Regulation of secreted protein production by filamentous fungi: recent developments and perspectives

MacKenzie, Donald; Jeenes, David J.; Belshaw, Nigel J.; Archer, David B.

doi:10.1099/00221287-139-10-2295

Cited by 67 publications

(24 citation statements)

References 130 publications

(72 reference statements)

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“…1). The 5Ј CAAT 3Ј sequence, shown to be involved in transcription activation in Saccharomyces and other fungi (24), was found at positions Ϫ38, Ϫ61, Ϫ112, and Ϫ373 (lowercase italic letters). No TATA box was found in the sequence immediately upstream of the start codon.…”

Section: Resultsmentioning

confidence: 99%

Molecular Cloning, Sequencing, and Heterologous Expression of the vaoA Gene from Penicillium simplicissimum CBS 170.90 Encoding Vanillyl-Alcohol Oxidase

Benen¹,

Sánchez-Torres²,

Wagemaker³

et al. 1998

Journal of Biological Chemistry

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The cDNA encoding vanillyl-alcohol oxidase (EC 1.1.3.7) was selected from a cDNA library constructed from mRNA isolated from Penicillium simplicissimum CBS 170.90 grown on veratryl alcohol by immunochemical screening. The vaoA-cDNA nucleotide sequence revealed an open reading frame of 1680 base pairs encoding a 560-amino acid protein with a deduced mass of 62,915 Da excluding the covalently bound FAD. The deduced primary structure shares 31% sequence identity with the 8␣-(O-tyrosyl)-FAD containing subunit of the bacterial flavocytochrome p-cresol methyl hydroxylase.The vaoA gene was isolated from a P. simplicissimum genomic library constructed in EMBL3 using the vaoA-cDNA as a probe. Comparison of the nucleotide sequence of the vaoA gene with the cDNA nucleotide sequence demonstrated that the gene is interrupted by five short introns.Aspergillus niger NW156 prtF pyrA leuA cspA transformed with the pyrA containing plasmid and a plasmid harboring the complete vaoA gene including the promoter and terminator was able to produce vaoA mRNA and active vanillyl-alcohol oxidase when grown on veratryl alcohol and anisyl alcohol. A similar induction of the vaoA gene was found for P. simplicissimum, indicating that similar regulatory systems are involved in the induction of the vaoA gene in these fungi.Introduction of a consensus ribosome binding site, AGAAGGAG, in the vaoA-cDNA resulted in elevated expression levels of active vanillyl-alcohol oxidase from the lac promoter in Escherichia coli TG2. The catalytic and spectral properties of the purified recombinant enzyme were indistinguishable from the native enzyme.Vanillyl-alcohol oxidase (EC 1.1.3.7) is a novel type of flavoprotein oxidase that was first isolated from Penicillium simplicissimum CBS 170.90 grown on veratryl alcohol (1). The enzyme is a homo-octamer with each 65-kDa subunit harboring an 8␣-(N 3 -histidyl)-FAD (2). Vanillyl-alcohol oxidase has a broad substrate specificity. In addition to the conversion of vanillyl alcohol to vanillin (Equation 1), the enzyme catalyzes the conversion of a wide range of phenolic compounds bearing side chains of variable size at the para-position of the aromatic ring (3, 4). Due to its broad substrate spectrum, vanillyl-alcohol oxidase may be applied in the fine chemical industry (5). Based on induction experiments, 4-(methoxymethyl)phenol has been proposed to represent the physiological substrate (6). Recently, from rapid reaction kinetics conclusive spectral evidence was obtained that the vanillyl-alcohol oxidase-mediated oxidative demethylation of 4-(methoxymethyl)phenol proceeds through the formation of a quinone-methide product intermediate (4). In the absence of oxygen, this intermediate is stabilized in the active site of the reduced enzyme. Upon flavin reoxidation, the quinone methide of 4-(methoxymethyl)phenol readily reacts with water, yielding 4-hydroxybenzaldehyde, methanol, and hydrogen peroxide as final products.Recently, the three-dimensional structure of vanillyl-alcohol oxidase was elucidated (7). The crystallo...

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Section: Resultsmentioning

confidence: 99%

Molecular Cloning, Sequencing, and Heterologous Expression of the vaoA Gene from Penicillium simplicissimum CBS 170.90 Encoding Vanillyl-Alcohol Oxidase

Benen¹,

Sánchez-Torres²,

Wagemaker³

et al. 1998

Journal of Biological Chemistry

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“…The importance of PL secretion during C. gloeosporioides attack in avocado fruits has been suggested by the inhibition of decay development during coinoculation of C. gloeosporioides spores with PL antibodies and also by the reduced pathogenicity of a Colletotrichum magna mutant with limited PL secretion (26,27). These results suggest that PL is a limiting factor during pathogenesis (27).Environmental conditions can affect protein production and secretion in various organisms (1,4,12). Dean and Timberlake (5) found that Aspergillus nidulans secretes PG at low pH values and PL when the medium pH increases and becomes conducive to PL activity.…”

mentioning

confidence: 90%

“…Environmental conditions can affect protein production and secretion in various organisms (1,4,12). Dean and Timberlake (5) found that Aspergillus nidulans secretes PG at low pH values and PL when the medium pH increases and becomes conducive to PL activity.…”

mentioning

confidence: 99%

pH Regulation of Pectate Lyase Secretion Modulates the Attack ofColletotrichum gloeosporioideson Avocado Fruits

Yakoby

Kobiler

Dinoor

et al. 2000

Appl Environ Microbiol

119

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Growth of Colletotrichum gloeosporioides in pectolytic enzyme-inducing medium (PEIM) increased the pH of the medium from 3.8 to 6.5. Pectate lyase (PL) secretion was detected when the pH reached 5.8, and the level of secretion increased up to pH 6.5. PL gene (pel) transcript production began at pH 5.0 and increased up to pH 5.7. PL secretion was never detected when the pH of the inducing medium was lower than 5.8 or when C. gloeosporioides hyphae were transferred from PL-secreting conditions at pH 6.5 to pH 3.8. This behavior differed from that of polygalacturonase (PG), where pg transcripts and protein secretion were detected at pH 5.0 and continued up to 5.7. Under in vivo conditions, the pH of unripe pericarp of freshly harvested avocado (Persea americana cv. Fuerte) fruits, resistant to C. gloeosporioides attack, was 5.2, whereas in ripe fruits, when decay symptoms were expressed, the pericarp pH had increased to 6.3. Two avocado cultivars, Ardit and Ettinger, which are resistant to C. gloeosporioides attack, had pericarp pHs of less than 5.5, which did not increase during ripening. The present results suggest that host pH regulates the secretion of PL and may affect C. gloeosporioides pathogenicity. The mechanism found in avocado may have equivalents in other postharvest pathosystems and suggests new approaches for breeding against and controlling postharvest diseases.Colletotrichum gloeosporioides (Penz.) Penz. & Sacc [teleomorph Glomerella cingulata (Stonem). Spauld et Schrenk] infects avocado fruits, via spore germination, appressorium formation, and penetration. The pathogen attacks fruits early in their development and remains as a germinated appressorium during fruit growth (quiescent infection) (17). After harvest and fruit ripening, fast-developing brown-black spots on the pericarp and soft rot in the mesocarp, the symptoms of anthracnose, appear (17). Prusky (17) has suggested several hypotheses to explain the resistance mechanism of unripe fruits: (i) nutrients available to the pathogen may be limited in unripe hosts, (ii) preformed antifungal compounds present in unripe fruits decline during ripening, (iii) inducible antifungal compounds in unripe fruits decline during ripening, and (iv) fungal pathogenicity factors may be activated mainly in ripening fruits. Antifungal compounds are implicated in the resistance of avocado fruits to C. gloeosporioides (19), but no reports have described the role of enzyme secretion as a factor in fungal attack.C. gloeosporioides produces an array of pectolytic enzymes, including polygalacturonase (PG) (20), pectin lyase (2), pectin methyl esterase (15), and pectate lyase (PL) (28). No reports regarding the involvement of PG or pectin methyl esterase in C. gloeosporioides attack have been published. However, targeted disruption of pectin lyase from C. gloeosporioides did not reduce virulence (2). The importance of PL secretion during C. gloeosporioides attack in avocado fruits has been suggested by the inhibition of decay development during coinoculation of C. glo...

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“…This is essentially a consequence of their saprophytic lifestyle. The nutrition of filamentous fungi depends on the secretion of a large array of extracellular enzymes in considerable quantities for the hydrolysis of complex polymers derived from plant and animal tissues (van den Hondel, et al, 1992;MacKenzie et al, 1993). The commercial implications of efficient protein secretion include the generation of high product titres during the course of a fermentation, compared to unicellular microorganisms, and a reduction in the extent of downstream processing required (Saunders et al, 1989;Peberdy, 1994).…”

Section: Introductionmentioning

confidence: 99%

“…Industrial strains of filamentous fungi possess a number of characteristics which make them attractive hosts for the production of heterologous proteins (MacKenzie et al, 1993;Peberdy, 1994). First of all, and most importantly, filamentous fungi have a naturally high capacity for protein secretion.…”

Section: Introductionmentioning

confidence: 99%

Effect of branch frequency inAspergillus oryzae on protein secretion and culture viscosity

Bocking

Wiebe

Robson

et al. 1999

Biotechnol. Bioeng.

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Regulation of secreted protein production by filamentous fungi: recent developments and perspectives

Cited by 67 publications

References 130 publications

Molecular Cloning, Sequencing, and Heterologous Expression of the vaoA Gene from Penicillium simplicissimum CBS 170.90 Encoding Vanillyl-Alcohol Oxidase

Molecular Cloning, Sequencing, and Heterologous Expression of the vaoA Gene from Penicillium simplicissimum CBS 170.90 Encoding Vanillyl-Alcohol Oxidase

pH Regulation of Pectate Lyase Secretion Modulates the Attack ofColletotrichum gloeosporioideson Avocado Fruits

Effect of branch frequency inAspergillus oryzae on protein secretion and culture viscosity

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