Heterologous protein secretion from Aspergillus niger in phosphate-buffered batch culture

Summary Aspergillus niger was grown in batch culture containing various initial concentrations of sodium phosphate buffer (pH 6.5). A wild-type strain of A. niger and a transformed strain producing hen egg-white lysozyme were studied. The maximum cell yield was attained in medium not supplemented with phosphate. In those cultures acidification of the medium resulted in a minimum of pH 2.0 before reverting to near neutrality. Increasing the initial levels of phosphate buffer reduced the fall in pH but lowered cell yields. Secreted levels of lysozyme were maximal in the 50–100 mm range of added phosphate buffer although mycelial yields were reduced by one third of mycelial yields in medium unsupplemented with phosphate buffer. Offprint requests to: D. B. Archer     

Growth of submerged mycelia of Aspergillus kawachii in solid-state culture

Submerged mycelial growth of Aspergillus kawachii IFO4308 in solid-state culture (SSC) was studied. From the result of Northern blot analysis, acid-stable α-amylase was found to be produced mainly by the submerged mycelia rather than the aerial mycelia. The submerged mycelia showed better growth in SSC using rice as the solid substrate (koji) than in agar plate culture in spite of low concentrations of dissolved oxygen in koji. Good growth in SSC suggested the existence of an effective oxygen transfer mechanism in koji which governed the mycelial growth. When koji was submerged in water, small bubbles were generated. This phenomenon indicated the formation of vacant spaces in koji during SSC. The submerged mycelia showed better growth in the koji having a larger number of vacant spaces. Considering these facts it was concluded that the vacant spaces participate in effecting an oxygen transfer mechanism in koji as air vents because the diffusivity of oxygen in an air is larger than in koji itself.     

Citric acid production in submerged and solid state culture of Aspergillus niger

In this work, the citric acid production in solid state culture was performed, evaluating the isolated effect and interactions of particle size and liquid phase employed, by means of the factorial design of first order. The results indicate that the particle size is the most determinant variable. An analysis comparing submerged and solid state in optimal conditions was performed. When solid state culture was used, the productivity of citric acid was doubled, reducing the fermentation time from 14 to 6 days, compared to the submerged culture, obtaining a maximum citric acid concentration of 21.24 g/l.List of Symbols B _s , B _v main effects - B _sv crossed effects - s cm particle size - S coded particle size - v ml liquid phase volume - V coded liquid phase volume     

Stimulation of the gibberellic acid synthesis by Aspergillus niger in submerged culture using a precursor

Stimulation of Aspergillus niger in submerged culture using a commonly known precursor, mevalonic acid (MVA), was investigated in terms of growth and gibberellic acid production. Increasing concentrations of MVA up to 60 M enhanced product and growth yields. Above this amount, gibberellic acid yields and growth were gradually decreased.     

Invertase production by Aspergillus niger in submerged and solid-state fermentation

Three Aspergillus nigerstrains were grown in submerged and solid state fermentation systems with sucrose at 100 g l^–1. Average measurements of all strains, liquid vs solid were: final biomass (g l^–1), 11 ± 0.3 vs 34 ± 5; maximal enzyme titres (U l^–1) 1180 ± 138 vs 3663 ± 732; enzyme productivity (U l^–1h^–1) 20 ± 2 vs 87 ± 33 and enzyme yields (U/gX) 128 ± 24 vs 138 ± 72. Hence, better productivity in solid-state was due to a better mould growth.     

Tannase enzyme production by entrapped cells of Aspergillus niger FETL FT3 in submerged culture system

The ability of immobilized cell cultures of Aspergillus niger FETL FT3 to produce extracellular tannase was investigated. The production of enzyme was increased by entrapping the fungus in scouring mesh cubes compared to free cells. Using optimized parameters of six scouring mesh cubes and inoculum size of 1 × 10⁶ spores/mL, the tannase production of 3.98 U/mL was obtained from the immobilized cells compared to free cells (2.81 U/mL). It was about 41.64% increment. The immobilized cultures exhibited significant tannase production stability of two repeated runs.     

Enhanced mannanase production by submerged culture of Aspergillus niger NCH-189 using defatted copra based media

Copra and other mannans including locust bean gum, guar gum, konjac flour, copra and defatted copra were used to produce extracellular mannanase by shaken flask cultures of Aspergillus niger NCH-189 in this study. The best carbon source for mannanase production was defatted copra, which provided more nitrogen source and mannan content. The peak mannanase activity at 28 U ml⁻¹ was obtained on the day 3 at 30 °C, which was four times of those obtained from other carbon sources. Presence of oil in copra depressed the mannanase production of the fungus and the amount should be less than 3% (w/w). The copra suspension could be sequentially treated by boiling and refrigeration, followed by using n-hexane to remove copra oil.     

Heterologous protein secretion by Candida utilis

The yeast Candida utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. utilis as a promising host for the secretory production of proteins.     

利用黑曲霉高效表达外源蛋白策略

黑曲霉Aspergillus niger作为一种广泛存在于自然界的丝状真菌,是极为重要的工业微生物,不仅是重要的柠檬酸生产菌,同时还在表达多种蛋白和生产次级代谢产物等方面应用广泛。虽然黑曲霉用于商业化生产外源蛋白已经有很长的历史,但大多数外源蛋白的表达水平不高,亟需研究高效表达外源蛋白的策略。文中概述了通过选用强启动子、增加基因拷贝数、使用蛋白酶缺陷菌株、采用基因融合表达、增加糖基化修饰等策略来增强黑曲霉表达外源蛋白的进展,旨在为深入开展该领域的研究工作提供参考。     

Fatty acid and lipid composition in mycelia from submerged or surface culture of Streptomyces viridochromogenes

Abstract Streptomyces viridochromogenes was grown both as submerged and surface culture. Mycelia from these cultures were analysed for the composition of lipids and fatty acids. An increase in ornithinolipid content according to incubation time was observed. The addition of phosphate inhibited the ornithinolipid synthesis. A mutant strain with bald phenotype did not exhibit the phosphate inhibition. At the same time, the mutant strain had a higher content of 12-methyltetradecanoic acid.     

A comparison of factors influencing citric acid production by Aspergillus niger grown in submerged culture and on filter paper

Summary A filter paper surface cultivation method (previously described as a tool for strain selection by Röhr et al. 1979) was adopted as a cultivation system for citric acid production on a small scale and compared with submerged cultivation. Citric acid production in the submerged system was optimal at defined low concentrations of zinc, ferrous and manganese ions, defined phosphate and nitrogen concentrations and within a defined initial pH. In constrast, citric acid production in the filter paper system was not at all influenced by any of these variables. On the other hand, optimal citrate production in both systems required a high (10%–14%, w/v) sucrose concentration. This identifies sugar concentration as a most significant parameter for citrate production, whereas all other nutritional effects are related to the cultivation system used.     

Conidiation of Aspergillus niger in continuous culture

Summary Conidiation of Aspergillus niger was studied in carbon-limited and nitrogen-limited chemostat culture. Under citrate-limitation conidiation intensity varied inversely with dilution rate. Conidiophores were less complex than in aerial conidiation and at high dilution rates conidia occasionally developed from modified hyphal tips. Conidiation was difficult to achieve under glucose-limitation. At the low dilution rates that allowed limited conidiation steady state could not be maintained due to onset of autolysis. At higher dilution rates when steady state was readily obtained conidiation did not occur. The maximum yield constants under citrate-limitation and glucose-limitation were respectively 0.145 and 0.4 mg dry weight/mg substrate, while the relative specific maintenance values were 0.045 and 0.018 mg substrate/mg dry weight/h. Under ammonium-limitation with citrate as the carbon source there was no conidiation. When nitrate became the limiting nitrogen source conidiophore initiation occurred but biomass production was low and wash-out occurred at D=0.034 h^-1.     

Cellulase production by Aspergillus niger in biofilm, solid-state, and submerged fermentations

Cellulase production by Aspergillus niger was compared in three different culture systems: biofilm, solid-state, and submerged fermentation. Biofilm and solid-state fermentations were carried out on perlite as inert support, and lactose was used as a carbon source in the three culture systems. In cryo-scanning electron microscopy, biofilm and solid-state cultures gave similar morphological patterns and confirmed that both spore first attachment and hyphal adhered growth are helped by the production of an adhesive extracellular matrix. Biofilm cultures produced higher cellulase activities than those in submerged and solid-state cultures (1,768, 1,165, and 1,174 U l⁻¹, respectively). Although biofilm cultures grew less than the other cultures, they produced significantly higher cellulase yields (370, 212, and 217 U g⁻¹ lactose, respectively) and volumetric productivities (24, 16, and 16 U l⁻¹ h⁻¹, respectively). Likewise, endoglucanase and xylanase activities were higher in biofilm cultures. Under the conditions tested, it seems that fungal attached growth on perlite may favor better enzyme production. Biofilms are efficient systems for cellulase production and may replace solid-state fermentation. Biofilm fermentation holds promise for further optimization and development. The results of this work reveal that fungal biofilms may be used for the commercial production of cellulase employing the technology developed for submerged fermentation at high cell densities.     

Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Aspergillus niger

The lignin peroxidase (isoenzyme H8) of Phanerochaete chrysosporium was expressed in Aspergillus niger under the control of plant nopaline synthase (NOS) promoter and terminator. H8 mRNA was produced in this heterologous system. Western blot analysis showed that the recombinant protein was of size similar to the native H8. The extracellular lignin peroxidase activity in these primary constructs was positive, though weak (1.12 nKat mg^–1).