From a ferulic-acid-degrading Pseudomonas fluorescens strain (BF13), we have isolated a transposon mutant, which retained the ability to bioconvert ferulic acid into vanillic acid but lost the ability to further degrade the latter acid. The mutant, BF13-97, was very stable, and therefore it was suitable to be used as a biocatalyst for the preparative synthesis of vanillic acid from ferulic acid. By use of resting cells we determined the effect on the bioconversion rate of several parameters, such as the addition of nutritional factors, the concentration of the biomass, and the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with cells pregrown on M9 medium containing p-coumaric acid ( Lignin-related aromatic acids, which contain phenylpropane (C 6 -C 3 ) type structures (such as ferulic acid and related compounds), are abundant molecules that play important functions in plant cells, as antimicrobial compounds, signaling molecules, and phytoalexins (23). They commonly occur, free or in combined form, in fruits, vegetables, grains, beans, leaves, seeds, nuts, grasses, flowers, and other types of vegetation and can be easily extracted from some agriculture by-products (22). The catabolism of these compounds is an important aspect for the mineralization of plant wastes because they are released during the breakdown of lignin and cell wall materials by white-rot fungii. Moreover, there is a growing interest in the potential use of ferulic acid as feedstock for the biocatalytic conversion into other valuable molecules such as styrenes, polymers, epoxydes, alkylbenzenes, vanillin and vanillic acid derivatives, guaiacol, cathecol, and protocatechuic-acid-related cathecols (22).We previously isolated a Pseudomonas fluorescens strain, named BF13, which utilized some phenylpropenoids (ferulic and m-and p-coumaric acids) as the sole carbon source (4). This strain, when degrading ferulic acid, transiently forms in the culture medium a certain amount of vanillic acid. The latter is a valuable product, used as a starting material in the chemical synthesis of oxygenated aromatic chemicals, such as vanillin, one of the most important flavor molecules (7,22). We studied the possibility of enhancing the formation of vanillic acid during ferulic acid degradation by using suspensions of BF13 cells at high density. We demonstrated that the use of cells not adapted to ferulic acid improved the production of vanillic acid, which remained in the medium for a long period, facilitating its recovery (4). Moreover, we observed that this strain tolerated, better than others, high concentrations of ferulic acid and vanillic acid, which is a relevant property in view of a bioconversion process (unpublished data).In this work our aim was to isolate a mutant of P. fluorescens BF13 which was unable to bioconvert vanillic acid into protocatechuic acid and use this mutant as a biocatalyst for the production of vanillic acid. The molecular characterization of this mutant allowed us to clone the genes fro...

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A cDNA coding for laccase was isolated from the white-rot fungus Trametes trogii 201. This cDNA corresponded to the lcc1 gene, which coded for a precursor protein of 517 amino acids with a 21 amino acid signal peptide. Comparison of the deduced sequence with known laccases showed that this enzyme was most closely related to Lac1 from basidiomycete PM1 and Trametes C30 (98% similarity). The expression of lcc1 was analysed under different growth conditions; transcription of this gene was enhanced by the addition of organic nitrogen to the medium. The level of lcc1 transcription was higher when T. trogii was grown on synthetic medium supplemented with yeast extract rather than mycological peptone or tryptone. The transcription data were in agreement with total laccase activity measured in the supernatant and suggested that laccase production and lcc1 transcription are coordinately regulated in this organism. The lcc1 cDNA was expressed in the methylotrophic yeast Pichia pastoris and the detection of laccase activity indicated that this cDNA encodes a laccase.

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