Investigation into biocatalytic routes towards monoterpene alcohols

The overall aim of this investigation was to identify novel bacterial and fungal
biocatalytic routes towards monoterpene alcohols such as enantiopure linalool and
menthol, and geraniol, each of which find many applications within the flavour and
fragrance industries.
The first part of the investigation targeted hydrolytic transformations of
racemic monoterpene esters. Enantioselective linalyl acetate esterase activity was
detected in Rhodococcus ruber DSM 43338, offering a potential route towards
enantiopure (R)- and (S)- linalool. Fractions displaying complementary
enantioselectivity for the reaction were isolated from anion-exchange
chromatography of cell extracts, giving enantiomeric excess values for linalool
ranging from 60% (R)- to 79% (S)-. Attempts to isolate the genes encoding relevant
esterases via polymerase chain reactions resulted in a gene fragment representing
75% of a putative tertiary alcohol esterase (TAE). An alternative gene putatively
encoding a TAB from a closely related Actinomycete Nocardia farcinica (NITAE)
was cloned and expressed in E. coli, and the enzyme purified and characterised.
NITAE was inactive towards linalyl acetate, but catalysed the hydrolysis of racemic
menthyl acetate to give the commercially-preferred 1(R)-enantiomer of menthol.
In the second part of the project, a strain of Rhodococcus erythropolis MLTl
was isolated by selective enrichment from soil surrounding hop plants using the hop
terpene f3-myrcene as sole carbon source. Resting-cell preparations transformed 13-
myrcene to the high-value monoterpene alcohol geraniol. While the relevant enzyme
remains to be identified, initial studies suggested an inducible operon for f3-myrcene
degradation in MLTl.