6 BiotransformationIBioconversion
Many volatile target compounds are not amenable to a microbial de novo synthesis
in anything like acceptable yields. Both constitutive or inducable microbial enzymes, however, turn over biotic intermediates and even xenobiotics in single step
(biotransformations) or multistep reactions (bioconversions) to products more
valuable from a flavor point of view. Biocatalysts can enlarge, degrade, or modify
the substrate, thereby supplementing or replacing chemosynthesis. Particularly
attractive is the breakdown of complex natural products to volatiles. Recent oil
tanker accidents near the Shetlands and in Prince William Sound, Alaska, have
unintentionally demonstrated to a broader public the impressive biodegradative
capabilities of the indigenous microflora (Venosa et al., 1992).
As outlined above (cf. Sect. 3.3), specificities and physiological conditions of
biochemical reactions favor their application in the field of volatile flavors. Biocatalysis competes best with chemical catalysis in the following types of reactions:
-
-
-
Functionalization of chemically inert carbons. Examples were regioselective
hydroxylations of terpenoids.
Selective modification of one functional group in multifunctional molecules.
Hydrogenation of one out of several double bonds was described above
(Fig. 3.2).
Introduction of chirality. Transformation of prochiral carbons, for example to
hydroxy carboxylic acids or volatile 2-alkanols, or the lipoxygenase induced
hydroperoxidation of fatty acids could be cited.
Resolution of racemates. This is industrially used to separate racemic <x-amino
acids and menthol acylates.
The beginning of modem transformation biotechnology is usually dated back to
Neuberg's classical work with yeast-catalyzed reactions of terpenes and benzaldehyde. The acyloin type condensation of benzaldehyde and acetaldehyde is still
used to produce an intermediate of ephedrin. After the first bioactive steroids were
structurally characterized, microbial dehydrogenation, side chain degradation, and
hydroxylation of steroid skeletons led to a cost-effective production of building
blocks for subsequent chemoderivatization to pharmaceuticals (Vischer and Wehstein, 1953). These findings had a strong impetus on further research. In 1960, the
group of Bhattacharyya launched a series of classical publications on the microbial
transformation of volatile terpenoids; Aspergillus niger and, later on, Pseudomonas strains were the catalysts of choice (Dhavalikar and Bhattacharyya, 1966).
This work used a variety of mono- and sesquiterpene hydrocarbons, sometimes as
R. G. Berger, Aroma Biotechnology
© Springer-Verlag Berlin Heidelberg 1995
6 BiotransformationIBioconversion
79
the sole source of carbon, to evidence a degradation to carbon dioxide and water
that was accompanied by the occurrence of partially oxidized, volatile compounds.
The following reaction steps were observed:
-
Oxidation of methyl groups, and of primary, secondary, tertiary (3-carene), and
phenolic (thymol) hydroxy groups
Hydroxylation of allylic carbons, sp2-carbon (to yield oxo compounds),
dihydroxylation of vicinal positions (limonene)
(De)hydrogenation, epoxidation, isomerization, and hydration of carbon double
bonds
Cleavage of carbon/carbon and carbon/oxygen bonds
In sharp contrast to the current use of biocatalysts in steroid transformation, the
possibility of applying the same technique to lower terpenoids has been largely
neglected by industry. Difficulties encountered are the incompatibility of lipophilic
substrates and aqueous bioprocess media, the generation of useless or difficult to
separate by-products, the lack of predictability if new strains are used, and the
sufficient availability of many volatiles terpenes from plant sources. Novel concepts to overcome some of the inherent drawbacks will be discussed later (cf.
Sect. 11.1.1)
A survey over the present literature shows that filamentous fungi and prokaryotes share a preference for converting volatile terpenes. The absolute number of
genera specialised in this metabolism is surprisingly small (Table 6.1 ). Yeasts,
with few exceptions, are not applicable to terpene bioconversions (cf. Sect. 2.3.1).
To improve the chances of success, an organism should be selected that is known
to perform the desired type of reaction on other compounds. If a mixed culture
isolated from a natural habitat is used instead of a commercial strain, the member
with the highest activity is easily selected by enrichment media containing the
substrate as a sole carbon source. Eukaryotic organisms are usually more sensitive
to terpenes and must be adapted stepwise. The surroundings of plant terpene producers should be considered as a particular ecological niche for adapted strains.
Much of the recent work on microbial transformations of volatile terpenes was
conducted by the group of Kieslich and Abraham (Kieslich et al., 1985). A database covering worldwide information on microbial transformations is currently being
set up (Kieslich, 'pers. comm.). Hence, discussion can be limited to representative
examples .
. Mycobacterium smegmatis was most suitable among more than 100 strains tested to convert the activated methyl groups of (+)-2- and (+)-3-carene to chaminic
acids, insect repellents difficult to obtain by chemosynthesis (Stumpf et al., 1990).
Carenones and a compound with a cleaved cyclopropane ring were identified as
volatile by-products (Fig. 6.1). Geranial, neral, and further alkadienals reacted to
hydroxyketones in fermentations of Mucor circinelloides (Stumpf and Kieslich,
1991). The acyloins were immediately reduced to (2S,3R)-diols. Reduction of
e--C, and 0:0 bonds, cyclizations, and further catabolic reactions yielded several
other products. Previously, acyloin formation ,was believed to be restricted to
yeasts. The spectrum of transformable aldehydes was greater in the Mucor culture