Cloning, characterization, and engineering of fungal L-arabinitol dehydrogenases

L-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD⁺ reduction in fungal L-arabinose catabolism. It is an important enzyme in the development of recombinant organisms that convert L-arabinose to fuels and chemicals. Here, we report the cloning, characterization, and engineering of four fungal LADs from Penicillium chrysogenum, Pichia guilliermondii, Aspergillus niger, and Trichoderma longibrachiatum, respectively. The LAD from P. guilliermondii was inactive, while the other three LADs were NAD⁺-dependent and showed high catalytic activities, with P. chrysogenum LAD being the most active. T. longibrachiatum LAD was the most thermally stable and showed the maximum activity in the temperature range of 55–65°C with the other LADs showed the maximum activity in the temperature range of 40–50°C. These LADs were active from pH 7 to 11 with an optimal pH of 9.4. Site-directed mutagenesis was used to alter the cofactor specificity of these LADs. In a T. longibrachiatum LAD mutant, the cofactor preference toward NADP⁺ was increased by 2.5 × 10⁴-fold, whereas the cofactor preference toward NADP⁺ of the P. chrysogenum and A. niger LAD mutants was also drastically improved, albeit at the expense of significantly reduced catalytic efficiencies. The wild-type LADs and their mutants with altered cofactor specificity could be used to investigate the functionality of the fungal L-arabinose pathways in the development of recombinant organisms for efficient microbial L-arabinose utilization.     

In vivo transport of the intermediates of the penicillin biosynthetic pathway in tailored strains of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>

Penicillium chrysogenum npe10 (Δpen; lacking the 56.8-kbp amplified region containing the penicillin gene cluster), complemented with one, two, or three penicillin biosynthetic genes, was used for in vivo studies on transport of benzylpenicillin intermediates. 6-Aminopenicillanic acid (6-APA) was taken up efficiently by P. chrysogenum npe10 unlike exogenous δ(l-α-aminoadipyl)-l-cysteinyl-d-valine or isopenicillin N (IPN), which were not taken up or were taken up very poorly. Internalization of exogenous IPN and 6-APA inside peroxisomes was tested by quantifying their peroximal conversion into benzylpenicillin in strains containing only the penDE gene. Exogenous 6-APA was transformed efficiently into benzylpenicillin, whereas IPN was converted very poorly into benzylpenicillin due to its weak uptake. IPN was secreted to the culture medium. IPN secretion decreased when increasing levels of phenylacetic acid were added to the culture medium. The P. chrysogenum membrane permeability to exogenous benzylpenicillin was tested in the npe10 strain. Penicillin is absorbed by the cells by an unknown mechanism, but its intracellular concentration is kept low. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and purification of O -acetyl-l-serine sulphhydrylase in Penicillium chrysogenum

We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-l-serine sulphhydrylase and O-acetyl-l-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-l-serine as substrate for the formation of l-cysteine. The purified␣enzyme did not catalyse the formation of l-homocysteine from O-acetyl-l-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyl-l-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl sulphate/polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K _m value for O-acetyl-l-serine and V _max of O-acetyl-l-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 μmol/mg protein⁻¹ h⁻¹ respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °C, which is much higher than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-l-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0–7.4. Received: 20 March 1998 / Received revision: 27 July 1998 / Accepted: 12 August 1998     

Measurement of airborne fungal spore dispersal from three types of flooring materials

Research was conducted in an experimental roomto measure the effect of human activity onairborne dispersal of settled fungal sporesfrom carpet and vinyl tile flooring. A seriesof experiments were conducted in whichcommercial loop pile carpet, residential cutpile carpet, or vinyl tile installed in theexperimental room were contaminated with Penicillium chrysogenum spores. The flooringmaterials were contaminated to two differentlevels (10⁶ and 10⁷ colony formingunits per square meter [c.f.u./m²] offlooring surface). Airborne culturable andtotal P. chrysogenum concentrations weremeasured using Andersen single-stage impactorsamplers and Burkard personal slide impactorsamplers, respectively. Bioaerosolconcentrations were measured at floor level, 1meter, and the adult breathing zone (1.5 meter)heights before and after human activityconsisting of walking in a prescribed patternfor 1 minute in the room. Airborne P.chrysogenum concentrations were greater withthe higher surface loading for all threeflooring materials. For all flooring materialsthere was no significant difference betweensampler locations, although the data from the1-meter location were the highest, followed bythe floor level and the breathing zonelocations, respectively. The data from theseexperiments indicate that while a very smallfraction of culturable P. chrysogenumspores present on flooring materials wereaerosolized by walking, relatively highairborne concentrations of spores maybere-entrained from contaminated materials. Theairborne P. chrysogenum concentrationswere significantly higher after walking on cutpile carpet than with the other two flooringmaterials at both contamination levels, withthe differences in concentration often 2orders of magnitude. No differences weremeasured in airborne culturable P.chrysogenum between vinyl flooring and looppile carpet at both contamination levels. Total spore data from the experiments with the10⁷ c.f.u./m² contamination levelindicated that walking on loop pile carpetproduced higher airborne spore concentrationsthan similarly contaminated vinyl tile althoughno significant difference was observed at the10⁶ c.f.u./m² level.     

The specific transport system for lysine is fully inhibited by ammonium in Penicillium chrysogenum: An ammonium-insensitive system allows uptake in carbon-starved cells

The regulation exerted by ammonium and other nitrogen sources on amino acid utilization was studied in swollen spores of Penicillium chrysogenum. Ammonium prevented the L-lysine, L-arginine and L-ornithine utilization by P. chrysogenum swollen spores seeded in complete media, but not in carbon-deficient media. Transport of L-[¹⁴C]lysine into spores incubated in presence of carbon and nitrogen sources was fully inhibited by ammonium ions (35 mM). However, in carbon-derepressed conditions (growth in absence of sugars, with amino acids as the sole carbon source) L-[¹⁴C]lysine transport was only partially inhibited. Competition experiments showed that L-lysine (1 mM) inhibits the utilization of L-arginine, and vice versa, L-arginine inhibits the L-lysine uptake. High concentrations of L-ornithine (100 mM) prevented the L-lysine and L-arginine utilization in P. chrysogenum swollen spores. In summary, ammonium seems to prevent the utilization of basic amino acids in P. chrysogenum spores by inhibiting the transport of these amino acids through their specific transport system(s), but not through the general amino acid transport system that is operative under carbon-derepression conditions.     

Resorcinol degradation by a <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> strain under osmotic stress: mono and binary substrate matrices with phenol

A phenol-degrading Penicillium chrysogenum strain previously isolated from a salt mine was able to grow at 1,000 mg l⁻¹ of resorcinol on solid medium. The aerobic degradation of resorcinol by P. chrysogenum CLONA2 was studied in batch cultures in minimal mineral medium with 58.5 g l⁻¹ of sodium chloride using resorcinol as the sole carbon source. The fungal strain showed the ability to degrade up to 250 mg l⁻¹ of resorcinol. Resorcinol and phenol efficiency degradation by P. chrysogenum CLONA2 was compared. This strain removes phenol faster than resorcinol. When phenol and resorcinol were in binary substrate matrices, phenol enhanced resorcinol degradation, and organic load decreased with respect to the mono substrate matrices. The acute toxicity of phenol and resorcinol, individually and in combination, to Artemia franciscana larvae has been verified before and after the bioremediation process with P. chrysogenum CLONA2. The remediation process was effective in mono and binary substrate systems.     

Combined Application of Rice Straw and Fungus Penicillium Chrysogenum to Remediate Heavy-Metal-Contaminated Soil

The search for cheap and environmentally friendly materials is essential for remediation of heavy-metal-contaminated agricultural soils. A pot experiment was undertaken to evaluate the application of rice straw and filamentous fungus Penicillium chrysogenum (P. chrysogenum) on the fractionation of copper (Cu) and cadmium (Cd), soil microbial properties, and Cu and Cd uptake by romaine lettuce (Lactuca sativa) in a contaminated agricultural soil. Rice straw was applied at three rates (0, 7.8, and 11.7 g kg^?1 soil), and in combinations with P. chrysogenum (1.0 × 10⁶ spores g^?1 soil). It was found that the combined treatment of rice straw and P. chrysogenum significantly decreased the acid-extractable Cu and Cd by 15.4–25.1% and 20.2–27.3%, and increased the oxidizable Cu and Cd by 16.1–18.0% and 72.1–98.4%, respectively. Soil microbial biomass and fresh weight of lettuce were also remarkably enhanced after rice straw plus P. chrysogenum addition. Rice straw combined with P. chrysogenum was more effective in reducing Cu and Cd uptake by lettuce than rice straw alone. The joint application of rice straw and P. chrysogenum remarkably reduced Cu and Cd concentrations in lettuce shoots by 13.6–21.9% and 32.9–41.7%, respectively. These results indicate that the combined application of P. chrysogenum and rice straw is a promising method to alleviate the bioavailability of metals, and to improve soil microbial properties and plant yield in heavy-metal-polluted agricultural soils.     

Optimization of the solvent-tolerant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12 as host for the production of <Emphasis Type="Italic">p</Emphasis>-coumarate from glucose

A Pseudomonas putida S12 strain was constructed that is able to convert glucose to p-coumarate via the central metabolite l-tyrosine. Efficient production was hampered by product degradation, limited cellular l-tyrosine availability, and formation of the by-product cinnamate via l-phenylalanine. The production host was optimized by inactivation of fcs, the gene encoding the first enzyme in the p-coumarate degradation pathway in P. putida, followed by construction of a phenylalanine-auxotrophic mutant. These steps resulted in a P. putida S12 strain that showed dramatically enhanced production characteristics with controlled l-phenylalanine feeding. During fed-batch cultivation, 10 mM (1.7 g l⁻¹) of p-coumarate was produced from glucose with a yield of 3.8 Cmol% and a molar ratio of p-coumarate to cinnamate of 85:1.     

Ribosomes from the blue-green alga Anabaena variabilis

Summary Extracts from the blue-green alga Anabaena variabilis were prepared by ultrasonic disintegration or by extrusion through a French pressure cell; examination by sucrose density gradient centrifugation and analytical ultracentrifugation indicated the existence of a procaryotic (70S) ribosome. However both the ribosomes and their sub-units were found to be relatively unstable after isolation and examination in tris buffer pH 7.4 but not in 10^-3 m-sodium phosphate buffer pH 7.0 containing 10^-1 m-potassium chloride. Experiments with ³²P and ³⁵S isotopes indicated that this instability was associated with the loss of ³⁵S labelled material, presumably therefore protein rather than nucleic acid. A comparison of behaviour with that of bacterial ribosomes is presented and the existence of certain similarities to ribosome preparations from chloroplasts of several plant species discussed.     

Iron requirement and chelator production of staphylococci,Streptococcus faecalis and enterobacteriaceae

The effect of iron deprivation on growth of 101 aerobic strains of gram-positive and gram-negative bacteria was studied on agar media in the presence of various concentrations of the synthetic iron chelator ethylene diamine diorthohydroxyphenyl acetic acid (EDDA) and the iron binding protein transferrin.Growth of Staphylococcus epidermidis was inhibited by 15mm EDDA and 1.5mm transferrin. Staphylococcus aureus was only inhibited by 44mm EDDA and not by transferrin. None of the strains of S. faecalis was inhibited. The majority of the enterobacteriaceae (E. coli, Salmonella spp, Klebsiella spp) was inhibited by 44mm EDDA and 1.5mm transferrin. The relation between susceptibility and concentration of EDDA and transferrin was expressed as S-value for each species. Iron supply with various iron compounds could restore the effects of inhibition.In all species except in S. faecalis iron chelator production could be demonstrated, using indicator plates of media containing EDDA and flooded with 10⁴–10⁵ colony forming units of indicator organisms.The iron chelator of both S. epidermidis and S. aureus could stimulate growth of S. epidermidis, but not that of enterobacteriaceae. Iron chelators from all gram-negative bacteria were functionally interchangeable, but did not stimulate growth of gram-positive bacteria.     

Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose‐Limited Cultivation of Penicillium chrysogenum

During glucose‐limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of β‐lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady‐state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose‐limited chemostat cultures at the dilution rates (D) of both 0.03 h^?1 and 0.05 h^?1 are determined to evaluate the quantitative impact on the physiology of a high‐yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h^?1, while this is not the case at D = 0.05 h^?1. In addition, a higher cytosolic redox status (NADH/NAD⁺), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h^?1, which are virtually absent at D = 0.03 h^?1. In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose‐limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial‐scale scenario.     

Novel Antifungal and Antifeedant Metabolites from Penicillium chrysogenum Co-Cultured with Nemania primolutea and Aspergillus fumigatus

The endophyte Nemania primolutea, inhibited the growth of Penicillium chrysogenum in the coculture system. Four new compounds, nemmolutines A–B ( 1–2 ), and penigenumin ( 3 ) from N. primolutea, penemin ( 4 ) from P. chrysogenum were isolated from the coculture. On the other hand, P. chrysogenum inhibited the Aspergillus fumigatus in the coculture. Induced metabolites ( 13–16 ) with monasone naphthoquinone scaffolds including a new one from P. chrysogenum were produced by the coculture of P. chrysogenum, and A. fumigatus. Interesting, cryptic metabolites penicichrins A–B isolated from wild P. chrysogenum induced by host Ziziphus jujuba medium were also found in induced P. chrysogenum cultured in PDB ordinary medium. So the induction of penicichrin production by supplementing with host extract occurred in the fungus P. chrysogenum not the host medium. The productions of penicichrins were the spontaneous metabolism, and the metabolites ( 13–16 ) were the culture driven. Compounds 4 , 6 , 8 , 10 , 11 , 14 , and 15 showed significant antifungal activities against the phytopathogen Alternaria alternata with MIC_S of 1–8 μg/mL, and compounds 7 , 9 , and 12 indicated significant antifeedant activities against silkworms with feeding deterrence indexes (FDIs) of 92 %, 66 %, and 64 %. The carboxy group in 4-(2-hydroxybutynoxy)benzoic acid derivatives, and xylabisboeins; the hydroxy group in mellein derivatives; and the quinoid in monasone naphthoquinone increased the antifungal activities.     

Biosorption of zinc by fungal mycelial wastes

Summary Waste mycelia from several industrial fermentation plants (Aspergillus niger, Penicillium chrysogenum, Claviceps paspali) were used as a biosorbent for zinc ions from aqueous environments, both batchwise as well as in a column mode. With all mycelia testes, biosorption per biomass dry weight was a function of pH (increasing with increasing pH between 1.0 and 9.0), biomass concentration (decreasing at high biomass concentrations) and the zinc concentration. Under optimized conditions, A. niger and C. paspali were superior to P. chrysogenum. Treatment of A. niger biomass with NaOH further increased its biosorbent capacity. Desorption of biosorbed zinc was achieved by elution with 0.1 m HCl, best results being obtained with NaOH-treated A. niger. Such treatment did not affect the capacity for biosorption in repeated experiments. NaOH-treated A. niger mycelia were also successfully used in removal of zinc from polluted waters in Austria, thereby showing that the simultaneous presence of other naturally occurring ions does not affect biosorption. Offprint requests to: C. P. Kubicek     

Development of a new transformant selection system for Penicillium chrysogenum: isolation and characterization of the P. chrysogenum acetyl-coenzyme A synthetase gene (facA) and its use as a homologous selection marker

A new transformation system for the filamentous fungus Penicillium chrysogenum is described, based on the use of the homologous acetyl-coenzyme A synthetase (facA) gene as a selection marker. Acetate-non-utilizing (Fac^–) strains of P. chrysogenum were obtained by positive selection for spontaneous resistance to fluoroacetate. Among these fac mutants putative facA strains were selected for a loss of acetyl-coenzyme A (CoA) synthetase activity. The facA gene, coding for the enzyme acetyl-CoA synthetase, was isolated from a P. chrysogenum genomic library using synthetic oligonucleotides derived from conserved regions from the corresponding genes of Aspergillus nidulans and Neurospora crassa. Vector pPC2-3, comprising a genomic 6.5 kb PstI fragment, was able to complement P. chrysogenum facA strains with frequencies up to 27 transformants·g^–1 DNA. Direct selection of transformants was accomplished using acetate and low amounts (0.001%) of glucose as carbon sources. About 50% of the transformants arose by integration of pPC2-3 DNA at the homologous facA locus and 50% by integration elsewhere in the genome. Determination of the nucleotide sequence of part of the cloned fragment showed the presence of an open reading frame of 2007 nucleotides, interrupted by five putative introns. Comparison of the nucleotide and the amino acid sequence of the facA gene of P. chrysogenum with the facA gene of A. nidulans reveals similarities of 80% and 89%, respectively. The putative introns present in the P. chrysogenum facA gene appear at identical positions as those in the A. nidulans facA gene, but show no significant sequence similarity. Correspondence to: R. F. M.van Gorcom     

Microelectrode characterization of the basolateral membrane of rabbit S3 proximal tubule

Summary The purpose of this study was to characterize the basolateral membrane of the S3 segment of the rabbit proximal tubule using conventional and ion-selective microelectrodes. When compared with results from S1 and S2 segments, S3 cells under control conditions have a more negative basolateral membrane potential (V _bl=–69 mV), a higher relative potassium conductance (t _K=0.6), lower intracellular Na⁺ activity (A _Na=18.4mm), and higher intracellular K⁺ activity (A _K=67.8mm). No evidence for a conductive sodium-dependent or sodium-independent HCO ₃ ^– pathway could be demonstrated. The basolateral Na–K pump is inhibited by 10^–4 m ouabain and bath perfusion with a potassium-free (0-K) solution. 0-K perfusion results inA _Na=64.8mm,A _K=18.5mm, andV _bl=–28 mV. Basolateral potassium channels are blocked by barium and by acidification of the bathing medium. The relative K⁺ conductance, as evaluated by increasing bath K⁺ to 17mm, is dependent upon the restingV _bl in both S2 and S3 cells. In summary, the basolateral membrane of S3 cells contains a pump-leak system with similar properties to S1 and S2 proximal tubule cells. The absence of conductive bicarbonate pathways results in a hyperpolarized cell and larger Na⁺ and K⁺ gradients across the cell borders, which will influence the transport properties and intracellular ion activities in this tubule segment.     

The NADP-dependent glutamate dehydrogenase gene from Penicillium chrysogenum and the construction of expression vectors for filamentous fungi

The gdhA gene encoding the NADP-dependent glutamate dehydrogenase activity from Penicillium chrysogenum has been isolated and characterized for its use in gene expression. The nucleotide sequence of a 2816-bp genomic fragment was determined, showing an open reading frame of 1600 bp interrupted by two introns, of 160 bp and 57 bp respectively, with fungal consensus splice-site junctions. The predicted amino acid sequence revealed a high degree of identity to glutamate dehydrogenase enzymes, especially to those from the fungi Aspergillus nidulans (82%) and Neurospora crassa (78%). The gdhA gene was found to be present in a single copy in the genome of several P. chrysogenum strains with different penicillin productivity. The use of the gdhA promoter for homologous and heterologous gene expression in fungi and Escherichia coli was analyzed. Heterologous gene expression was ascertained by the construction of gene fusions with the lacZ gene from E. coli and the bleomycin-resistance determinant (ble ^R) from Streptoalloteichus hindustanus. Homologous gene expression was shown through the use of the penicillin-biosynthetic genes pcbC and penDE from P. chrysogenum and the cephalosporin biosynthetic genes cefEF and cefG from Acremonium chrysogenum. Received: 2 November 1998 / Received revision: 15 January 1999 / Accepted: 5 March 1999     

Characterization of a 52 kDa Exoantigen of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> and Monoclonal Antibodies Suitable for its Detection

The indoor clade of Penicillium chrysogenum, the so-called Fleming clade, is the most common species of Penicillium on moldy building materials. In a previous study, we identified a 52 kDa human antigen characteristic of the indoor clade of P. chrysogenum not present in a taxonomically diverse selection of fungi. Further investigations revealed that it is a modestly glycosylated mature protein with a pI 5.3. The protein is apparently identical to a glucoamylase previously reported from an aluminum-tolerant P. chrysogenum mutant. Based on sequence similarity, molecular weight, and pI, it is distinct from a number of other glucoamylases from domesticated strains of Aspergillus oryzae and A. niger used to produce industrial enzymes. Surprisingly, it had not been reported as an allergen. The monoclonal antibodies developed have the potential for use in assays of P. chrysogenum antigens in spores and spore/mycelial fragments in dust.     

The potential effect of high atmospheric CO2 on soil fungi–invertebrate interactions

Litter mixtures of four meadow plant species, Cardamine hirsuta, Poa annua, Senecio vulgaris, and Spergula arvensis, were produced from laboratory model terrestrial ecosystems maintained at either ambient or enriched (ambient + 200 µmol mol^?1) CO₂ concentrations. The effect of litter source on the oviposition attractivity of fungi to the sciarid fly Lycoriella ingenua was tested for seven fungal species (Absidia glauca, Cladosporium cladosporioides, C. herbarum, Fusarium oxysporum, Penicillium arenicola, P. chrysogenum, and P. janthinellum). For all species, except F. oxysporum, oviposition attractivity increased when the fungi were growing on litter derived from CO₂‐enriched environments. The relative increase of the oviposition attractiveness of fungi growing on CO₂‐enriched litter differed substantially and resulted in a shift in sciarid fly oviposition preference. For example, when P. chrysogenum and C. herbarum grew on ambient litter, P. chrysogenum was more attractive; the opposite was true for mycelia growing on enriched litter. The effect of litter source on the suitability of four fungal species for larval development was also tested. In two species of fungi (A. glauca and C. herbarum) suitability was significantly higher if growing on CO₂‐enriched litter. With P. chrysogenum the opposite was true. The consequences of these rarely considered CO₂‐induced trophic interactions on ecosystem processes such as nutrient feedback cycles between plants and soil decomposition are considered.