Characterization of three GH35 ß-galactosidases, enzymes able to shave galactosyl residues linked to rhamnogalacturonan in pectin, from Penicillium chrysogenum 31B.

Three recombinant ß-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and 1H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze ß-1,4-galactosyl residues linked to L-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-ß-D-galactopyranoside and ß-D-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the ß-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked ß-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.

Identification of a novel Penicillium chrysogenum rhamnogalacturonan rhamnohydrolase and the first report of a rhamnogalacturonan rhamnohydrolase gene.

Rhamnogalacturonan (RG) I is one of the main components of pectins in the plant cell wall. We recently reported two RG I-degrading enzymes, endo-RG and exo-RG lyases, secreted by Penicillium chrysogenum 31B. Here, our aims were to purify a RG rhamnohydrolase (PcRGRH78A) from the culture filtrate of this strain and to characterize this enzyme. On the basis of the internal amino acid sequences, the encoding gene, Pcrgrh78A, was cloned and overexpressed in Aspergillus oryzae. The deduced amino acid sequence of PcRGRH78A is highly similar to an uncharacterized protein belonging to glycoside hydrolase family 78. Pfam analysis revealed that PcRGRH78A contains a bacterial α-l-rhamnosidase (PF05592) domain. PcRGRH78A shows optimal activity at 45°C and pH 5. The specificity of PcRGRH78A toward rhamnose (Rha)-containing substrates was compared with that of a P. chrysogenum α-l-rhamnosidase (PcRHA78B) belonging to glycoside hydrolase family 78. PcRGRH78A specifically hydrolyzes RG oligosaccharides that contain Rha at their nonreducing ends, releasing the Rha, but has no activity toward naringin, hesperidin, or rutin. In contrast, PcRHA78B effectively degrades p-nitrophenyl α-l-rhamnopyranoside and the three flavonoids, but not RG oligosaccharides. When galactosyl RG oligosaccharides were used as the substrate, PcRGRH78A released Rha in 3.5-fold greater amounts in the presence of ß-galactosidase than in its absence, indicating that PcRGRH78A preferentially acts on Rha residues without the galactose moiety at nonreducing ends. To our knowledge, this is the first report of a gene encoding a RG rhamnohydrolase.

A novel low-temperature-active pectin methylesterase from Penicillium chrysogenum F46 with high efficiency in fruit firming.

A pectin methylesterase gene (pe8F46) was cloned from Penicillium chrysogenum F46 and successfully expressed in Pichia pastoris. The full-length cDNA consists of 969 bp and encodes a 322-residue polypeptide with the calculated molecular weight of 34.1 kDa. Deduced PE8F46 belongs to family 8 of carbohydrate esterases and shares 54% identity with a functionally characterised counterpart from Myceliophthora thermophile. Purified recombinant PE8F46 showed the optimal activity at pH 5.0 and 40°C, and remained 52% maximum activity even at 10°C. An orthogonal experiment was employed to determine the best conditions for firming pineapple dices. After incubation with 0.75% (w/v) PE8F46 and 0.4% calcium lactate (w/v) for 20 min, the firmness of pineapple dices was improved by 47.6%, 13.7% higher than that of a commercial pectinase complex. These results suggest that PE8F46 has application potential in the food industry.

A novel GH43 α-l-arabinofuranosidase of Penicillium chrysogenum that preferentially degrades single-substituted arabinosyl side chains in arabinan.

We previously described three α-l-arabinofuranosidases (ABFs) secreted by Penicillium chrysogenum 31B. Here, we purified a fourth ABF, termed PcABF43A, from the culture filtrate. The molecular mass of the enzyme was estimated to be 31kDa. PcABF43A had the highest activity at 35°C and at around pH 5. The enzyme activity was strong on sugar beet l-arabinan but weak on debranched arabinan and arabinoxylan. Low molecular-mass substrates such as p-nitrophenyl α-l-arabinofuranoside, α-1,5-l-arabinooligosaccharides, and branched arabinotriose were highly resistant to the action of PcABF43A. (1)H-NMR analysis revealed that PcABF43A hydrolyzed arabinosyl side chains linked to C-2 or C-3 of single-substituted arabinose residues in l-arabinan. Reports concerning enzymes specific for l-arabinan are quite limited. Pcabf43A cDNA encoding PcABF43A was isolated by in vitro cloning. The deduced amino acid sequence of the enzyme shows high similarities with the sequences of other fungal uncharacterized proteins. Semi-quantitative RT-PCR analysis indicated that the Pcabf43A gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was induced with pectic components such as l-arabinose, l-rhamnose, and d-galacturonic acid. Analysis of enzymatic characteristics of PcABF43A, GH51 ABF (AFQ1), and GH54 ABF (AFS1) from P. chrysogenum suggested that PcABF43A and AFS1 function as debranching enzymes and AFQ1 plays a role of saccharification in the degradation of l-arabinan by this fungus.

Quantitative PCR study on the mode of action of oligosaccharide elicitors on penicillin G production by Penicillium chrysogenum.

AIM: To investigate the effects of single and multiple additions of the oligosaccharide elicitors, obtained from alginate and locust bean gum, on penicillin G production and the transcript level of penicillin G biosynthetic genes. METHODS AND RESULTS: The transcript copy numbers and penicillin G concentration in liquid cultures of Penicillium chrysogenum grown under control and elicited conditions were compared using quantitative PCR and HPLC assay respectively. An increase in the penicillin G production rate and transcript copy numbers of the three major penicillin G biosynthetic genes pcbAB, pcbC and penDE was observed in the elicited cultures compared to control cultures. The effects were observed to be higher in multiple elicitor added cultures compared to single elicitor supplemented and control cultures. CONCLUSIONS: The results show, for the first time in bioreactor cultures, the enhancement of penicillin G transcript copy number of the penicillin biosynthetic genes using qPCR with a corresponding increase in the penicillin G production upon multiple elicitor addition of two different types of elicitors. SIGNIFICANCE AND IMPACT OF THE STUDY: Establishment of the effect of multiple elicitor addition on penicillin G production and investigating the role of oligosaccharide elicitors as transcriptional activators has wide spread impact for antibiotic industry.

Exploring and dissecting genome-wide gene expression responses of Penicillium chrysogenum to phenylacetic acid consumption and penicillinG production.

BACKGROUND: Since the discovery of the antibacterial activity of penicillin by Fleming 80 years ago, improvements of penicillin titer were essentially achieved by classical strain improvement through mutagenesis and screening. The recent sequencing of Penicillium chrysogenum strain Wisconsin1255-54 and the availability of genomics tools such as DNA-microarray offer new perspective. RESULTS: In studies on beta-lactam production by P. chrysogenum, addition and omission of a side-chain precursor is commonly used to generate producing and non-producing scenarios. To dissect effects of penicillinG production and of its side-chain precursor phenylacetic acid (PAA), a derivative of a penicillinG high-producing strain without a functional penicillin-biosynthesis gene cluster was constructed. In glucose-limited chemostat cultures of the high-producing and cluster-free strains, PAA addition caused a small reduction of the biomass yield, consistent with PAA acting as a weak-organic-acid uncoupler. Microarray-based analysis on chemostat cultures of the high-producing and cluster-free strains, grown in the presence and absence of PAA, showed that: (i) Absence of a penicillin gene cluster resulted in transcriptional upregulation of a gene cluster putatively involved in production of the secondary metabolite aristolochene and its derivatives, (ii) The homogentisate pathway for PAA catabolism is strongly transcriptionally upregulated in PAA-supplemented cultures (iii) Several genes involved in nitrogen and sulfur metabolism were transcriptionally upregulated under penicillinG producing conditions only, suggesting a drain of amino-acid precursor pools. Furthermore, the number of candidate genes for penicillin transporters was strongly reduced, thus enabling a focusing of functional analysis studies. CONCLUSION: This study demonstrates the usefulness of combinatorial transcriptome analysis in chemostat cultures to dissect effects of biological and process parameters on gene expression regulation. This study provides for the first time clear-cut target genes for metabolic engineering, beyond the three genes of the beta-lactam pathway.

NADPH-dependent glutamate dehydrogenase in Penicillium chrysogenum is involved in regulation of beta-lactam production.

The interactions between the ammonium assimilatory pathways and beta-lactam production were investigated by disruption of the NADPH-dependent glutamate dehydrogenase gene (gdhA) in two industrial beta-lactam-producing strains of Penicillium chrysogenum. The strains used were an adipoyl-7-ADCA- and a penicillin-producing strain. The gdhA gene disruption caused a decrease in maximum specific growth rate of 26 % and 35 % for the adipoyl-7-ADCA-producing strain and the penicillin-producing strain, respectively, compared to the corresponding reference strains. Interestingly, no beta-lactam production was detected in either of the DeltagdhA strains. Supplementation with glutamate restored growth but no beta-lactam production was detected for the constructed strains. Cultures with high ammonium concentrations (repressing conditions) and with proline as nitrogen source (de-repressed conditions) showed continued beta-lactam production for the reference strains whereas the DeltagdhA strains remained non-productive under all conditions. By overexpressing the NAD-dependent glutamate dehydrogenase, the specific growth rate could be restored, but still no beta-lactam production was detected. The results indicate that the NADPH-dependent glutamate dehydrogenase may be directly or indirectly involved in the regulation of beta-lactam production in industrial strains of P. chrysogenum.

Penicillium chrysogenum Pex5p mediates differential sorting of PTS1 proteins to microbodies of the methylotrophic yeast Hansenula polymorpha.

We have isolated the Penicillium chrysogenum pex5 gene encoding the receptor for microbody matrix proteins containing a type 1 peroxisomal targeting signal (PTS1). Pc-pex5 contains 2 introns and encodes a protein of approximately 75 kDa. P. chrysogenum pex5 disruptants appear to be highly unstable, show poor growth, and are unable to sporulate asexually. Furthermore, pex5 cells mislocalize a fluorescent PTS1 reporter protein to the cytosol. Pc-pex5 was expressed in a PEX5 null mutant of the yeast Hansenula polymorpha. Detailed analysis demonstrated that the PTS1 proteins dihydroxyacetone synthase and catalase were almost fully imported into microbodies. Surprisingly, alcohol oxidase, which also depends on Pex5p for import into microbodies, remained mainly in the cytosol. Thus, P. chrysogenum Pex5p has a different specificity of cargo recognition than its H. polymorpha counterpart. This was also suggested by the observation that Pc-Pex5p sorted a reporter protein fused to various functional PTS1 signals with different efficiencies.

Inactivation of the lys7 gene, encoding saccharopine reductase in Penicillium chrysogenum, leads to accumulation of the secondary metabolite precursors piperideine-6-carboxylic acid and pipecolic acid from alpha-aminoadipic acid.

Pipecolic acid serves as a precursor of the biosynthesis of the alkaloids slaframine and swainsonine (an antitumor agent) in some fungi. It is not known whether other fungi are able to synthesize pipecolic acid. Penicillium chrysogenum has a very active alpha-aminoadipic acid pathway that is used for the synthesis of this precursor of penicillin. The lys7 gene, encoding saccharopine reductase in P. chrysogenum, was target inactivated by the double-recombination method. Analysis of a disrupted strain (named P. chrysogenum SR1-) showed the presence of a mutant lys7 gene lacking about 1,000 bp in the 3'-end region. P. chrysogenum SR1- lacked saccharopine reductase activity, which was recovered after transformation of this mutant with the intact lys7 gene in an autonomously replicating plasmid. P. chrysogenum SR1- was a lysine auxotroph and accumulated piperideine-6-carboxylic acid. When mutant P. chrysogenum SR1- was grown with L-lysine as the sole nitrogen source and supplemented with DL-alpha-aminoadipic acid, a high level of pipecolic acid accumulated intracellularly. A comparison of strain SR1- with a lys2-defective mutant provided evidence showing that P. chrysogenum synthesizes pipecolic acid from alpha-aminoadipic acid and not from L-lysine catabolism.

Cloning and characterization of an aromatic amino acid and leucine permease of Penicillium chrysogenum.

The gene encoding the amino acid permease ArlP (Aromatic and leucine Permease) was isolated from the filamentous fungus Penicillium chrysogenum after PCR using degenerated oligonucleotides based on conserved regions of fungal amino acid permeases. The cDNA clone was used for expression of the permease in Saccharomyces cerevisiae M4054, which is defective in the general amino acid permease Gap1. Upon overexpression, an increase in the uptake of L-tyrosine, L-phenylalanine, L-tryptophan and L-leucine was observed. Further competition experiments indicate that ArlP recognizes neutral and aromatic amino acids with an unbranched beta-carbon atom.

Conversion of pipecolic acid into lysine in Penicillium chrysogenum requires pipecolate oxidase and saccharopine reductase: characterization of the lys7 gene encoding saccharopine reductase.

Pipecolic acid is a component of several secondary metabolites in plants and fungi. This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. In Penicillium chrysogenum pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the lys1, lys2, or lys3 genes allowing a slow growth of these auxotrophs. We have isolated two P. chrysogenum mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine. These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine. The 10.25 mutant was unable to grow in Czapek medium supplemented with alpha-aminoadipic acid. A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named lys7) revealed that it encoded a protein with high similarity to the saccharopine reductase from Neurospora crassa, Magnaporthe grisea, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that lys7 encodes a functional saccharopine reductase. Our data suggest that in P. chrysogenum the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.

The manganese superoxide dismutase from the penicillin producer Penicillium chrysogenum.

The antioxidant enzyme superoxide dismutase has been studied in order to define mechanisms for the influence of oxygen on penicillin production. Manganese-containing SOD activity was purified from penicillin-producing cultures of the filamentous fungus Penicillium chrysogenum and reverse genetics was used to identify full-length cDNA and genomic clones. Sequence analysis revealed a 630-bp ORF containing three exons and two introns with fungal consensus splice-site junctions. The deduced amino-acid sequence (210 amino acids; 23.13 kDa) includes conserved residues required for enzymatic activity and metal binding, and shares significant similarity with Mn- and Fe-containing superoxide dismutases. The sod gene is present as a single copy in the genome of different P. chrysogenum strains and its expression level is not correlated with penicillin-G productivity.

NRE, the major nitrogen regulatory protein of Penicillium chrysogenum, binds specifically to elements in the intergenic promoter regions of nitrate assimilation and penicillin biosynthetic gene clusters.

NRE, the nitrogen regulatory protein of Penicillium chrysogenum, contains a single Cys2/Cys2-type zinc-finger motif followed immediately by a highly basic region. The zinc-finger domain was expressed to Escherichia coli as a fusion protein with beta-galactosidase. In order to test the putative DNA-binding ability of NRE, the intergenic promoter region of the nitrate reductase/nitrite reductase gene cluster (niiA-niaD) of Penicillium was sequenced. Our results show that NRE is a DNA-binding protein and binds to the intergenic promoter regions of the P. chrysogenum niiA-niaD and acvA-pcbC gene cluster, encoding the first two enzymes in penicillin biosynthesis. Three of the four high-affinity NRE-binding sites contained two GATA core elements. In one of the recognition sites for NRE, one GATA motif was replaced by GATT. The two GATA elements showed all possible orientations, head-to-head, head-to-tail and tail-to-tail, and were separated by between 4 and 27 bp. Missing-contact analysis showed that all three purines in both of the GATA core sequences and the single adenine residue in each of the complementary TATC sequences were involved in the binding of NRE. Moreover, loss of purines in the flanking regions of the GATA elements also affect binding of NRE, as their loss causes reduced affinity.

Expression of the penDE gene of Penicillium chrysogenum encoding isopenicillin N acyltransferase in Cephalosporium acremonium: production of benzylpenicillin by the transformants.

No DNA sequence homologous to the penDE gene of Penicillium chrysogenum was found in the genome of three different strains of Cephalosporium acremonium. The pcbC-penDE gene cluster of P. chrysogenum complemented the isopenicillin N synthase deficiency of C. acremonium mutant N2 and resulted in the production of penicillin, in addition to cephalosporin, in cultures supplemented with phenylacetic acid. The penicillin formed was identified as benzylpenicillin by HPLC and NMR studies. The penDE gene of P. chrysogenum is expressed in C. acremonium forming a transcript of 1.15 kb. The transcript is processed and translated in C. acremonium resulting in the formation of acyl CoA: isopenicillin N acyl transferase. When the penDE gene was introduced into a cephalosporin producing strain, the total titre of beta-lactam antibiotics comprised distinct proportions of penicillin and cephalosporin in different transformants. Analysis of the hybridization patterns of the DNA of C. acremonium transformed with the pcbC or penDE genes indicated that integration occurs by non-homologous recombination.