responsible for this conversion has been purified (Wolken 2003). Some enzymes
which are involved in terpene biosynthesis have been sequenced and characterized.
The fragrance of lemon-scented sweet basil (Ocimum basilicum) is due to geraniol,
produced from geranyl diphosphate, catalysed by geraniol synthase (GES). Iijima
et al. (2004) isolated gene for GES, sequenced and expressed in Escherichia coli.
Similarly, geraniol synthase gene (CtGES) present in geraniol chemotypes of
Cinnamomum tenuipilum has also been cloned and expressed in E. coli (Yang
et al. 2004).
Limonene, a cyclic monoterpene, is used widely in citrus-flavored products such
as soft drinks and candies, and also in fragrant household cleaning products and
perfumes (Duetz et al. 2003). It is the principal precursor in number of biotechnological monoterpenoids production; used for a variety of fine chemicals such as
perillyl alcohol or carvone (Marmulla and Harder 2014). Both chiral forms of
limonene have different aroma characteristics and hence differ in their applications;
(+)-Limonene (also called R- or d-limonene) has a pleasant, orange-like odour
whereas the (À)-form (also called S- or l-limonene) has a more harsh turpentinelike odour with a lemon note (Friedman and Miller 1971). d-limonene is present in
the oils of citrus fruits (70–98%), it is chiefly produced as a side product from the
citrus juice industry (Ciriminna et al. 2014). The production and cost of limonene
mainly depends on the availability of citrus oil, which is usually alleviated by
various environmental factors such as bacterial disease in citrus plants. In plants,
limonene is bio-synthesized from the precursor geranyl diphosphate (GPP) by the
enzymatic biotransformation with d- or l-limonene synthetase. In microorganisms,
GPP is produced via the methylerythritol phosphate (MEP) pathway from pyruvate
and glyceraldehyde-3-phosphate or by mevalonate pathway (Carter et al. 2003;
Jongedijk et al. 2016). Due to the less amounts of geranyl diphosphate (GPP) in
microorganisms, lower yield of limonene is obtained. Hence, the availability of GPP
and consequently the yield of limonene in microorganisms have to be increased
using metabolic engineering approaches. Willrodt et al. (2014) optimized the synthesis of (S)-limonene from glycerol and glucose as carbon sources in a two liquid
phase fed batch setup using recombinant Escherichia coli. A fourfold increase in the
yield of limonene in E. coli was observed by limiting the amount of magnesium
sulphate in the fermentation medium (Willrodt et al. 2016).
Another strategy includes the use of a truncated version of 3-hydroxy-3methylglutaryl-CoA reductase (tHMGR), a key regulatory enzyme of the
mevalonate pathway to increase limonene production by mevalonate pathway
(Willrodt et al. 2014; Zebec et al. 2016). A genetically engineered yeast, Yarrowia
lipolytica, was used to produce d-limonene and l-limonene by heterologous expression of 10 genes which included d-limonene synthase gene and l-limonene synthase
gene, respectively. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to
be the key rate-limiting enzyme in the mevalonate (MVA) pathway for improving
limonene synthesis in Y. lipolytica. It significantly increased the titres of both
d-limonene and l-limonene upon overexpression (Pang et al. 2019). Heterologous
expression of codon optimized neryl diphosphate synthase-1 (NDPS1) and limonene
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T. Malik and S. Rawat
which are involved in terpene biosynthesis have been sequenced and characterized.
The fragrance of lemon-scented sweet basil (Ocimum basilicum) is due to geraniol,
produced from geranyl diphosphate, catalysed by geraniol synthase (GES). Iijima
et al. (2004) isolated gene for GES, sequenced and expressed in Escherichia coli.
Similarly, geraniol synthase gene (CtGES) present in geraniol chemotypes of
Cinnamomum tenuipilum has also been cloned and expressed in E. coli (Yang
et al. 2004).
Limonene, a cyclic monoterpene, is used widely in citrus-flavored products such
as soft drinks and candies, and also in fragrant household cleaning products and
perfumes (Duetz et al. 2003). It is the principal precursor in number of biotechnological monoterpenoids production; used for a variety of fine chemicals such as
perillyl alcohol or carvone (Marmulla and Harder 2014). Both chiral forms of
limonene have different aroma characteristics and hence differ in their applications;
(+)-Limonene (also called R- or d-limonene) has a pleasant, orange-like odour
whereas the (À)-form (also called S- or l-limonene) has a more harsh turpentinelike odour with a lemon note (Friedman and Miller 1971). d-limonene is present in
the oils of citrus fruits (70–98%), it is chiefly produced as a side product from the
citrus juice industry (Ciriminna et al. 2014). The production and cost of limonene
mainly depends on the availability of citrus oil, which is usually alleviated by
various environmental factors such as bacterial disease in citrus plants. In plants,
limonene is bio-synthesized from the precursor geranyl diphosphate (GPP) by the
enzymatic biotransformation with d- or l-limonene synthetase. In microorganisms,
GPP is produced via the methylerythritol phosphate (MEP) pathway from pyruvate
and glyceraldehyde-3-phosphate or by mevalonate pathway (Carter et al. 2003;
Jongedijk et al. 2016). Due to the less amounts of geranyl diphosphate (GPP) in
microorganisms, lower yield of limonene is obtained. Hence, the availability of GPP
and consequently the yield of limonene in microorganisms have to be increased
using metabolic engineering approaches. Willrodt et al. (2014) optimized the synthesis of (S)-limonene from glycerol and glucose as carbon sources in a two liquid
phase fed batch setup using recombinant Escherichia coli. A fourfold increase in the
yield of limonene in E. coli was observed by limiting the amount of magnesium
sulphate in the fermentation medium (Willrodt et al. 2016).
Another strategy includes the use of a truncated version of 3-hydroxy-3methylglutaryl-CoA reductase (tHMGR), a key regulatory enzyme of the
mevalonate pathway to increase limonene production by mevalonate pathway
(Willrodt et al. 2014; Zebec et al. 2016). A genetically engineered yeast, Yarrowia
lipolytica, was used to produce d-limonene and l-limonene by heterologous expression of 10 genes which included d-limonene synthase gene and l-limonene synthase
gene, respectively. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to
be the key rate-limiting enzyme in the mevalonate (MVA) pathway for improving
limonene synthesis in Y. lipolytica. It significantly increased the titres of both
d-limonene and l-limonene upon overexpression (Pang et al. 2019). Heterologous
expression of codon optimized neryl diphosphate synthase-1 (NDPS1) and limonene
138
T. Malik and S. Rawat