Anthony P. J. Trinci

Fungal siderophores: structures, functions and applications

Siderophores are low molecular weight, iron-chelating ligands produced by nearly all microorganisms. Fungi synthesize a wide range of hydroxamate siderophores. This review considers the chemical and biological aspects of these siderophores, their distribution amongst fungal genera and their possible applications. Siderophores function primarily as iron transport compounds. Expression of siderophore biosynthesis and the uptake systems is regulated by internal iron concentrations. Transport of siderophores is an energy-dependent process and is stereoselective, depending on recognition of the metal ion coordination geometry. In addition to transporting iron, siderophores have other functions and effects, including enhancing pathogenicity, acting as intracellular iron storage compounds and suppressing growth of other microorganisms. Siderophores can complex other metals apart from iron, in particular the actinides. Because of their metal-binding ability there are potential applications for siderophores in medicine, reprocessing of nuclear fuel, remediation of metal-contaminated sites and the treatment of industrial waste.

Growth of Anaerobic Rumen Fungi on Defined and Semi-defined Media Lacking Rumen Fluid

Summary: Anaerobic fungi were isolated from rumen digesta of sheep and cattle and were purified using a plate culture technique. The isolates were successfully cultured on a semi-defined medium which lacked rumen fluid, and on a defined medium.

Anaerobic fungi in herbivorous animals

The conviction that all fungi required oxygen for growth was summarized in 1949 by J. N. Foster when he wrote, ‘One of the major metabolic differences between moulds and bacteria is that there are no anaerobic moulds, either obligate or facultative’. For obligate anaerobes, this view held until 1975, when C. G. Orpin demonstrated that certain motile cells in the rumen of sheep, previously believed to be protozoan flagellates, were in fact zoospores of an obligately anaerobic fungus. Prior to this discovery it was assumed that only anaerobic bacteria and protozoa were involved in the hydrolysis of plant biomass in the rumen, but now it is acknowledged that anaerobic fungi participate in this process both in ruminants such as sheep and hindgut-fermenting herbivores such as horses. This review describes the life-cycles and physiology of anaerobic fungi, details their interactions with other rumen micro-organisms and assesses their contribution to the digestion of plant material in herbivores.

Glucoamylase : green fluorescent protein fusions to monitor protein secretion in Aspergillus niger

A glucoamylase::green fluorescent protein fusion (GLA::sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA::sGFP which employed slightly different lengths of the glucoamylase protein (GLA499 and GLA514). Expression of GLA::sGFP revealed that fluorescence was localized in the hyphal cell walls and septa, and that fluorescence was most intense at hyphal apices. Extracellular GLA::sGFP was detectable by Western blotting only in the supernatant of young cultures grown in soya milk medium. In older cultures, acidification of the medium and induction of proteases were probably responsible for the loss of extracellular and cell wall fluorescence and the inability to detect GLA::sGFP by Western analysis. A strain containing the GLA::sGFP construct was subjected to UV mutagenesis and survivors screened for mutations in the general secretory pathway. Three mutants were isolated that were unable to form a halo on either starch or gelatin medium. All three mutants grew poorly compared to the parental strain. Fluorescence microscopy revealed that for two of the mutants, GLA::sGFP accumulated intracellularly with no evidence of wall fluorescence, whereas for the third mutant, wall fluorescence was observed with no evidence of intracellular accumulation. These results indicate that the GLA::sGFP fusion constructs can be used as convenient fluorescent markers to study the dynamics of protein secretion in vivo and as a tool in the isolation of mutants in the general secretory pathway.

Identification and characterization of anaerobic gut fungi using molecular methodologies based on ribosomal ITS1 and 18S rRNA.

The gut fungi are an unusual group of zoosporic fungi occupying a unique ecological niche, the anaerobic environment of the rumen. They exhibit two basic forms, with nuclear migration throughout the hyphal mass for polycentric species and with concentration of nuclear material in a zoosporangium for monocentric species. Differentiation between isolates of these fungi is difficult using conventional techniques. In this study, DNA-based methodologies were used to examine the relationships within and between two genera of monocentric gut fungi gathered from various geographical locations and host animals. The ribosomal ITS1 sequence from 16 mono- and 4 polycentric isolates was PCR-amplified and sequenced; the sequences obtained were aligned with published sequences and phylogenetic analyses were performed. These analyses clearly differentiate between the two genera and reflect the previously published physiological conclusions that Neocallimastix spp. constitute a more closely related genus than the relatively divergent genus Piromyces. The analyses place two type species N. frontalis and N. hurleyensis together but, contrary to a recent suggestion in the literature, place them apart from the other agreed species N. patriciarum. In situ hybridization and slot-blotting were investigated as potential methods for detection of and differentiation between monocentric gut fungi. DNA slot-blot analysis using ribosomal sequences is able to differentiate between gut fungal genera and thus has considerable potential for use in ecological studies of these organisms.

Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 +/- 8 mg (mean +/- S.E.) glucoamylase (GAM) L-1 in batch culture and 373 +/- 9 mg GAM L-1 in maltodextrin-limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (qp) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (qp, 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 +/- 12 to 496 +/- 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 +/- 0.4 to 16.4 +/- 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production.

Myco-protein: A twenty-year overnight success story

Fusarium graminearum for production of myco-protein is currently grown on a glucose-ammonia-biotin-mineral salts medium in a 40 m3 air-lift fermenter. The resulting biomass is RNA reduced, harvested, texturized and sold for human consumption, either directly as a food or as meat or poultry alternatives in pre-prepared meals. Originally intended in the 1960s to combat the worlds flagging supply of protein foods, Quorn® myco-protein was marketed in the middle 1980s as a low-calorie, high-fibre, food containing no cholesterol or animal fats. The continuous flow culture system currently used for myco-protein production is described, together with details of the ‘evolution’ of the fungus in prolonged culture.

A glucoamylase::GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger.

Although Aspergillus niger is used as a host for heterologous protein production, yields are generally lower than those obtained for homologous proteins. Mechanisms of protein secretion and the secretory pathway in filamentous fungi are poorly characterised, although there is evidence to suggest that secretion occurs by a mechanism similar to that in other eukaryotes, but with proteins destined for secretion being directed to the hyphal tip. We report on a method using a glucoamylase: GFP gene fusion which allows us for the first time to monitor, in vivo, protein secretion in A. niger at the single hyphal level. A synthetic green fluorescent protein (sGFP(S65T)) was fused to truncated A. niger glucoamylase (GLA:499). Southern blot analysis of transformants confirmed that the gene fusion had successfully integrated into the A. niger genome. Confocal and fluorescence microscopy revealed that the GLA::GFP fusion protein is fluorescent in A. niger and appears to be directed to the hyphal tip. In young mycelia, hyphal cell wall fluorescence is apparent and immunogold labelling of GFP confirmed that GFP was partially localised within the hyphal cell wall. Using Western blotting, extracellular GLA::GFP was detected only in culture filtrates of young mycelia grown in a soya milk medium. The actin inhibitor latrunculin B was used to disrupt the secretion process, and its effects on the distribution of GLA::GFP were monitored.

Distribution of anaerobic fungi in the digestive tract of cattle and their survival in faeces

A most probable numbers procedure was used to enumerate populations of anaerobic fungi in the digesta and faeces of cattle. Anaerobic fungi were isolated from the rumen, omasum, abomasum, small intestine, caecum, large intestine and faeces. By determining the amount of digesta in each organ of the digestive tract, it was possible to estimate the total population of anaerobic fungi in cattle and make comparisons between populations in different organs. In addition to enumerating anaerobic fungi in freshly collected samples, they were quantified in digesta and faeces which had been dried at ambient temperature and stored in air for up to 9 months. These experiments showed that a higher proportion of the anaerobic fungi present in the hindgut and faeces were able to withstand desiccation than those present within the gastric and pre-gastric organs. Our results support the hypothesis that the life cycle of anaerobic fungi consists of three stages; the motile zoospore, the vegetative thallus and an aero-tolerant survival stage (cyst or resistant zoosporangium).

Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity.

Highly branched mutants of two strains of Aspergillus oryzae (IFO4177, which produces alpha-amylase, and a transformant of IFO4177 [AMG#13], which produces heterologous glucoamylase in addition to alpha-amylase) were generated by UV or nitrous acid mutagenesis. Four mutants of the parental strain (IFO4177), which were 10 to 50% more branched than the parental strain, were studied in stirred batch culture and no differences were observed in either the amount or the rate of enzyme production. Five mutants of the transformed parental strain (AMG#13), which were 20 to 58% more branched than the parental strain, were studied in either batch, fed-batch or continuous culture. In batch culture, three of the mutants produced more glucoamylase than the transformed parental strain, although only two mutants produced more glucoamylase and alpha-amylase combined. No increase in enzyme production was observed in either chemostat or fed-batch culture. Cultures of highly branched mutants were less viscous than those of the parental and transformed parental strains. A linear relationship was found between the degree of branching (measured as hyphal growth unit length) and culture viscosity (measured as the torque exerted on the rheometer impeller) for these strains. DOT-controlled fed-batch cultures (in which the medium feed rate was determined by the DOT) were thus inoculated with either the transformed parent or highly branched mutants of the transformed parent to determine whether the reduced viscosity would improve aeration and give higher enzyme yields. The average rate of medium addition was higher for the two highly branched mutants (ca. 8.3 g medium h(-1)) than for the parental strain (5.7 g medium h(-1)). Specific enzyme production in the DOT controlled fed-batch cultures was similar for all three strains (approx. 0.24 g alpha-amylase and glucoamylase [g of biomass](-1)), but one of the highly branched mutants made more total enzyme (24.3 +/- 0.2 g alpha-amylase and glucoamylase) than the parental strain (21.7 +/- 0.4 g alpha-amylase and glucoamylase).