Dry entrapment of enzymes by epoxy or polyester resins hardened on different solid supports.

Embedding of enzymes was performed with epoxy or polyester resin by mixing in a dried enzyme preparation before polymerization was started. This fast and low-cost immobilization method produced enzymatically active layers on different solid supports. As model enzymes the well-characterized Thermomyces lanuginosus lipase and a new threonine aldolase from Ashbya gossypii were used. It was shown that T. lanuginosus lipase recombinantly expressed in Aspergillus oryzae is a monomeric enzyme with a molecular mass of 34kDa, while A. gossypii threonine aldolase expressed in Escherichia coli is a pyridoxal-5'-phosphate binding homotetramer with a mass of 180kDa. The enzymes were used freeze dried, in four different preparations: freely diffusing, adsorbed on octyl sepharose, as well as cross-linked enzyme aggregates or as suspensions in organic solvent. They were mixed with standard two-component resins and prepared as layers on solid supports made of different materials e.g. metal, glass, polyester. Polymerization led to encapsulated enzyme preparations showing activities comparable to literature values.

A developmental stage of hyphal cells shows riboflavin overproduction instead of sporulation in Ashbya gossypii.

The hemiascomycete Ashbya gossypii develops a mycelium. Nutritional stress leads to its differentiation into sporangia. These generate spores. In parallel, the yellow pigment riboflavin is produced. Intracellularly accumulated riboflavin, made visible as a bright green fluorescence, was observed in only 60% of the hyphal cells. For the remaining 40%, it was unclear whether these cells simply export riboflavin or its biosynthesis remains down-regulated in contrast to the accumulating cells. The approach followed in this work was to convert the hyphae into protoplasts by enzymatic degradation of the cell wall. Afterwards, the protoplasts were sorted by fluorescence-activated cell sorting on the basis of riboflavin accumulation. When a reporter strain expressing lacZ under the control of the most important riboflavin biosynthesis promoter, RIB3, was used, green protoplasts were found to have more than tenfold greater reporter activity than hyaline protoplasts. This was true on the basis of total protein as well as on the basis of hexokinase specific activity, a marker for constitutive expression. These results allow the conclusion that hyphal cells of A. gossypii differ in phenotype regarding riboflavin overproduction and accumulation.

Quantitative turbidity assay for lipolytic enzymes in microtiter plates.

A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers were stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction. Dependencies of the signal kinetics on the substrate and enzyme concentrations were studied. For 0.5-1% tributyrin content the reaction rate is not substrate-limited. An initial slope of the signal kinetics is proportional to the lipase activity. A detailed characterization of the assay was performed. Lipolysis of tributyrin was confirmed by glycerol detection. Various gel-forming agents were compared and diffusion conditions in these gels were analyzed. Agar and agarose were found to be the most suitable gel-forming agents, which do not affect enzyme diffusion whereas polyacrylamide gels block lipase diffusion and therefore are not suitable for the assay. The optimized assay prepared from 1% tributyrin emulsion in 2% agar gel was tested with six microbial lipases and porcine pancreatic lipase. The detection limit is 20-60 ng/well which is equivalent to 30 µU/well for T. lanuginosus lipase.

Growth stress triggers riboflavin overproduction in Ashbya gossypii.

The filamentous fungus Ashbya gossypii is used for riboflavin biosynthesis on an industrial scale, but even the wild type displays overproduction. Because riboflavin overproduction was known to start at the transition between growth and stationary phase, it was suspected that overproduction was induced at low growth rates. However, chemostatic cultivations performed at different growth rates did not result in any detectable riboflavin formation. In this study, we report that it was not the final growth rate that triggered riboflavin overproduction but a decline in growth rate. Therefore, continuous fermenter cultivations with dilution rate shifts were performed. Peaks of riboflavin overproduction were observed in the wild type and in a RIB3placZ reporter strain after downshifts in dilution rate. Accumulation of riboflavin correlated with an increased expression of lacZ reporter activity. The step size of the downshifts corresponded to the peak size of riboflavin formation and reporter activity. Expression of further RIB genes encoding riboflavin biosynthetic enzymes was analyzed by RT-PCR. RIB mRNA levels of the ribulose-5-phosphate branch of the divided riboflavin biosynthesis pathway (RIB3, RIB4, and RIB5) were found to increase in the riboflavin production phase, whereas the RIB2 and RIB7 mRNA levels belonging to the GTP branch remained constant. We propose that a decline in growth rate triggers the increased expression of RIB3, RIB4, and RIB5 resulting in riboflavin overproduction. Because although a reduction in oxygen supply, temperature increase or decrease, or salt stress did affect growth, but neither did lead to riboflavin overproduction nor did induce RIB3 reporter expression, we conclude that declining nutrition must be the stress stimulus. Because about half of the cells in the hyphae of Ashbya gossypii did not accumulate riboflavin, the regulatory response on the cellular level can be estimated to be at least twice as great in comparison to what we detected as overall signals.

Alanine : glyoxylate aminotransferase of Saccharomyces cerevisiae-encoding gene AGX1 and metabolic significance.

Alanine : glyoxylate aminotransferase is one of three different enzymes used for glycine synthesis in Saccharomyces cerevisiae. The open reading frame YFL030w (named AGX1 in the following), encoding this enzyme, was identified by comparing enzyme specific activities in knockout strains. While 100% activity was detectable in the parental strain, 2% was found in a YFL030w::kanMX4 strain. The ORF found at that locus was suspected to encode alanine : glyoxylate aminotransferase because its predicted amino acid sequence showed 23% identity to the human homologue. Since the YFL030w::kanMX4 strain showed no glycine auxtrophic phenotype, AGX1 was replaced by KanMX4 in a Delta GLY1 Delta SHM1 Delta SHM2 background. These background mutations, which cause inactivation of threonine aldolase, mitochondrial and cytosolic serine hydroxymethyltransferase, respectively, lead to a conditional glycine auxotrophy. This means that growth is not possible on glucose but on ethanol as the sole carbon source. Additional disruption of AGX1 revealed a complete glycine auxotrophy. Complementation was observed by transformation with a plasmid-encoded AGX1.

Disruption of the SHM2 gene, encoding one of two serine hydroxymethyltransferase isoenzymes, reduces the flux from glycine to serine in Ashbya gossypii.

Riboflavin overproduction in the ascomycete Ashbya gossypii is limited by glycine, a precursor of purine biosynthesis, and therefore an indicator of glycine metabolism. Disruption of the SHM 2 gene, encoding a serine hydroxymethyltransferase, resulted in a significant increase in riboflavin productivity. Determination of the enzyme's specific activity revealed a reduction from 3 m-units/mg of protein to 0.5 m-unit/mg protein. The remaining activity was due to an isoenzyme encoded by SHM 1, which is probably mitochondrial. A hypothesis proposed to account for the enhanced riboflavin overproduction of SHM 2-disrupted mutants was that the flux from glycine to serine was reduced, thus leading to an elevated supply with the riboflavin precursor glycine. Evidence for the correctness of that hypothesis was obtained from (13)C-labelling experiments. When 500 microM 99% [1-(13)C]threonine was fed, more than 50% of the label was detected in C-1 of glycine resulting from threonine aldolase activity. More than 30% labelling determined in C-1 of serine can be explained by serine synthesis via serine hydroxymethyltransferase. Knockout of SHM 1 had no detectable effect on serine labelling, but disruption of SHM 2 led to a decrease in serine (2-5%) and an increase in glycine (59-67%) labelling, indicating a changed carbon flux.

Transcriptional regulation of 3,4-dihydroxy-2-butanone 4-phosphate synthase.

The filamentous hemiascomycete Ashbya gossypii is a strong riboflavin overproducer. A striking but as yet uninvestigated phenomenon is the fact that the overproduction of this vitamin starts when growth rate declines, which means that most of the riboflavin is produced in the stationary phase, the so-called production phase. The specific activity of 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase, the first enzyme in the biosynthetic pathway for riboflavin, was determined during cultivation and an increase during the production phase was found. Furthermore, an increase of RIB3 mRNA, encoding DHBP synthase, was observed by competitive RT-PCR in the production phase. The mRNAs of two housekeeping genes, ACT1 (encoding actin) and TEF (encoding translation elongation factor-1 alpha), served as standards in the RT-PCR. Reporter studies with a RIB3 promoter-lacZ fusion showed an increase of beta-galactosidase specific activity in the production phase. This investigation verified that the increase of RIB3 mRNA in the production phase is caused by an induction of promoter activity. These data suggest that the time course of riboflavin overproduction of A. gossypii is correlated with a transcriptional regulation of the DHBP synthase.

Riboflavin, overproduced during sporulation of Ashbya gossypii, protects its hyaline spores against ultraviolet light.

Riboflavin (vitamin B2), essential in tiny amounts as a precursor for oxidoreductase coenzymes, is a yellow pigment. Although it causes cytotoxicity via photoinduced damage of macromolecules, several microorganisms are striking overproducers. A question, unanswered for decades, is whether riboflavin overproducers can benefit from this property. Here, we report an ultraviolet (UV) protective effect of riboflavin. The spores of Ashbya gossypii, a riboflavin-overproducing fungus, are more sensitive to UV than those of Aspergillus nidulans. The addition of riboflavin to suspensions improves the UV resistance of both spore types. Interestingly, we show that regulation of sporulation and riboflavin overproduction in A. gossypii are linked. In batch culture, both were elevated when growth ceased. At constant growth rates, obtained in a chemostat culture, neither was elevated. Supplementation of cultures by cAMP, a known stress signal, negatively affected sporulation as well as riboflavin overproduction, establishing a second, independent argument for the linkage.

Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production.

Chemical riboflavin production, successfully used for decades, is in the course of being replaced by microbial processes. These promise to save half the costs, reduce waste and energy requirements, and use renewable resources like sugar or plant oil. Three microorganisms are currently in use for industrial riboflavin production. The hemiascomycetes Ashbya gossypii, a filamentous fungus, and Candida famata, a yeast, are naturally occurring overproducers of this vitamin. To obtain riboflavin production with the gram-positive bacterium Bacillus subtilis requires at least the deregulation of purine synthesis and a mutation in a flavokinase/FAD-synthetase. It is common to all three organisms that riboflavin production is recognizable by the yellow color of the colonies. This is an important tool for the screening of improved mutants. Antimetabolites like itaconate, which inhibits the isocitrate lyase in A. gossypii, tubercidin, which inhibits purine biosynthesis in C. famata, or roseoflavin, a structural analog of riboflavin used for B. subtilis, have been applied successfully for mutant selections. The production of riboflavin by the two fungi seems to be limited by precursor supply, as was concluded from feeding and gene-overexpression experiments. Although flux studies in B. subtilis revealed an increase both in maintenance metabolism and in the oxidative part of the pentose phosphate pathway, the major limitation there seems to be the riboflavin pathway. Multiple copies of the rib genes and promoter replacements are necessary to achieve competitive productivity.

Glycine metabolism in Candida albicans: characterization of the serine hydroxymethyltransferase (SHM1, SHM2) and threonine aldolase (GLY1) genes.

Genes encoding the mitochondrial (SHM1) and cytosolic (SHM2) serine hydroxymethyltransferases, and the L-threonine aldolase gene (GLY1) from Candida albicans were cloned and sequenced. All three genes are involved in glycine metabolism. The C. albicans Shm1 protein is 82% identical to that from Saccharomyces cerevisiae and 56% identical to that from Homo sapiens. The corresponding identities for the Shm2 proteins are 68% and 53%. The Gly1 protein shares significant identity with the S. cerevisiae L-threonine aldolase (55%) and also with threonine aldolases from Aeromonas jandiae (36%) and Escherichia coli (36%). Genetic ablation experiments show that GLY1 is a non-essential gene in C. albicans and that L-threonine aldolase plays a lesser role in glycine metabolism than it does in S. cerevisiae. GenBank Accession Nos of the C. albicans SHM1 and SHM2 are AF009965 and AF009966, respectively. Accession No. for C. albicans GLY1 is AF009967.

The multiply-regulated gabA gene encoding the GABA permease of Aspergillus nidulans: a score of exons.

We describe the cloning, sequence and expression of gabA, encoding the gamma-amino-n-butyrate (GABA) permease of the fungus Aspergillus nidulans. Sequence changes were determined for three up-promoter (gabI ) and six gabA loss-of-function mutations. The predicted protein contains 517 residues and shows 30.3% overall identity with a putative GABA permease of Arabidopsis thaliana, 29.6% identity with the yeast choline transporter and 23.4% identity with the yeast UGA4 GABA permease. Structural predictions favour 11-12 transmembrane domains. Comparison of the genomic and cDNA sequences shows the presence of 19 introns, an unusually large number of introns for, we believe, any fungal gene. In agreement with the wealth of genetic data available, transcript level analyses demonstrate that gabA is subject to carbon catabolite and nitrogen metabolite repression, omega-amino acid induction and regulation in response to ambient pH (being acid-expressed). In agreement with this, we report consensus binding sites 5' to the coding region, six each for CreA and AREA and one for PacC, the transcription factors mediating carbon catabolite and nitrogen metabolite repression and response to ambient pH respectively.

Isocitrate lyase of Ashbya gossypii--transcriptional regulation and peroxisomal localization.

The isocitrate lyase-encoding gene AgICL1 from the filamentous hemiascomycete Ashbya gossypii was isolated by heterologous complementation of a Saccharomyces cerevisiae icl1d mutant. The open reading frame of 1680 bp encoded a protein of 560 amino acids with a calculated molecular weight of 62584. Disruption of the AgICL1 gene led to complete loss of AgIcl1p activity and inability to grow on oleic acid as sole carbon source. Compartmentation of AgIcl1p in peroxisomes was demonstrated both by Percoll density gradient centrifugation and by immunogold labeling of ultrathin sections using specific antibodies. This fitted with the peroxisomal targeting signal AKL predicted from the C-terminal DNA sequence. Northern blot analysis with mycelium grown on different carbon sources as well as AgICL1 promoter replacement with the constitutive AgTEF promoter revealed a regulation at the transcriptional level. AgICL1 was subject to glucose repression, derepressed by glycerol, partially induced by the C2 compounds ethanol and acetate, and fully induced by soybean oil.

Threonine aldolase overexpression plus threonine supplementation enhanced riboflavin production in Ashbya gossypii.

Riboflavin production in the filamentous fungus Ashbya gossypii is limited by glycine, an early precursor required for purine synthesis. We report an improvement of riboflavin production in this fungus by overexpression of the glycine biosynthetic enzyme threonine aldolase. The GLY1 gene encoding the threonine aldolase of A. gossypii was isolated by heterologous complementation of the glycine-auxotrophic Saccharomyces cerevisiae strain YM13 with a genomic library from A. gossypii. The deduced amino acid sequence of GLY1 showed 88% similarity to threonine aldolase from S. cerevisiae. In the presence of the GLY1 gene, 25 mU of threonine aldolase specific activity mg-1 was detectable in crude extracts of S. cerevisiae YM13. Disruption of GLY1 led to a complete loss of threonine aldolase activity in A. gossypii crude extracts, but growth of and riboflavin production by the knockout mutant were not affected. This indicated a minor role of the enzyme in glycine biosynthesis of A. gossypii. However, overexpression of GLY1 under the control of the constitutive TEF promoter and terminator led to a 10-fold increase of threonine aldolase specific activity in crude extracts along with a 9-fold increase of riboflavin production when the medium was supplemented with threonine. This strong enhancement, which could not be achieved by supplementation with glycine alone, was attributed to an almost quantitative uptake of threonine and its intracellular conversion into glycine. This became evident by a subsequent partial efflux of the glycine formed.

Identification of Saccharomyces cerevisiae GLY1 as a threonine aldolase: a key enzyme in glycine biosynthesis.

Determination of enzyme-specific activities revealed that GLY1 encodes a threonine aldolase (TA) in Saccharomyces cerevisiae. A knock-out mutant auxotrophic for glycine lacked detectable activity. After transformation with YEp24GLY1 glycine prototrophy was restored and TA-specific activity was 16-fold higher than in the wild type. Growth experiments using glucose as the sole carbon source showed that GLY1 is more important for glycine biosynthesis than SHM1 and SHM2 encoding alternative serine hydroxymethyltransferases. On ethanol as carbon source simultaneous disruption of GLY1, SHM1 and SHM2 did not lead to glycine auxotrophy because glycine biosynthesis proceeds via alanine glyoxylate aminotransferase.

Regulation and properties of a fungal lipase showing interfacial inactivation by gas bubbles, or droplets of lipid or fatty acid.

Ashbya gossypii can grow on triacyglycerol as carbon source. A degradation rate of 0.05 g x g-1 mycelial dry mass x h-1 was detected for soybean oil. Although this rate was within the sensitivity range of lipase assays no activity was detectable. On the other hand, extracellular lipase activity could be visualized by clearance halos round the growing mycelium when trioleoylglycerol was emulsified as the sole carbon source in agar plates. Variation of the culture conditions revealed that reduced shaking speed and decreased fat content in the medium led to detectable amounts of lipase in the supernatant of flask cultures. A maximal activity of 800 U x l-1 was obtained after 32 h of cultivation in flasks containing 1% yeast extract and incubated at 60 rpm. Because of its pI of 9.0, the enzyme could be purified in a single step by preparative isoelectric focusing. It appeared as a homogeneous protein in analytical isoelectric focusing and SDS/PAGE (M 35,000). The lipase was inactivated within minutes in stirred gas/water, trioleoylglycerol/water or oleic acid/water mixtures. These effects suggested an interface inactivation. This idea was supported by a stability modulation observed with the surfactant Pluronic F-68. Inactivation by oleic acid led to an aggregation of the lipase shown by gel filtration. Growth experiments performed under lipase-stabilizing conditions revealed a negative influence of glucose, glycerol or oleic acid on detectable lipase activity, probably due to a regulation of lipase formation. Inactivation and regulation thus explained the lack of detectable lipase activity in cultures of A. gossypii growing on triacylglycerol.

Inhibition of purified isocitrate lyase identified itaconate and oxalate as potential antimetabolites for the riboflavin overproducer Ashbya gossypii.

A specific isocitrate lyase (ICL) activity of 0.17 U (mg protein)-1 was detected in cultures of the riboflavin-producing fungus Ashbya gossypii during growth on soybean oil. Enzyme activity was not detectable during growth on glucose [<0.005 U (mg protein)-1], indicating a regulation. The enzyme was purified 108-fold by means of ammonium sulphate fractionation, gel filtration and cation-exchange chromatography. SDS-PAGE of the purified protein showed a homogeneous band with an M r of 66000. The M r of 254000 determined by gel-filtration chromatography indicated a tetrameric structure of the native protein. The enzyme was found to have a pH optimum for the isocitrate cleavage of 7.0, and the K m for threo-DL-isocitrate was determined as 550 µ. Enzyme activity was Mg2+- dependent. In regulation studies ICL was weakly inhibited by central metabolites. A concentration of 10 mM phosphoenolpyruvate or 6-phosphogluconate revealed a residual activity of more than 40%. On the other hand, oxalate (K i: 4 µM) and itaconate (K i: 170 µM) showed a strong inhibition and may therefore be interesting as antimetabolites.

Correlation of isocitrate lyase activity and riboflavin formation in the riboflavin overproducer Ashbya gossypii.

Isocitrate lyase (ICL) was assayed during batch cultivations of Ashbya gossypii on soybean oil or glucose as carbon source. On soybean oil, a correlation between enzyme activity and riboflavin synthesis was observed. On glucose as carbon source, riboflavin overproduction started in the late growth phase when glucose was exhausted. ICL activity appeared in parallel and reached a maximum of 0.41 U (mg protein)-1. This suggested synthesis of vitamin B2 from the intracellular reserve fat. ICL specific activity correlated with the enzyme concentration detected by specific antibodies. Itaconate, an efficient inhibitor of ICL, was used as an antimetabolite to screen mutants with enhanced ICL activity. Cultivations of an itaconate-resistant mutant on soybean oil revealed a 15% increase in enzyme specific activity and a 25-fold increase in riboflavin yield compared to the wild-type. On the other hand, growth experiments on glucose resulted in an eightfold increase in riboflavin yield but showed a 33% reduction in ICL specific activity compared to the wild-type grown on the same medium. These results support the idea of an ICL bottleneck in the riboflavin overproducer A. gossypii when plant oil is used as the substrate.

Structural properties of native and sonicated cinerean, a beta- (1-->3) (1-->6)-D-glucan produced by Botrytis cinerea.

Cinerean, the extracellular beta-(1-->3) (1-->6)-D-glucan of the fungus Botrytis cinerea was studied. Electron micrographs of the native polysaccharide revealed quasi-endless fibrils with an estimated diameter of ca. 1.5 nm. A particle mass of 10(9)-10(10) daltons was determined from dilute solutions by low-angle laser light scattering. Sonication of increasing duration led to fragmentation of the native polymer with an approximately exponential decrease of mass in the range of average molecular masses between 250,000 and 50,000 daltons. Shadowed by platinum, cinerean fibril fragments with a weight-average molecular mass of 172,000 +/- 3000 daltons could be characterized from electron micrographs as a distribution of rods of most probable length of 45 nm and an average length of 72 nm. Small-angle X-ray scattering confirmed the fibrillar structure of the native cinerean and the rodlike structure of sonicated cinerean. A rod diameter of 1.9 +/- 0.2 nm and a mass per unit length of 2250 +/- 490 daltons/nm were found. The latter is in agreement with the value of 1830 daltons/nm calculated from the length distribution determined from the electron micrographs. These data-especially the mass per unit length-suggest a quaternary structure for the polysaccharide. Such a structure would explain the rigidity of the rods which, in turn, is responsible for the characteristic phase separation behaviour in aqueous solutions observed by nephelometry and viscometry.

Degradation of Extracellular beta-(1,3)(1,6)-d-Glucan by Botrytis cinerea.

During growth on glucose, Botrytis cinerea produced extracellular beta-(1,3)(1,6)-d-glucan (cinerean), which formed an adhering capsule and slime. After glucose was exhausted from the medium, cinereanase activity increased from <0.4 to 30 U/liter, effecting a striking loss in the viscosity of the culture. Cinerean was cleaved into glucose and gentiobiose. Gentiobiose was then hydrolyzed to glucose. While cinereanase activity was strongest in the culture supernatant, gentiobiase activity was located mainly in the cell wall fraction. The addition of extra glucose or cycloheximide prevented the cinerean degradation caused by an effect on cinereanase formation. Cinerean degradation was accompanied by microconidiation and sclerotium formation. B. cinerea was found to grow on cinerean with the latter as its single carbon and energy source. In this case, cinerean degradation occurred during hyphal growth, and no microconidiation or sclerotium formation was observed. Growth experiments with various carbon sources indicated that cinerean had a positive effect on the formation of cinerean-degrading enzymes.