Lipase production by recombinant strains of Aspergillus niger expressing a lipase-encoding gene from Thermomyces lanuginosus.

Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Y(xp total)), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7+/-0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3+/-0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (r(p total), the sum of extracellular and intracellular lipase productivity) was found to be 1.60+/-0.81 KU/g DW/h in maltose-limited chemostats at a dilution rate of 0.08 h(-1), compared with a total specific lipase productivity of 1.10+/-0.41 KU/g DW/h in glucose-limited chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall.

Protein kinase A is involved in the control of morphology and branching during aerobic growth of Mucor circinelloides.

The cAMP signal transduction pathway controls many processes in fungi. The Mucor circinelloides pkaR and pkaC genes, encoding the regulatory (PKAR) and catalytic (PKAC) subunit of the cAMP-dependent protein kinase A (PKA), have been cloned recently. Expression analysis during the dimorphic shift and colony morphology suggested a role for PKAR in the control of morphology and branching. Here strain KFA121, which overexpresses the M. circinelloides pkaR gene, was used to quantify growth and branching under different aerobic growth conditions in a flow-through cell by computerized image analysis. An inverse relationship between the pkaR expression level in KFA121 and the hyphal growth unit length was observed in KFA121, suggesting a central role for PKAR in branching. A biochemical analysis of PKAR using antibodies and enzyme assay demonstrated that the level of PKAR is higher in KFA121 under inducing conditions, i.e. in the presence of high glucose, than in the vector control strain KFA89. Measurement of cAMP binding demonstrated a significant increase (two- to threefold) in PKAR level for KFA121 at the time of germ-tube emission in medium containing 10 g glucose l(-1). The level of PKA activity was determined using kemptide in the same crude cell extracts used to determine cAMP binding. Strain KFA121 showed a twofold increase in PKA activity. An excess of free PKAR subunit over PKA holoenzyme was determined using sucrose gradient centrifugation of extracts from KFA89 and KFA121. The data indicate that cAMP-dependent PKA in M. circinelloides might be down-regulated during hyphal-tube emergence and that an increase in PKAR levels results in increased branching.

Morphology and physiology of the dimorphic fungus Mucor circinelloides (syn. M. racemosus) during anaerobic growth.

The dimorphic Mucor circinelloides requires an anaerobic atmosphere and the presence of 30% CO2 to grow as a multipolar budding yeast, otherwise hyphal growth predominates. Establishing other means to control the morphology would be a distinct advantage in the development of a fermentation process for this organism for the production of heterologous proteins. Thus, conditions suppressing polarised growth while at the same time abolishing the CO2 requirement were investigated in submerged cultivations. It was found that supplementing cultures with mixtures of ergosterol and Tween 80 resulted in yeast-like growth under 100% N2. Their impact on growth and morphological development was assessed at a range of concentrations. Maximum biomass levels and the specific growth rate decreased at elevated levels of ergosterol and Tween 80. Possible effects of carbon dioxide and the added fatty acid/sterol mixture on supporting yeast growth by influencing the fluidity of the plasma membrane or affecting polarised growth are discussed.

Fungal genomics beyond Saccharomyces cerevisiae?

Fungi are used extensively in both fundamental research and industrial applications. Saccharomyces cerevisiae has been the model organism for fungal research for many years, particularly in functional genomics. However, considering the diversity within the fungal kingdom, it is obvious that the application of the existing methods of genome, transcriptome, proteome and metabolome analysis to other fungi has enormous potential, especially for the production of food and food ingredients. The developments in the past year demonstrate that we have only just started to exploit this potential.

The autolysis of industrial filamentous fungi.

Fungal autolysis is the natural process of self-digestion of aged hyphal cultures, occurring as a result of hydrolase activity, causing vacuolation and disruption of organelle and cell wall structure. Previously, authors have considered individual aspects of fungal lysis, in terms of either an enzyme, a process or an organism. This review considers both the physiology and morphology of fungal autolysis, with an emphasis on correlations between enzymological profiles and the morphological changes occurring during culture degeneration. The involvement of the main groups of autolytic hydrolases is examined (i.e., proteases, glucanases, and chitinases), in addition to the effects of autolysis on the morphology and products of industrial bioprocesses. We call for a concerted approach to the study of autolysis, as this will be fundamental for research to progress in this field. Increased understanding will allow for greater control of the prevention, or induction of fungal autolysis. Such advances will be applicable in the development of antifungal medicines and enable increased productivity and yields in industrial bioprocesses. Using paradigms in existing model systems, including mammalian cell death and aging in yeast, areas for future study are suggested in order to advance the study of fungal cell death.

The influence of carbon sources and morphology on nystatin production by Streptomyces noursei.

Carbon source nutrition and morphology were examined during cell growth and production of nystatin by Streptomyces noursei ATCC 11455. This strain was able to utilise glucose, fructose, glycerol and soluble starch for cell growth, but failed to grow on media supplemented with galactose, xylose, maltose, sucrose, lactose and raffinose. Utilisation of glucose had a negative influence on production of nystatin independent of the specific growth rate when phosphate and ammonium was in excess. Consumption of carbon sources was related to the specific growth rate. S. noursei ATCC 11455 formed mainly mycelial clumps during cultivation, while pellet growth dominated the culture of the morphologically altered high producing mutant S. noursei NG7.19. When the pellet size increased above a critical size, cell growth and nystatin production terminated. Fluorescent staining of hyphae revealed that this coincided with loss of activity inside the core of the pellets, probably due to diffusion limitation of oxygen or other nutrients.

Metabolic engineering of the morphology of Aspergillus oryzae by altering chitin synthesis.

Morphology and alpha-amylase production during submerged cultivation were examined in a wild-type strain (A1560) and in strains of Aspergillus oryzae in which chitin synthase B (chsB) and chitin synthesis myosin A (csmA) have been disrupted (ChsB/G and CM101). In a flowthrough cell, the growth of submerged hyphal elements was studied online, making it possible to examine the growth kinetics of the three strains. The average tip extension rates of the CM101 and ChsB/G strains were 25 and 88% lower, respectively, than that of the wild type. The branching intensity in the CM101 strain was 25% lower than that in the wild type, whereas that in the ChsB/G strain was 188% higher. During batch cultivation, inseparable clumps were formed in the wild-type strain, while no or fewer large inseparable clumps existed in the cultivations of the ChsB/G and CM101 strains. The alpha-amylase productivity was not significantly different in the three strains. A strain in which the transcription of chsB could be controlled by the nitrogen source-regulated promoter niiA (NiiA1) was examined during chemostat cultivation, and it was found that the branching intensity could be regulated by regulating the promoter, signifying an important role for chsB in branching. However, the pattern of branching responded very slowly to the change in transcription, and increased branching did not affect alpha-amylase productivity. alpha-Amylase residing in the cell wall was stained by immunofluorescence, and the relationship between tip number and enzyme secretion is discussed.