Shifting the biotransformation pathways of L-phenylalanine into benzaldehyde by Trametes suaveolens CBS 334.85 using HP20 resin

AIMS: The biotransformation of L-phenylalanine into benzaldehyde (bitter almond aroma) was studied in the strain Trametes suaveolens CBS 334.85. METHODS AND RESULTS: Cultures of this fungus were carried out in the absence or in the presence of HP20 resin, a highly selective adsorbent for aromatic compounds. For the identification of the main catabolic pathways of L-phenylalanine, a control medium (without L-phenylalanine) was supplemented with each of the aromatic compounds, previously detected in the culture broth, as precursors. Trametes suaveolens CBS 334.85 was shown to biosynthesize benzyl and p-hydroxybenzyl derivatives, particularly benzaldehyde, and large amounts of 3-phenyl-1-propanol, benzyl and p-hydroxybenzyl alcohols as the products of both cinnamate and phenylpyruvate pathways. CONCLUSION: The addition of HP20 resin, made it possible to direct the catabolism of L- phenylalanine to benzaldehyde, the desired target compound, and to trap it before its transformation into benzyl alcohol. In these conditions, benzaldehyde production was increased 21-fold, from 33 to 710 mg l-1 corresponding to a molar yield of 31%. SIGNIFICANCE AND IMPACT OF THE STUDY: These results showed the good potential of Trametes suaveolens as a biotechnological agent to synthesize natural benzaldehyde which is one of the most important aromatic aldehydes used in the flavour industry.     

Release of ferulic acid from agroindustrial by-products by the cell wall-degrading enzymes produced by Aspergillus niger I-1472

Aspergillus niger I-1472 was grown on sugar beet pulp to produce cell wall polysaccharide-degrading enzymes, including feruloyl esterases. Compared to enzymatic activities measured in commercially available mixtures previously used for the release of ferulic acid, the A. niger enzymes were more various. These enzymes were tested to release ferulic acid from sugar beet pulp, maize bran, or autoclaved maize bran. They were as efficient as the commercial mixture to release ferulic acid from sugar beet pulp. On the other hand, they were much more efficient to release ferulic acid from maize bran after autoclaving pretreatment, as 95% of ferulic acid ester were solubilized. Thus, A. niger enzymes exhibited a high interest in the release of ferulic acid from various agro-industrial by-products.     

Evaluation of the potential of Aspergillus niger species for the bioconversion of L-phenylalanine into 2-phenylethanol

Aspergillus niger was explored, for the first time, for the production of 2-phenylethanol (a rose-like aroma) using L-phenylalanine as precursor. Among the strains screened, A. niger CMICC 298302 was shown to produce, in a culture medium containing 6 g L-phenylalanine l^–1 and 60 g glucose l^–1, 1375 mg 2-phenylethanol l^–1 with a productivity of 153 mg l^–1 day^–1 and a molar yield of 74%. 2-Phenylethanol concentrations of 1 to 2 g l^–1 led to a two-fold and ten-fold decrease, respectively, in the mycelial radial growth rate. However, 2-phenylethanol was synthesized as the sole aromatic product and accumulated in the culture broth.     

Mass balance modeling of vanillin production from vanillic acid by cultures of the fungus Pycnoporus cinnabarinus in bioreactors.

A systematic two-step procedure for the structural identification of bioprocesses is followed in order to establish a mechanistic model for vanillin production by Pycnoporus cinnabarinus. The first step is devoted to the identification of the underlying reaction structure and the development of a validated mass balance model for the growth of P. cinnabarinus and the biotransformation of vanillic acid into vanillin. The second step is devoted to the kinetic modeling, namely, the estimation of the reaction rates and the calibration of the kinetic parameters. The whole procedure leads to the final set up of a simulation model of the process. The results are supported by the data from five cultures of P. cinnabarinus in bioreactors.     

By-passing of unwanted vanillyl alcohol formation using selective adsorbents to improve vanillin production with Phanerochaete chrysosporium

World Journal of Microbiology and Biotechnology -     

FunGene-DB: A web-based tool for Polyporales strains authentication

Polyporales are extensively studied wood-decaying fungi with applications in white and green biotechnologies and in medicinal chemistry. We developed an open-access, user-friendly, bioinformatics tool named FunGene-DB (http://www.fungene-db.org). The goal was to facilitate the molecular authentication of Polyporales strains and fruit-bodies, otherwise subjected to morphological studies. This tool includes a curated database that contains ITS1-5.8S-ITS2 rDNA genes screened through a semi-automated pipeline from the International Nucleotide Sequence Database (INSD), and the similarity search BLASTn program. Today, the web-accessible database compiles 2379 accepted sequences, among which 386 were selected as reference sequences (most often fully identified ITS sequences for which a voucher, strain or specimen, has been deposited in a public-access collection). The restriction of the database to one reference sequence per species (or per clade for species complex) allowed most often unequivocal analysis. We conclude that FunGene-DB is a promising tool for molecular authentication of Polyporales. It should be especially useful for scientists who are not expert mycologists but who need to check the identity of strains (e.g. for culture collections, for applied microbiology).     

Gene Overexpression and Biochemical Characterization of the Biotechnologically Relevant Chlorogenic Acid Hydrolase from Aspergillus niger

The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter⁻¹, i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 × 10⁶ M⁻¹ s⁻¹ toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.     

Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger

The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter(-1), i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 x 10(6) M(-1) s(-1) toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.     

Evidence of a new biotransformation pathway of p-coumaric acid into p-hydroxybenzaldehyde in Pycnoporus cinnabarinus

Pycnoporus cinnabarinus MUCL39533 was shown to be able to convert p-coumaric acid into p-hydroxybenzaldehyde, a component of high organoleptic note present in natural vanilla aroma. Use of phospholipid-enriched medium led to high-density cultures of P. cinnabarinus, since dry mycelial biomass was increased three-fold as compared to glucose medium. In the presence of phospholipids, 155 mg l(-1) p-hydroxybenzaldehyde was produced as the major compound on culture day 13 with a molar yield of 26%. The degradation pathways of p-coumaric acid were investigated. Based on the different metabolites identified, an oxidative side-chain degradation pathway of p-coumaric acid conversion to p-hydroxybenzoic acid was suggested. This acid was further reduced to p-hydroxybenzaldehyde and p-hydroxybenzyl alcohol, or hydroxylated and reduced to protocatechyl derivatives. Additionally, a reductive pathway of p-coumaric acid with 3-(4-hydroxyphenyl)-propanol as the terminal product occurred.     

Enhanced benzaldehyde formation by a monokaryotic strain of Pycnoporus cinnabarinus using a selective solid adsorbent in the culture medium.

A monokaryotic strain of the white-rot fungus Pycnoporus cinnabarinus was shown to produce, in a 2-L bioreactor culture, 100 mg.L-1 benzaldehyde (bitter almond aroma) from L-phenylalanine with a productivity of 33 mg.L-1.day-1. The addition of HP20 resin, a styrene divinylbenzene copolymer highly selective for benzaldehyde, enabled an eightfold increase in the production of benzaldehyde and a twofold increase in productivity. In the presence of HP20 resin, the production of 790 mg.L-1 benzaldehyde was concomitant with the synthesis of cinnamic acid derivatives of high organoleptic notes such as cinnamaldehyde, cinnamyl alcohol, and methyl cinnamate.