and Jandoust 2016). PE disguises the aroma of rose flower in such a realistic manner
that the popular saying by William Shakespeare, ‘A rose by any other name would
smell as sweet’, can be reframed as ‘A rose by any other name is phenylethanol’. It is
widely used in perfumes, cosmetics, pharmaceuticals, foods and beverages
(Carlquist et al. 2015). It is also used as a raw material to produce other important
flavour compounds, such as 2-phenylethylacetate (Etschmann et al. 2002) and
phenylacetaldehyde (Guo et al. 2017).
Kluyveromyces marxianus has been determined to be a promising candidate for
industrial production of 2-PE (Fabre et al. 1997; Wittman et al. 2002). Genome
engineering has been done in Saccharomyces by introducing allele variation through
sequential oligonucleotide recombination. Designer synthetic DNA oligonucleotides
have been introduced in the original genome, which allow the combinatorial alteration of pathway genes of aromatic compounds. In the successive rounds of transformation, the yeast genome is gradually re-modelled towards the production of a
flavoured metabolite (Mitchell et al. 2015). In a targeted metabolic footprinting
method, at low initial nitrogen concentrations, Saccharomyces cerevisiae strain
KU1 produced higher quantities of esters and fatty acids, whereas M522 produced
higher concentrations of isoacids, γ-butyrolactone, higher alcohols and
3-methylthio-1-propanol (Carrau et al. 2008).
7.3.2.3 Esters
Among esters, acetate esters are usually relevant from flavour viewpoint. These are
ethyl acetate (apple aroma), isoamyl acetate (banana-like aroma) and
2-phenylethylacetate (honey- and rose-like aroma) (Verstrepen et al. 2003). Sachsia
suaveolens and Oidium suaveolens are known to produce certain fruity odours due to
methylbutanols and other esters (Hattori et al. 1974). The yeasts Hanseniaspora
guilliermondii and Pichia anomala are potent 2-phenylethyl acetate and isoamyl
acetate producers, respectively (Rojas et al. 2001). The alleles which confer superior
production of phenylethyl acetate have been identified to be wild-type TOR1 allele
and a superior FAS2BTCD allele in Saccharomyces cerevisiae using polygenic
analysis. A hybrid diploid Saccharomyces cerevisiae yeast strain was developed
by crossing two descendants from the unrelated industrial yeast strains, ‘ale’ yeast
and the other was a bioethanol production yeast. Exchange of both superior alleles in
the ER18 parent strain increased 2-PEAc production to 70%. The polygenic analysis
combined with CRISPR/Cas9-mediated allele exchange comprises a novel strategy
which could be used for the creation of cis-genic yeasts having a novel flavour
profile which could be used for the production of alcoholic beverages (de Carvalho
et al. 2017). Lomascolo et al. (2001) have selected S. cerevisiae mutants which can
convert L-phenylalanine added in the medium via deamination, decarboxylation and
subsequent reduction into 2-phenylethanol. A high yield of the fragrant ester (>2 g/
L) was obtained by solvent extraction of the fermentation broth. Double coupled
system was used to identify non-Saccharomyces yeasts from Agave duranguensis,
which showed that Pichia fermentans can be used for the biotechnological production of isoamyl acetate (Hernández-Carbajal et al. 2013). E. coli, genetically
engineered for the synthesis of banana flavour, nicknamed as ‘Eau d’coli’, was
7 Biotechnological Interventions for Production of Flavour and Fragrance. . .
141
that the popular saying by William Shakespeare, ‘A rose by any other name would
smell as sweet’, can be reframed as ‘A rose by any other name is phenylethanol’. It is
widely used in perfumes, cosmetics, pharmaceuticals, foods and beverages
(Carlquist et al. 2015). It is also used as a raw material to produce other important
flavour compounds, such as 2-phenylethylacetate (Etschmann et al. 2002) and
phenylacetaldehyde (Guo et al. 2017).
Kluyveromyces marxianus has been determined to be a promising candidate for
industrial production of 2-PE (Fabre et al. 1997; Wittman et al. 2002). Genome
engineering has been done in Saccharomyces by introducing allele variation through
sequential oligonucleotide recombination. Designer synthetic DNA oligonucleotides
have been introduced in the original genome, which allow the combinatorial alteration of pathway genes of aromatic compounds. In the successive rounds of transformation, the yeast genome is gradually re-modelled towards the production of a
flavoured metabolite (Mitchell et al. 2015). In a targeted metabolic footprinting
method, at low initial nitrogen concentrations, Saccharomyces cerevisiae strain
KU1 produced higher quantities of esters and fatty acids, whereas M522 produced
higher concentrations of isoacids, γ-butyrolactone, higher alcohols and
3-methylthio-1-propanol (Carrau et al. 2008).
7.3.2.3 Esters
Among esters, acetate esters are usually relevant from flavour viewpoint. These are
ethyl acetate (apple aroma), isoamyl acetate (banana-like aroma) and
2-phenylethylacetate (honey- and rose-like aroma) (Verstrepen et al. 2003). Sachsia
suaveolens and Oidium suaveolens are known to produce certain fruity odours due to
methylbutanols and other esters (Hattori et al. 1974). The yeasts Hanseniaspora
guilliermondii and Pichia anomala are potent 2-phenylethyl acetate and isoamyl
acetate producers, respectively (Rojas et al. 2001). The alleles which confer superior
production of phenylethyl acetate have been identified to be wild-type TOR1 allele
and a superior FAS2BTCD allele in Saccharomyces cerevisiae using polygenic
analysis. A hybrid diploid Saccharomyces cerevisiae yeast strain was developed
by crossing two descendants from the unrelated industrial yeast strains, ‘ale’ yeast
and the other was a bioethanol production yeast. Exchange of both superior alleles in
the ER18 parent strain increased 2-PEAc production to 70%. The polygenic analysis
combined with CRISPR/Cas9-mediated allele exchange comprises a novel strategy
which could be used for the creation of cis-genic yeasts having a novel flavour
profile which could be used for the production of alcoholic beverages (de Carvalho
et al. 2017). Lomascolo et al. (2001) have selected S. cerevisiae mutants which can
convert L-phenylalanine added in the medium via deamination, decarboxylation and
subsequent reduction into 2-phenylethanol. A high yield of the fragrant ester (>2 g/
L) was obtained by solvent extraction of the fermentation broth. Double coupled
system was used to identify non-Saccharomyces yeasts from Agave duranguensis,
which showed that Pichia fermentans can be used for the biotechnological production of isoamyl acetate (Hernández-Carbajal et al. 2013). E. coli, genetically
engineered for the synthesis of banana flavour, nicknamed as ‘Eau d’coli’, was
7 Biotechnological Interventions for Production of Flavour and Fragrance. . .
141