Improving 2-phenylethanol and 6-pentyl-α-pyrone production with fungi by microparticle-enhanced cultivation (MPEC).

Trichoderma atroviride IMI 206040 synthesizes the coconut lactone 6-pentyl-α-pyrone (6-PAP) de novo and Aspergillus niger DSM 821 produces the rose-like flavour compound 2-phenylethanol (2-PE) from the precursor l-phenylalanine. Here, microparticles of different chemical composition and nominal particle diameter in the range 5-250 µm were added to shake-flask cultures of both fungi to investigate the particles' effect on product formation. Maximum 2-PE concentration increased by a factor of 1.3 to 1430 mg/l with the addition of 2% w/v talc (40 µm diameter). Maximum 6-PAP concentration increased by a factor of 2 to 40 mg/l with the addition of 2% w/v iron (II, III) oxide. The influence of ions leaching out of the particles was investigated by cultivating the fungi in leached particle medium. For the first time, the positive effect of the microparticle-enhanced cultivation (MPEC) technique on the microbial production of volatile metabolites, here flavour compounds from submerged fungal cultures, is demonstrated. The effect is strain- and particle-specific.

Production of the aroma chemicals 3-(methylthio)-1-propanol and 3-(methylthio)-propylacetate with yeasts.

Yeasts can convert amino acids to flavor alcohols following the Ehrlich pathway, a reaction sequence comprising transamination, decarboxylation, and reduction. The alcohols can be further derivatized to the acetate esters by alcohol acetyl transferase. Using L: -methionine as sole nitrogen source and at high concentration, 3-(methylthio)-1-propanol (methionol) and 3-(methylthio)-propylacetate (3-MTPA) were produced with Saccharomyces cerevisiae. Methionol and 3-MTPA acted growth inhibiting at concentrations of >5 and >2 g L(-1), respectively. With the wild type strain S. cerevisiae CEN.PK113-7D, 3.5 g L(-1) methionol and trace amounts of 3-MTPA were achieved in a bioreactor. Overexpression of the alcohol acetyl transferase gene ATF1 under the control of a TDH3 (glyceraldehyde-3-phosphate dehydrogenase) promoter together with an optimization of the glucose feeding regime led to product concentrations of 2.2 g L(-1) 3-MTPA plus 2.5 g L(-1) methionol. These are the highest concentrations reported up to now for the biocatalytic synthesis of these flavor compounds which are applied in the production of savory aroma compositions such as meat, potato, and cheese flavorings.

An aqueous-organic two-phase bioprocess for efficient production of the natural aroma chemicals 2-phenylethanol and 2-phenylethylacetate with yeast.

The natural aroma chemicals 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEAc) are of high industrial relevance and can be produced from L-phenylalanine in a yeast-based process with growth-associated product formation. Due to product inhibition, in situ product removal is mandatory to obtain economically interesting concentrations. A fed-batch approach using polypropylene glycol 1200 as in situ extractant and the precursor in a saturated concentration led to the highest 2-PE productivity reported for a bioprocess so far. With Kluyveromyces marxianus CBS 600, 26.5 g/l 2-PE and 6.1 g/l 2-PEAc in the organic phase were obtained, corresponding to space-time yields of 0.33 and 0.08 g/l h, respectively.

Applied biocatalysis for the synthesis of natural flavour compounds--current industrial processes and future prospects.

The industrial application of biocatalysis for the production of natural flavour compounds is illustrated by a discussion of the production of vanillin, gamma-decalactone, carboxylic acids, C6 aldehydes and alcohols ('green notes'), esters, and 2-phenylethanol. Modern techniques of molecular biology and process engineering, such as heterologous expression of genes, site-directed mutagenesis, whole-cell biocatalysis in biphasic systems, and cofactor regeneration for in vitro oxygenation, may result in more biocatalytic processes for the production of flavour compounds in the future.

Biotechnological production of 2-phenylethanol.

2-Phenylethanol (2-PE) is an important flavour and fragrance compound with a rose-like odour. Most of the world's annual production of several thousand tons is synthesised by chemical means but, due to increasing demand for natural flavours, alternative production methods are being sought. Harnessing the Ehrlich pathway of yeasts by bioconversion of L-phenylalanine to 2-PE could be an option, but in situ product removal is necessary due to product inhibition. This review describes the microbial production of 2-PE, and also summarizes the chemical syntheses and the market situation.