John F. Peberdy

The Molecular Biology of Secreted Enzyme Production by Fungi

Enzymes from filamentous fungi are already widely exploited, but new applications for known enzymes and new enzymic activities continue to be found. In addition, enzymes from less amenable non-fungal sources require heterologous production and fungi are being used as the production hosts. In each case there is a need to improve production and to ensure quality of product. While conventional, mutagenesis-based, strain improvement methods will continue to be applied to enzyme production from filamentous fungi the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis of fungal enzyme production and this knowledge is now being applied both in the laboratory and commercially. We review the current state of knowledge on the molecular basis of enzyme production by filamentous fungi. We focus on transcriptional and post-transcriptional regulation of protein production, the transit of proteins through the secretory pathway and the structure of the proteins produced including glycosylation.

Protein secretion in filamentous fungi — trying to understand a highly productive black box

Protein secretion is important in all fungi. The majority of proteins by fungi are thought to be glycosylated and many of them are structurally associated with the cell envelope, the plasma membrane and the cell wall. Many of the enzymes secreted by fungi have been incorporated into commercial processes and used in a range of industries. The existence of strains producing very high levels of secreted enzymes stimulated interest in the use of fungi as hosts for the expression of recombinant proteins. Despite the attention that protein secretion in fungi has attracted, and the multifaceted importance of the process, our understanding of the cellular mechanisms involved is still minimal and, for the most part, it is necessary to extrapolate from other eukaryotic organisms. However, current research suggests that protein secretion in filamentous fungi is intimately associated with the process of growth at the hyphal tip. Such unique features merit a detailed study of this important phenomenon.

Efficient protoplast isolation from fungi using commercial enzymes

Abstract Several commercial polysaccharases have been compared for their ability to liberate protoplasts from fungi. These enzymes were found to contain side activities capable of hydrolysing fungal cell walls. Protoplasts have been commonly isolated from fungi using enzyme systems prepared by workers in their own laboratories. However, these procedures are time consuming and considerable variation may be found between different batches of enzyme. The present study shows that high yields of protoplasts can be prepared from a variety of fungi using relatively cheap commercial enzymes. The yields obtained were normally as good as or better than those previously produced.

Isolation of endophytic actinomycetes from selected plants and their antifungal activity

The isolation of endophytic actinomycetes from surface-sterilized tissues of 36 plant species was made using humic acid–vitamin (HV) agar as a selection medium. Of the 330 isolates recovered, 212 were from roots, 97 from leaves and 21 isolates from stems with a prevalence of 3.9, 1.7 and 0.3%, respectively. Identification of endophytic actinomycetes was based on their morphology and the amino acid composition of the whole-cell extract. Most isolates were classified as Streptomyces sp. (n = 277); with the remainder belonging to Microbispora sp. (n = 14), Nocardia sp. (n = 8) and Micromonospora sp. (n = 4). Four isolates were unclassified and 23 were lost during subculture. The most prevalent group of isolates were the Streptomyces sp. occurring in 6.4% of the tissue samples of Zingiber officinale. Scanning electron microscopy investigation of this plant revealed that 7.5% of the root and 5% of the leaf samples contained endophytes. Three of the Streptomyces sp. isolates strongly inhibited Colletotrichum musae, five were very active against Fusarium oxysporum and two strongly inhibited growth of both test fungi.

The nematophagous fungus Verticillium chlamydosporium produces a chymoelastase-like protease which hydrolyses host nematode proteins in situ.

The nematophagous fungus Verticillium chlamydosporium secreted several proteases in submerged culture in which soya peptone was the sole carbon and nitrogen source. One protease, VCP1 (M(r) 33,000, pI 10.2), was purified 14-fold from culture filtrates to apparent homogeneity using preparative isoelectric focusing in free solution, and shown to rapidly hydrolyse the chymotrypsin substrate Suc-(Ala)2-Pro-Phe-pNA and elastin. VCP1 had a Km for Suc-(Ala)2-Pro-Phe-pNA of 4.3 x 10(-5) M and a kcat of 5.8 s-1. It was highly sensitive to PMSF and TPCK, but only moderately sensitive to chicken egg-white and soya bean trypsin inhibitors. VCP1 degraded a wide range of polymeric substrates, including Azocoll, hide protein, elastin, casein and albumin, and accounted for most of the non-specific protease activity detected in culture filtrates. The purified enzyme hydrolysed proteins in situ from the outer layer of the egg shell of the host nematode Meloidogyne incognita and exposed its chitin layer. VCP1 was secreted by several isolates of V. chlamydosporium and V. lecanii, pathogens of nematodes and insects respectively, but not plant-pathogenic species of Verticillium. These observations suggest that VCP1 or similar enzyme(s) may play a role in the infection of invertebrates.

Can leaf degrading enzymes provide evidence that endophytic fungi becoming saprobes

The diversity of endophytic and saprobic fungi from Magnolia liliifera leaves were observed and analyzed to establish relationships. Nine endophytes were morphologically and phylogenetically similar to the saprobes; Colletotrichum gloeosporioides, Colletotrichum sp. 2, Corynespora cassiicola, Fusarium sp. 1, Guignardia mangiferae, Leptosphaeria sp., Phomopsis sp. 2, Phomopsis sp. 6, and Phomopsis sp. 10. The endophytes were found to produce the same degrading enzymes as their saprobic counterparts. The isoform of β-mannanase produced from each of endophyte and saprobe counterparts were similar. Fungal succession and enzyme production patterns during leaf decomposition were correlated. The occurrence of saprobes was found to be related to the enzymes that the fungi produce. The study provides further compelling evidence that endophytes can switch lifestyle to saprobes.

Regulation of chitinase synthesis in Trichoderma harzianum

The production of chitinase by Trichoderma species is of interest in relation to their use in biocontrol and as a source of mycolytic enzymes. Fourteen isolates of the genus were screened to identify the most effective producer of chitinase. The best strain for chitinase was Trichoderma harzianum 39.1, and this was selected for study of the regulation of enzyme synthesis. Washed mycelium of T. harzianum 39.1 was incubated with a range of carbon sources. Chitinase synthesis was induced on chitin-containing medium, but repressed by glucose and N-acetylglucosamine. Production of the enzyme was optimal at a chitin concentration of 0.5%, at 28 degrees C, pH 6.0 and was independent of the age of the mycelium. The synthesis of chitinase was blocked by both 8-hydroxyquinoline and cycloheximide, inhibitors of RNA and protein synthesis, respectively. The mode of chitinase synthesis in this fungus is discussed.

Protoplast fusion — a tool for genetic manipulation and breeding in industrial microorganisms

Abstract Protoplasts can be isolated from microbial cells by enzymatic digestion of the cell wall, in the presence of an osmotic stabilizer. Such protoplasts can be induced to fuse in the presence of agents such as the poly (ethylene glycols). When suitably selected auxotrophic strains are used, the fusion products can be recovered by selection on the basis of nutritional complementation. Cultivation of the protoplasts on a hypertonic growth medium induces regeneration of new cell wall material and their subsequent reversion to the normal cell form of the organism. The protoplast fusion technique has been applied sucessfully to both bacterial and fungal systems leading to the recovery of recombinant progeny. In the fungi, the recovery of non-parental segregants from inter-species crosses has also been demonstrated. In assessing the value of the fusion technique, caution may be necessary at this stage in its application to genetic mapping in bacteria. The behaviour of protoplasts, especially with respect to reversion, could be an additional factor that operates during selection, distorting recombination frequencies. However, the fusion technique, in providing a mechanism by which genetic recombination can be readily achieved, should be of great potential in empirical breeding and strain improvement. These aspects are reviewed.

The role of the proteinase VCP1 produced by the nematophagous Verticillium chlamydosporium in the infection process of nematode eggs

The infection process of the nematophagous fungus Verticillium chlamydosporium (isolate Vc1O) was studied in two nematode species that differed in susceptibility according to standardized in vitro assays. After 14 d, V. chlamydosporium infected 9 and 80% of Globodera rostochiensis and Meloidogyne incognita eggs, respectively. The purified proteinase VCPl, from V. chlamydosporium, removed the protein coat from eggs of M. incognita but not of G. rostochiensis. VCPl was more effective at hydrolysing proteins from M. incognita eggs than the related enzymes Prl and proteinase K, whereas the latter two were slightly more active against G. rostochiensis eggs than VCPl. Pretreatment of eggs with VCPl increased susceptibility to V. chlamydosporium from 80–100% in M. incognita eggs and from 9 to 29% in G. rostochiensis eggs. These observations suggest that VCPl is a host specificity-determining factor and also confirm it to be a pathogenicity determinant which works best in concert with other enzymes secreted by the actively growing fungus. Fungal growth on the surface of eggs of both species was commensurate with the infection frequencies observed; growth was limited and fewer appressoria were observed on eggs of G. rostochiensis compared with those of M. incognita. Eggs of M. incognita denatured by heat or killed with sodium azide, like eggs treated with VCPl, were more susceptible than untreated, healthy eggs. In contrast heat and azide treatment of G. rostochiensis eggs did not increase the number of infections. Appressoria were observed on the eggs of both nematode species in all treatments, but the numbers and types varied greatly. On M. incognita, the dominant type was swollen and branched laterally from hyphae, while on G. rostochiensis lateral inconspicuous appressoria prevailed. Differentiation of appressoria on live and dead nematode eggs showed that their production was not limited to the parasitic state of the fungus.

Characterization of isolates of the entomopathogenic fungus Metarhizium anisopliae by RAPD-PCR

Twenty isolates of the entomopathogenic fungus Metarhizium anisopliae from Australia, Brazil, Finland and the Philippines were characterized using RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction). Distinct RAPD-PCR profiles were seen for most of these isolates. More similarities were seen between profiles of isolates from the same countries than from different countries.