Paul Broda

The identification, molecular cloning and characterisation of a gene from Phanerochaete chrysosporium that shows strong homology to the exo-cellobiohydrolase I gene from Trichoderma reesei

We have identified a genomic DNA fragment from the lignin-degrading fungus Phanerochaete chrysosporium (P.c.) that hybridizes to a DNA probe encoding part of the exo-cellobiohydrolase I (CBHI) gene of Trichoderma reesei (T.r.). This fragment has been subcloned and its nucleotide sequence determined. We demonstrate that it could encode a 516 residue protein that shows strong homology with the known protein sequence of CBHI from T.r. Comparison of the two nucleotide sequences identifies two regions within the P.c. sequence that are not represented in T.r. Further inspection of these regions reveals sequences closely related to the conserved elements of filamentous fungal introns. We conclude that the P.c. genomic sequence contains the same number of introns (2) as found in T.r. but that these are located at different relative positions within the two genes. The transcript from the P.c. sequence is induced in the presence of cellulose but not by glucose and we therefore conclude that this sequence represents the first cellulase gene to have been described from this organism.

Screening for lignin-degrading actinomycetes and characterization of their activity against [14C]lignin-labelled wheat lignocellulose

SUMMARY: A sedimentation chamber and Andersen sampler were used to isolate a range of actinomycetes on selective and non-selective media. The occurrence of different actinomycete groups in natural substrates was compared and strains were screened for the ability to degrade ball-milled straw or to grow on lignin-related phenolic compounds. Evidence for ligninolytic activity in representatives of several genera was obtained by assaying 14CO2 evolution from [14C]lignin-labelled wheat lignocellulose. Most of the straw-degrading isolates were assigned to the genera Thermomonospora and Micromonospora, but only representatives of the latter were found to be active against [14C]lignin. Of the non-straw-degrading strains also examined, some which could utilize phenolic substrates produced 14CO2 from the [lignin-14C]lignocellulose, and two of these were selected for further study. These strains, Thermomonospora mesophila and a Streptomyces sp., attacked [14C]lignin yielding 14CO2 and water-soluble 14C-labelled compounds during primary growth. This activity was not accounted for by the utilization of phenolic acids linked to the carbohydrate fraction of wheat lignocellulose and was unaffected by cultural parameters known to influence lignin degradation by white-rot fungi.

Studies on the extracellular xylanase activity of some thermophilic actinomycetes

SummaryAn agar plate-clearing assay was used to screen 37 thermophilic actinomycete strains for extracellular xylanase production. The xylanase activity in culture supernatants of strains representing Saccharomonospora viridis and three Thermomonospora spp. was characterised by measurement of reducing sugar released from oat spelt xylan and analysis of degradation products by thin-layer chromatography. In all four species, xylanase activity was optimal within the temperature range 60–75°C and between pH 5 and pH 8. While culture supernatants of Thermomonospora strains incubated at 70°C for 60 min retained >80% of their activity, that of S. viridis was almost, totally inactivated.All of the culture supernatants initially hydrolysed xylan to a mixture of oligomeric products, indicating that the main activity was of the endoxylanase type. Prolonged incubation for 24h resulted in the hydrolysis of xylan to d-xylose by T curvata and T. fusca preparations, indicating the additional presence of exoxylanase or β-xylosidase activity. Xylanase production was induced by growth on xylan although low levels of activity were also detected in glucose-grown cultures. Thermomonospora curvata MT815 culture supernatant was the most active and produced d-xylose from milled wheat straw in yields approximately 10% of those from oat spelt xylan.

Multiple ligninase-related genes from Phanerochaete chrysosporium

We have investigated four distinct sequences in genomic DNA of Phanerochaete chrysosporium that show strong homology to a lignin peroxidase cDNA sequence [Tien and Tu: Nature 236 (1987) 520-523, and 328 (1987) 742]. The complete nucleotide sequence of one of these genes is presented here. Comparison of the sequenced gene, LIG1, with the published cDNA sequence reveals the presence of eight short intervening sequences. The open reading frame encodes a polypeptide of 372 amino acids (aa), which includes a 28-aa residue hydrophobic leader sequence. The predicted Mr of the mature protein product is 37,000. Partial sequencing of the other three genes reveals substantial nucleotide and amino acid homology.

Comparison of the lignin-degrading white rot fungi Phanerochaete chrysosporium and Sporotrichum pulverulentum at the DNA level.

SummaryDNA-hybridisation studies showed a close relationship between Phanerochaete chrysosporium ME446, most used in lignin degradation studies, and Sporotrichum pulverulentum Novobranova, the other standard lignin degrading strain. Two other strains of P. chrysosporium were both less related. We show that P. chrysosporium ME446 and S. pulverulentum Novobranova both have a GC-content of 59% for chromosomal DNA with the rRNA genes present as an AT-rich satellite; the mitochondrial DNA has a GC-content of 33%. The genome size estimated for P. chrysosporium ME446 is about 4–5 × 107 bp.

Lignocellulose degradation by Phanerochaete chrysosporium: Gene families and gene expression for a complex process

Phanerochaete chrysosporium completely degrades lignocellulose. The most recalcitrant component, lignin, is oxidized by the radical products of lignin and manganese peroxidases, whereas cellulose and hemicellulose are hydrolysed. Both peroxidases and cellulases exist as complex families at both the DNA and protein levels. The lignin peroxidases may function principally when mycelium‐bound and, therefore, undetectable in culture supernatants. Moreover, methods for the study of P. chrysosporium must be applicable to solid substrate as well as liquid‐culture conditions. For these reasons, detailed studies of gene expression, made possible by the reverse transcriptase–polymerase chain reaction method, are essential. Such studies reveal that gene families are subject to differential expression. The cellulase system has some differences from that of Trichoderma reesei; the distinction made between the activities of exocellobiohydrolases and endoglucanases needs to be re‐appraised in both species. Current studies also seek to reconstruct the systems of degradation of lignocellulose and its individual components by heterologous expression of individual proteins in recombinant systems, and their use in mechanistic studies singly and in combinations.

Excision of the 40kb segment of the TOL plasmid from Pseudomonas putida mt-2 involves direct repeats

SummaryThe TOL plasmid pWWO gives rise to the Tol− plasmid pWWO-8 due to a specific excision event involving 40 kb of TOL DNA. This event occurs by means of reciprocal recombination involving a pair of directly repeated 1.4 kb sequences. This 40 kb segment is not involved as such in the formation in vivo of hybrid plasmids between TOL and resistance plasmids, notably RP4.

Production and initial characterisation of the xylan-degrading system of Phanerochaete chrysosporium

We report the optimum conditions for the degradation of oat spelt arabinoxylan and a preliminary characterisation of the inducible xylan-degrading system of the lignin-degrading white-rot fungus Phanerochaete chrysosporium. Xylanase activity was optimal at pH 5.0 and 50°C; see attached sheet the maximum reaction velocity (Vmax) of the system was 3.86 units (U) mg−1 protein with arabinoxylan as substrate and the substrate concentration giving half Vmax (S0.5) was 0.52 mg ml−1. At concentrations of arabinoxylan greater than 15 mg ml−1 excess substrate inhibition was observed. Xylose at 0.9 mm inhibited activity to the extent of 50%. Xylanase activity increased as a function of the dilution of the enzyme preparation prior to assay. It was resolved into four peaks by using a DEAE-Biogel column; the material in these peaks differed with respect to xylan solubilisation and the formation of reducing sugars. Electrofocusing gels allowed visualisation of several bands of activity corresponding to each peak. The arabinoxylan degradation system of P. chrysosporium is therefore composed of multiple components.

RFLP-based genetic map of Phanerochaete chrysosporium ME446: lignin peroxidase genes occur in clusters

We describe the construction of an RFLP‐based genetic map of the white rot fungus Phanerochaete chrysosporium ME446. The map is deduced from the allele distributions of 38 RFLP markers in a test set of 53 meiotically derived haploid recombinants of this strain. The map includes a cellulase gene, a mating‐type locus and a family of lignin peroxidase (and related) genes that are arranged in two unlinked clusters.

Differential expression of multiple exo-cellobiohydrolase I-like genes in the lignin-degrading fungus Phanerochaete chrysosporium.

The genome of Phanerochaete chrysosporium strain ME446 contains multiple, non‐allelic, cellobiohydrolase I (CSHI)‐like sequences, at least two of which are expressed in a cellulose‐dependent manner. Each of the expressed genes contains two identically positioned introns within its coding region. The lengths and sequences of these introns are different and one is not excised from all transcripts, raising the possibility that subtly different protein products may be expressed from a common gene. Introns are also present upstream of both genes but these differ in number and position, as well as sequence and length. Endoglucanase‐like sequences could not be identified and it is suggested that variant CBHI‐like proteins may provide endoglucanase activity in this fungus.