Microbial biotransformations

Microorganisms are extremely versatile and have captured practicallyevery ecological niche. Therefore it's not surprising that a multitude ofbacteria and fungi can also be isolated from habitats rich in terpenoids andare capable of metabolizing terpenes by specific enzymes. Some of theseorganisms use terpenoids as sole energy and carbon source and can degrade themcompletely. In contrast other microbes only modify these substances to some extent, e.g. by introduction of oxygen atoms. Such biotransformations can be applied in biotechnological processes to convert a specific terpene substrate into an interesting derivative of higher value. Expecially the phyla of ascomycetes and basidiomycetes contain many species capable to biotransform terpenoids.

Our group performs screenings with microorganism collections to identify strains that show strong abilities to transform terpene structures of industrial importance. Current projects mainly address mono-, sesqui- and triterpenes whose oxyfunctionalized derivatives are of high interest to the aroma, cosmetics and pharma industries. For the first time we found lilac aldehydes and lilac alcohols as conversion products of the monoterpene linalool by specific filamentous fungi. Certain yeast strains able to efficiently hydroxylate (+)-limonene with high regio- and stereoselectivities are also currently investigated.

After identification of a microorganism suitable for a specific biotransformation experiments in lab scale bioreactors are carried out to evaluate the potential of the biocatalyst and to optimize process parameters. Another strategy can be the heterologous overexpression of genes encoding the biotransformation enzymes in well established host organisms such as Escherichia coli or Saccharomyces cerevisiae .<

Bacterial biotransformations

Due to its pronounced tolerance against organic solvents the bacterium Pseudomonas putida is very well suited for the oxidation of the monocyclic monoterpene (+)-limonene to (+)-perillic acid and is able to grow even in high concentrations of the cytotoxic precursor. The biotransformation of (+)-limonene to (+)-perillic acid is known from literature. High concentrations of the inhibiting product perillic acid can only be obtained if a fed-batch bioprocess is coupled with   in-situ product removal (ISPR) based on anion exchange resins. Currently, the applicability of this bacterium for other terpene biotransformations is being investigated. Recombinant E. coli cells harboring a genetically evolved P450 monooxygenase from Bacillus megaterium can convert the bicyclic monoterpene α-pinene to value-added natural flavor compounds or precursors thereof. An aqueous-organic two-phase bioprocess for simultaneous in-situ substrate delivery and product removal was developed. Further improvement of the efficiency of the whole-cell biocatalyst could be established by cloning of a recombinant intracellular regeneration system for the cofactor NADPH. In the future this recombinant strain will serve as platform host for the expression and biocatalytic utilization of other NAD(P)H-dependant oxidoreductases.

The methylotrophic bacterium Methylobacterium extorquens naturally synthesizes carotenoids. These chromophoric isoprenoids impart an intensive red color to the culture. Furthermore, under adequate process conditions the bacterium is capable to grow to high cell densities with methanol as sole carbon and energy source. This feature, along with established molecular biological techniques, renders M. extorquens an interesting alternative to sugar-based fermentation processes for industrial biotechnology.

Yeasts

Yeasts are mostly associated with aroma generation in traditional food processing and thus have a great potential for industrial flavor and fragrance generation. For example under certain conditions they convert amino acids to the corresponding higher alcohols and their acetate esters via the Ehrlich pathway.

Optimizing the medium and process conditions can make yeasts such asKluyveromyces marxianus produce unphysiologically high concentrations of these aroma compounds. However, the inhibiting effect of the products is quickly triggered and can only be alleviated by the application of in-situ product removal methods such as liquid-organic two-phase systems or organophilic pervaporation. Both can be used for the generation of the rose-like 2-phenylethanol and 2-phenylethylacetate from L-phenylalanine in high concentrations and high purity.
Other valuable products are the potato-like methionol and 3-methylthiopropylacetate, which can be synthesized by genetically modified Saccharomyces cerevisiae strains in an optimized bioprocess on the grams per litre scale.

Further flavor compounds are derived from unsaturated fatty acids. Overexpressing the respective genes of the plant fatty acid metabolism in S. cerevisiae makes the yeast produce C6 aldehydes and alcohols from fatty acid hydroperoxides. Known as "green notes" they are used as flavors in food and beverages.
Current research focuses on metabolic engineering of S. cerevisiae for multistep biotransformations and the development of substrate- and product specific integrated bioprocesses. Thus yeast shall be used as a model whole-cell biocatalyst for the synthesis of a multitude of flavors, fragrances and bioactives in general.