrose yeast extract agar (SYA) medium shifted to normal conidiation when the medium was supplemented with sucrose, nitrate, or phosphate. By linking changes in nutrients with the conidiation pattern shift and transcriptional changes, we obtained conidiation pattern shift libraries by Solexa/Illumina deep-sequencing technology. A comparative analysis demonstrated that the expression of 137 genes was up-regulated during the shift to normal conidiation, while the expression of 436 genes was up-regulated at the microcycle conidiation stage. A comparison of subtractive libraries revealed that 83, 216, and 168 genes were related to sucrose-induced, nitrate-induced, and phosphate-induced conidiation pattern shifts, respectively. The expression of 217 genes whose expression was specific to microcycle conidiation was further analyzed by the gene expression profiling via multigene concatemers method using mRNA isolated from M. acridum grown on SYA and the four normal conidiation media. The expression of 142 genes was confirmed to be up-regulated on standard SYA medium. Of these 142 genes, 101 encode hypothetical proteins or proteins of unknown function, and only 41 genes encode proteins with putative functions. Of these 41 genes, 18 are related to cell growth, 10 are related to cell proliferation, three are related to the cell cycle, three are related to cell differentiation, two are related to cell wall synthesis, two are related to cell division, and seven have other functions. These results indicate that the conidiation pattern shift in M. acridum mainly results from changes in cell growth and proliferation.ConclusionsThe results indicate that M. acridum shifts conidiation pattern from microcycle conidiation to normal conidiation when there is increased sucrose, nitrate, or phosphate in the medium during microcycle conidiation. The regulation of conidiation patterning is a complex process involving the cell cycle and metabolism of M. acridum. This study provides essential information about the molecular mechanism of the induction of the conidiation pattern shift by single nutrients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2971-0) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Transcriptional analysis of the conidiation pattern shift of the entomopathogenic fungus Metarhizium acridum in response to different nutrients

2016

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“…We also obtained some proteins that are involved in virulence, antagonism other fungi, and defense, including serine carboxypeptidase, superoxide dismutase, disulphide isomerase, and lactonohydrolase. Some of these proteins were predicted and/or reported to function as virulence factors ( Data S1 and S2 ), such as peptidase C1 [30] , carboxypeptidase [31] , [32] , glucanosyltransferase [33] , [34] , S-adenosylmethionine synthetase [35] , peroxiredoxin [36] , chitinase 1 [37] , catalase-peroxidase [38] , and amidase [39] . These results indicated that signal peptides–containing proteins and virulence factors might be important contributors to the strong virulence seen in Fusarium-induced banana wilt.…”

Section: Discussionmentioning

confidence: 99%

“…This peptidase is widely observed in fungi, plants, and animals and has diverse biological functions [31] . In Aspergillus oryzae , this enzyme is required for normal hyphal growth, and the deletion strain formes fewer conidia on agar plates [32] . We identified two protein spots in F4 as carboxypeptidase, indicating that carboxypeptidase might be important for hyphal growth and conidiation of F. oxysporum .…”

Section: Discussionmentioning

confidence: 99%

Proteomics of Fusarium oxysporum Race 1 and Race 4 Reveals Enzymes Involved in Carbohydrate Metabolism and Ion Transport That Might Play Important Roles in Banana Fusarium Wilt

Sun

Peng

et al. 2014

PLoS ONE

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Banana Fusarium wilt is a soil–spread fungal disease caused by Fusarium oxysporum. In China, the main virulence fungi in banana are F. oxysporum race 1 (F1, weak virulence) and race 4 (F4, strong virulence). To date, no proteomic analyses have compared the two races, but the difference in virulence between F1 and F4 might result from their differentially expressed proteins. Here we report the first comparative proteomics of F1 and F4 cultured under various conditions, and finally identify 99 protein species, which represent 59 unique proteins. These proteins are mainly involved in carbohydrate metabolism, post-translational modification, energy production, and inorganic ion transport. Bioinformatics analysis indicated that among the 46 proteins identified from F4 were several enzymes that might be important for virulence. Reverse transcription PCR analysis of the genes for 15 of the 56 proteins revealed that their transcriptional patterns were similar to their protein expression patterns. Taken together, these data suggest that proteins involved in carbohydrate metabolism and ion transport may be important in the pathogenesis of banana Fusarium wilt. Some enzymes such as catalase-peroxidase, galactosidase and chitinase might contribute to the strong virulence of F4. Overexpression or knockout of the genes for the F4-specific proteins will help us to further understand the molecular mechanism of Fusarium-induced banana wilt.

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“…AF095574). Aspergillus oryzae also has an ortholog of Kex1 termed KexA and displays a serine-type carboxypetidase activity and a broader substrate [12]. Amino acid comparison of P. pastoris and S. cerevisiae Kex1protein shows only 36% identity and 43.7% similarity [13].…”

Section: Introductionmentioning

confidence: 99%

Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain Insulin glargine in Pichia pastoris

Sreenivas

Krishnaiah

Mohan

et al. 2016

Protein Expression and Purification

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a b s t r a c tInsulin glargine is a slow acting analog of insulin used in diabetes therapy. It is produced by recombinant DNA technology in different hosts namely E. coli and Pichia pastoris. In our previous study, we have described the secretion of fully folded two-chain Insulin glargine into the medium by over-expression of Kex2 protease. The enhanced levels of the Kex2 protease was responsible for the processing of the glargine precursor with in the host. Apart from the two-chain glargine product we observed a small proportion of arginine clipped species. This might be due to the clipping of arginine present at the Cterminus of the B-chain as it is exposed upon Kex2 cleavage. The carboxypeptidase precursor Kex1 is known to be responsible for clipping of C-terminal lysine or arginine of the proteins or peptides. In order to address this issue we created a Kex1 knock out in the host using Cre/loxP mechanism of targeted gene deletion. When two-chain glargine was expressed in the Kex1 knock out host of P. pastoris GS115 the Cterminal clipped species reduced by~80%. This modification further improved the process by reducing the levels of product related impurities.

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Serine-Type Carboxypeptidase KexA of Aspergillus oryzae Has Broader Substrate Specificity than Saccharomyces cerevisiae Kex1 and Is Required for Normal Hyphal Growth and Conidiation

Cited by 11 publications

References 17 publications

Transcriptional analysis of the conidiation pattern shift of the entomopathogenic fungus Metarhizium acridum in response to different nutrients

Transcriptional analysis of the conidiation pattern shift of the entomopathogenic fungus Metarhizium acridum in response to different nutrients

Proteomics of Fusarium oxysporum Race 1 and Race 4 Reveals Enzymes Involved in Carbohydrate Metabolism and Ion Transport That Might Play Important Roles in Banana Fusarium Wilt

Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain Insulin glargine in Pichia pastoris

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