WO2019092388A1 - Synthèse de composés d'ester monoterpénoïde - Google Patents

Synthèse de composés d'ester monoterpénoïde Download PDF

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WO2019092388A1
WO2019092388A1 PCT/GB2017/053390 GB2017053390W WO2019092388A1 WO 2019092388 A1 WO2019092388 A1 WO 2019092388A1 GB 2017053390 W GB2017053390 W GB 2017053390W WO 2019092388 A1 WO2019092388 A1 WO 2019092388A1
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monoterpenoid
synthase
geraniol
nucleic acid
producing
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PCT/GB2017/053390
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English (en)
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Matthew Quinn STYLES
Micaela Gabrielle CHACON
David Jonathan Leak
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University Of Bath
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • DMAPP mevalonate-dependent pyrophosphate
  • GPP geranyl pyrophosphate
  • MEP methylerythritol 4-phosphate
  • Geraniol trans-3,7-dimethyl-2,6-octadien- l-ol
  • geraniol is an acyclic monoterpene alcohol found in the essential oils of plants such as lemongrass, rose, and geranium (Talapatra, S.K., and Talapatra, B., 2015). Due to its distinctly sweet and floral aroma, it has played a significant role in the flavour, fragrance, and cosmetic industries. More recently, however, several pharmaceutical applications for geraniol have been suggested, including use as anti-cancer, antiinflammatory, and pain relief agents.
  • geraniol has also been proposed as a promising gasoline alternative, superior to other biofuels such as ethanol, by virtue of its low volatility, low hygroscopicity and high energy density (Peralta-Yahya and Keasling, 2010; Guimaraes et al, 2013; de Cassia da Silveira e Sa et al, 2013; Cho et al, 2016; Liu et al, 2016).
  • the worldwide demand for geraniol has surpassed 1000 metric tons/year (Lapczyski, 2008).
  • the predominant methods for obtaining geraniol include extraction from plant material and chemical synthesis; both of which are costly and inefficient (Sell, 2003; Liu et al, 2015).
  • Geraniol toxicity has been attributed to its amphiphilic nature, endowing it with the ability to interact with cell membranes impacting integrity and permeability, as well as interact with intracellular components (Trombetta et al, 2005; Brennan et al, 2012).
  • Shah et al, (2013) demonstrated that geraniol exposure in E. coli causes DNA damage, adding further evidence that this compound has a multifaceted mode of toxicity.
  • several groups have reported loss of geraniol in E.
  • US20110160501 discloses the production of the alkane 2,6-dimethyloctane biofuel using biosynthetic and/or chemical steps. US20110160501 discloses producing the intermediate geraniol using biological strains cultured in commercial aqueous growth medium as a step to producing the biofuel. The geraniol is reportedly produced in relatively small production titres of 1.84 ⁇ (approximately 360
  • US20ll0l6050l also discloses that the geraniol maybe converted to a further intermediate geranyl acetate using an acetyltransferase in cells grown in a commercial aqueous growth medium before being chemically hydrogenated to the biofuel.
  • the present invention seeks to overcome problem(s) associated with the prior art.
  • the present inventors found that toxicity can be reduced and product specificity significantly improved by biosynthesising a monoterpenoid alcohol and
  • the bulk of the monoterpenoid alcohol geraniol was deliberately converted to geranyl acetate which was shown to reduce geraniol toxicity, with the added advantage that this stops further, endogenous metabolism.
  • a modified E. coli strain was engineered co-expressing a heterologous mevalonate pathway and a geraniol synthase (GES) from Ocim um basilicum for the production of geraniol and additionally expressing an alcohol acyltransferase (AAT) from Rosa hybrida (Fig. 1) to esterify geraniol to its acetate ester.
  • GES geraniol synthase
  • AAT alcohol acyltransferase
  • Rosa hybrida Rosa hybrida
  • the present invention provides method for producing a monoterpenoid compound comprising a step of culturing at least one microbial organism co-expressing genes encoding a metabolic pathway for the production of isopentenyl pyrophosphate and dimethylallyl pyrophosphate and:
  • component (i), component (ii) and optional component (iv) expressed in the microorganism converts a carbon source to produce a monoterpenoid comprising an alcohol, which is then converted by component (iii) expressed in the microorganism to a monoterpenoid ester, wherein these conditions comprise a two- phase aqueous-organic system.
  • the present invention provides a method for producing a
  • monoterpenoid compound comprising a step of culturing at least one microbial organism co-expressing genes encoding a mevalonate pathway and:
  • component (i), component (ii) and optional component (iv) expressed in the microorganism converts a carbon source to produce a monoterpenoid comprising an alcohol, which is then converted by component (iii) expressed in the microorganism to a monoterpenoid ester, wherein these conditions comprise a two- phase aqueous-organic system.
  • the present invention provides a nucleic acid construct comprising genes encoding a metabolic pathway for the production of isopentenyl pyrophosphate and dimethylallyl pyrophosphate and: (i) geranyl pyrophosphate synthase; (ii) monoterpenoid synthase; (iii) an alcohol acyltransferase; and optionally
  • the present invention provides a nucleic acid construct comprising genes encoding a mevalonate pathway and: (i) geranyl pyrophosphate synthase; (ii) monoterpenoid synthase; (iii) an alcohol acyltransferase; and optionally
  • the present invention provides a combination of two nucleic acid constructs, wherein (a) the first nucleic acid construct comprises genes encoding a metabolic pathway for the production of isopentenyl pyrophosphate and dimethylallyl pyrophosphate and (i) geranyl pyrophosphate synthase and (ii) a monoterpenoid synthase and optionally (iv) a hydroxylase or a reductase; and (b) the second nucleic acid construct comprises (iii) an alcohol acyltransferase; and optionally (iv) a hydroxylase or a reductase.
  • the present invention provides a combination of two nucleic acid constructs, wherein (a) the first nucleic acid construct comprises genes encoding a mevalonate pathway and (i) geranyl pyrophosphate synthase and (ii) a monoterpenoid synthase and optionally (iv) a hydroxylase or a reductase; and (b) the second nucleic acid construct comprises (iii) an alcohol acyltransferase; and optionally (iv) a hydroxylase or a reductase.
  • the present invention provides a host cell comprising the nucleic acid construct as described herein or the combination of two nucleic acid constructs as described herein, suitably, wherein the alcohol acyltransferase is heterologous to the host cell.
  • the present invention provides a microbial organism comprising genes encoding a metabolic pathway for the production of isopentenyl pyrophosphate and dimethylallyl pyrophosphate and:
  • the present invention provides a microbial organism comprising genes encoding a mevalonate pathway and:
  • the present invention provides an expression system comprising
  • a first nucleic acid construct or a first microbial organism comprising genes encoding a metabolic pathway for the production of isopentenyl pyrophosphate and dimethylallyl pyrophosphate and (i) geranyl pyrophosphate synthase and (ii) a monoterpenoid synthase; and optionally (iv) a hydroxylase or a reductase; and
  • a second nucleic acid construct or a second microbial organism comprising (iii) a heterologous alcohol acyltransferase; and optionally (iv) a hydroxylase or a reductase.
  • the present invention provides an expression system comprising (a) a first nucleic acid construct or a first microbial organism comprising genes encoding a mevalonate pathway and (i) geranyl pyrophosphate synthase and (ii) a monoterpenoid synthase; and optionally (iv) a hydroxylase or a reductase; and
  • a second nucleic acid construct or a second microbial organism comprising (iii) a heterologous alcohol acyltransferase; and optionally (iv) a hydroxylase or a reductase.
  • the present invention provides a method for producing a
  • the present invention provides the use of the microbial organism as described herein or the nucleic acid construct as described herein or the combination as described herein or the host cell as described herein or the or the expression system as described herein for producing a monoterpenoid compound.
  • Carbon source as used herein generally refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth.
  • Carbon sources can be in various forms, including, but not limited to carboxylic acids (such as succinic acid, lactic acid, acetic acid), alcohols (e.g., ethanol), sugar alcohols (e.g., glycerol), aldehydes, amino acids, carbohydrates, saturated or unsaturated fatty acids, ketones, peptides, proteins, and mixtures thereof.
  • the carbon substrate may also be one-carbon substrates such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated.
  • methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity.
  • methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth Ci-Compd., [Int. Symp.], 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept,
  • the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
  • Lignocellulosic material carbon dioxide (CO2), and coal are also contemplated as suitable carbon sources.
  • Culture medium as used herein includes any medium which supports microorganism life (i.e. a microorganism that is actively metabolizing carbon).
  • a culture medium usually contains a carbon source.
  • the carbon source can be anything that can be utilized, with or without additional enzymes, by the microorganism for energy.
  • Enzyme Commission Number (EC) as used herein are derived from the International Union of Biochemistry and Molecular Biology.
  • heterologous is used to refer to a nucleic acid or polypeptide sequence from another species or genera, i.e., a species different from the host cell species or genera different from the host genera.
  • Isoprene is 2-methyl-i,3-butadiene (C 5 H 5 ) and is the monomer building block for terpenes.
  • DMAPP dimethylallyl pyrophosphate
  • IPP isopentenyl pyrophosphate
  • pyrophosphate and "diphosphate” are used herein interchangeably.
  • DMAPP and IPP are isomers of each other.
  • MEV pathway is used herein to refer to the biosynthetic pathway that converts acetyl-CoA to IPP and DMAPP.
  • “mevalonate pathway enzyme” is an enzyme, or functional fragment or variant thereof, that catalyzes one or more steps in the MEV pathway (upper or lower MEV pathway).
  • the term “monoterpenoid synthase” refers to any enzyme that catalyzes the conversion of geranyl pyrophosphate (GPP) to a monoterpenoid.
  • GPP geranyl pyrophosphate
  • monoterpenoid synthase (the "monoterpenoid synthase substrate") is GPP.
  • the term “monoterpenoid synthase” includes both linear and cyclic monoterpenoid synthases. Cyclic monoterpenoid synthases are also referred to as monoterpenoid cyclases.
  • “Monoterpenes” as used herein generally refers to a class of terpenes that consist of two isoprene units. Monoterpenes may be linear (acyclic) or contain rings.
  • “Monoterpenoid compounds” as used herein generally refers to compounds that comprise monoterpenes and derivatives thereof that can be prepared by biochemical modifications such as oxidation, reduction or rearrangement. Some steps in the synthesis of monoterpenoid compounds may include chemical modifications.
  • An "expression vector” is a nucleic acid vehicle that comprises a combination of nucleic acid components for enabling the expression of nucleic acid. Suitable expression vectors include episomes capable of extra-chromosomal replication such as circular, double-stranded nucleic acid plasmids; linearized double-stranded nucleic acid plasmids; and other functionally equivalent expression vectors of any origin.
  • An expression vector comprises at least a promoter positioned upstream and operably- linked to a nucleic acid or nucleic acid construct.
  • construct refers to a double-stranded, recombinant nucleic acid fragment comprising one or more polynucleotides.
  • the construct comprises a "template strand” base-paired with a complementary "sense or coding strand.”
  • a given construct can be inserted into a vector in two possible orientations, either in the same (or sense) orientation or in the reverse (or anti-sense) orientation with respect to the orientation of a promoter positioned within a vector - such as an expression vector.
  • vector refers to a nucleic acid vehicle that comprises a combination of nucleic acid components for enabling the transport of nucleic acid, nucleic acid constructs and nucleic acid conjugates and the like. Suitable vectors include episomes capable of extra-chromosomal replication such as circular, double-stranded nucleic acid plasmids; linearized double-stranded nucleic acid plasmids; and other vectors of any origin.
  • identity refers to the degree of sequence similarity between two
  • polypeptides or between two nucleic acid molecules compared by sequence alignment The degree of identity between two discrete nucleic acid sequences being compared is a function of the number of identical, or matching, nucleotides at comparable positions.
  • the percent identity may be determined by visual inspection and mathematical calculation.
  • the percent identity of two nucleic acid sequences maybe determined by comparing sequence information using a computer program such as - ClustalW, BLAST, FASTA or Smith-Waterman.
  • the percentage identity for two sequences may take different values depending on: (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (for example, BLOSUM62, PAM250, Gonnet etc.), and gap- penalty, for example, functional form and constants. Having made the alignment, there are different ways of calculating percentage identity between the two sequences.
  • percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
  • the popular multiple alignment program ClustalW ⁇ Nucleic Acids Research (1994) 22, 4673-4680; Nucleic Acids Research (1997), 24, 4876-4882) is a suitable way for generating multiple alignments of polypeptides or polynucleotides.
  • calculation of percentage identities is then calculated from such an alignment as (N/T), where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.
  • the metabolic pathway for the production of isopentenyl pyrophosphate and dimethylallyl pyrophosphate is chosen from a mevalonate pathway and a
  • the metabolic pathway for the production of isopentenyl pyrophosphate and dimethylallyl pyrophosphate is a methylerythritol phosphate pathway.
  • the metabolic pathway for the production of isopentenyl pyrophosphate and dimethylallyl pyrophosphate is a mevalonate pathway.
  • the "mevalonate (MEV) pathway” converts acetyl-CoA to IPP and DMAPP and comprises the following enzymatic reactions: (a) condensing two molecules of acetyl- CoAto acetoacetyl-CoA; (b) condensing acetoacetyl-CoA with acetyl-CoA to form 3- hydroxy-3-methyl-glutaryl-CoA (HMG-CoA); (c) converting HMG-CoA to mevalonate; (d) phosphorylating mevalonate to mevalonate 5-phosphate; (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate (also known as mevalonate-5- diphosphate); (f) converting mevalonate 5-pyrophosphate to IPP; and (g) conversion of IPP to DMAPP. Enzymes that carry out these reactions include acetyl-CoA
  • acetyltransferase also known as acetoacetyl-CoA thiolase
  • HMGS 3-hydroxy-3-methyl- glutaryl-CoA synthase
  • HMGR 3-hydroxy-3-methyl-glutaryl-CoA reductase
  • MK mevalonate kinase
  • PMK phosphomevalonate kinase
  • diphosphomevalonate decarboxylase also known as mevalonate pyrophosphate decarboxylase or as mevalonate-5-diphosphate decarboxylase
  • the upper MEV pathway includes enzymes responsible for the conversion of acetyl-CoA to mevalonate.
  • the lower MEV pathway includes enzymes responsible for the conversion of mevalonate to IPP, which can in step (g) be converted to its isomer, dimethylallyl pyrophosphate [also known as diphosphomevalonate or dimethylallyl diphosphate] (DMAPP), by isopentenyl pyrophosphate isomerase [also known as isopentyl diphosphate isomerase].
  • Eukaryotic cells other than plant cells use the MEV pathway to convert acetyl-CoA to IPP, which is subsequently isomerized to DMAPP.
  • the genes encoding the mevalonate pathway encode acetyl-CoA
  • the host cell will typically be genetically engineered to incorporate the genes encoding the mevalonate pathway.
  • One or more of the genes encoding the mevalonate pathway will typically be heterologous to the host cell.
  • One or more of the genes encoding the mevalonate pathway will typically be homologous to the host cell.
  • the "methylerythritol phosphate pathway" converts pyruvate and glyceraldehyde 3- phosphate to IPP and DMAPP and comprises the following enzymatic reactions (i) converting glyceraldehyde-3-phosphate and pyruvate i-deoxy-D-xylulose-5-phosphate (DOXP); (ii) converting DOXP to 2-C-methylerythritol 4-phosphate (MEP); (iii) converting MEP to 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME); (iv) converting CDP-ME to 4-diphosphocytidyl-2C-methylerythritol 2-phosphate (CDP-MEP); (v) converting CDP-MEP to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP); (vi) converting MEcPP to i-
  • the genes encoding the methylerythritol phosphate pathway encode l-deoxy- xylulose 5-phosphate synthase , i-deoxy-D-xylulose-5-phosphate reductoisomerase, 4- diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2-C-methyl- D-erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, 1- hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase and i-hydroxy-2-methyl-2- (E)-butenyl-4-diphosphate reductase.
  • the host cell will typically be genetically engineered to incorporate the genes encoding the methylerythritol phosphate pathway.
  • One or more of the genes encoding the methylerythritol phosphate pathway will typically be heterologous to the host cell.
  • One or more of the genes encoding the methylerythritol phosphate pathway will typically be homologous to the host cell.
  • the IPP and/ or the DMAPP can be acted on by prenyl transferases to produce polyprenyl pyrophosphates.
  • IPP or DMAPP can be modified by prenyl transferases to generate the polyprenyl diphosphates such as geranyl diphosphate [also known as geranyl pyrophosphate] (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP).
  • GPP geranyl diphosphate
  • FPP farnesyl diphosphate
  • GGPP geranylgeranyl diphosphate
  • the GPP may be further modified by monoterpenoid synthases to generate specific monoterpenoid compounds.
  • Step a The conversion of 2 molecules of acetyl-CoA to acetoacetyl-CoA is catalyzed by acetyl-CoA acetyltransferase (E.C. 2.3.1.9), examples of sequences encoding this enzyme include, but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): P76461, P44873, Q9I2A8, Q12598, Q04677, Q8S4Y1, Q9FIK7, Q9BWD1, Q8CAY6, Q5XI22, P45369, Q9ZHI1, P24752, Q8HXY6, Q8QZT1, P66927, P46707, P66926, P54810, P14610, P14611, P17764, P50174, P10551, P45363, Q6L8K7, P41338, P07097, P45359, Q18AR0, Q2FJQ
  • Step b The conversion of acetoacetyl-CoA and acetyl-CoA to 3-hydroxy-3-methyl- glutaryl-CoA (HMG-CoA) is catalyzed by 3-hydroxy-3-methyl-glutaryl-CoA synthase (E.C.
  • HMGS 2.3.3.10
  • sequences include, but are not limited to genes that encode the following proteins (indicated below as GenPept accession numbers): P54961, P23228, P13704, Q01581, Q8JZK9, Q5R7Z9, P17425, P54870, Q2KIE6, P54868, P54869, O02734, P22791, P54873, P54871, P54872, P54874, and ⁇ 54839 ⁇
  • the HMGS is derived from Staphylococcus aureus. Step c.
  • HMGR sequences encoding this enzyme include, but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): P14891, P34135, ⁇ 64966, P29057, A2X8W3, Q0DY59, P48020, P12683, P43256, Q9XEL8, P34136, ⁇ 64967, P29058, P48022, Q41437, P12684, Q00583, Q9XHL5, Q41438, Q9YAS4, O76819, ⁇ 28538, Q9Y7D2, Q0C8L9, P54960, P48021, Q03163, P00347, P14773, Q12577, Q59468, P04035, ⁇ 24594, P09610, Q58116, ⁇ 26662, Q01237, Q01559, Q12649, O74164, Q1W675, Q5R6N3, Q9V1R3, ⁇ 59469, Q29512,
  • the membrane binding domain of HMG-CoA reductase is deleted to cause overexpression of a cytosolic form of the enzyme. This may be achieved, for example, by deleting the DNA sequence encoding amino acids 1-552 from the 5. cerevisiae HMGi gene.
  • Step d The conversion of mevalonate (MEV) to mevalonate-5-phosphate (MEV-P) is catalyzed by mevalonate kinase (E.C. 2.7.1.36) [MK], examples of sequences encoding this enzyme include, but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): Q9Y946, P46086, ⁇ 27995, Q5E9T8, Q03426, Q58487, Q50559, Q9R008, Q9V187, Q8U0F3, O59291, Q5JJC6, P17256, Q09780, and P07277.
  • Step e The conversion of mevalonate-5-phosphate (MEV-P) to mevalonate-5- diphosphate (MEV-PP) is catalyzed by phosphomevalonate kinase (E.C. 2.7.4.2)
  • PMK examples of sequences encoding this enzyme include, but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): P24521, Q2KIU2, Q9VIT2, Q15126, Q9D1G2, and Q29081.
  • the PMK derived from Saccharomyces cerevisiae.
  • Step f The conversion of mevalonte-5-diphosphate (MEV-PP) to isopentenyl diphosphate (IPP) is catalyzed by mevalonate-5-diphosphate decarboxylase (E.C.
  • PMD 4.1.1.33
  • sequences encoding this enzyme include, but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): P53602, Q99JF5, Q62967, and P32377.
  • the PMD derived from Saccharomyces cerevisiae.
  • Step g The conversion of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) is catalyzed by isopentenyl diphosphate isomerase (E.C. 5.3.3.2) [IDI], examples of sequences encoding this enzyme include but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): Q38929, Q5P011, Q1LZ95, ⁇ 48964, Q39472, Q13907, Q4R4W5, ⁇ 35586, P58044, O42641, Q7N1V4, Q5R8R6, O35760, Q10132, P15496, Q9YB30, Q8YNH4, Q3MAB0, Q42553, ⁇ 27997, Q5NWG5, Q81SX4, Q73AZ6, Q81FS0, Q63DN3, Q6HL56, Q65I10, P50740, Q6MMK2, O51627, Q660I6, ⁇
  • the IDI is derived from Escherichia coli.
  • Step (i) The conversion of glyceraldehyde-3-phosphate and pyruvate to l-deoxy-D- xylulose-5-phosphate (DOXP) is catalyzed by i-deoxy-xylulose 5-phosphate synthase (E.C.
  • sequences encoding this enzyme include but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): Q39UB1, Q74FC3, Q28WA7, Q9F1V2, Q2RYD6, Q3J1A8, Q16DV7, Q82ML4, Q9X7W3, Q5NN52, Q39RT4, Q74CBO, Q28W25, Q8VUR8, Q2RR29, Q3IYR6, Q16CP0, Q82 KW8, Q8CJP7, Q5NM38, Q6F7N5, Q8UHD7, Q0VMI4, Q2IPZ2, Q8YZ80, Q3M4F6, O67036, Q38854, Q5P228, Q81M54, Q731B7, Q818R9, Q635A7, Q5LH44, Q64Y02, Q9K971, Q6HDY8, Q65HJ2, Q5WF63, P54523, Q8A
  • Step (ii) The conversion of DOXP to 2-C-methylerythritol 4-phosphate (MEP) is catalyzed by i-deoxy-D-xylulose-5-phosphate reductoisomerase (E.C. 1.1.1.267).
  • sequences encoding this enzyme include but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): Q81N10, Q81B49, Q638M6, Q6HG59, Q81WL4, Q819Y3, Q636K5,
  • Step (iii) The conversion of MEP to 4-diphosphocytidyl-2-C-methylerythritol (CDP- ME) is catalyzed by 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (E.C.
  • sequences encoding this enzyme include but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): Q92F40, Q724H7, Q8YAB5, Q2YV76, Q5HJC5, P65176, P65177, Q6GK63, Q6GCM3, Q8NYI0, Q87LQ2, Q92CV0, Q720Y7, Q8Y832, Q2YV73, Q5HJC1, Q99WW8, Q7A7V0, Q6GK57, Q6GCL7, Q7A1W0, Q87Q30, Q8UFF4, Q2IQG8, A1USA2, Q6G3Z8, Q6G164, Q89LQ8, A5EIY9, A4YUQ7, Q2YPW1, Q57D18, Q8YHD8, Q8G0H4, A0RN28, Q9PM68, A1W1K9, Q5HSI4, Q9
  • Step (iv) The conversion of CDP-ME to 4-diphosphocytidyl-2C-methylerythritol 2- phosphate (CDP-MEP) is catalyzed by 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (E.C. 2.7.1.148), examples of sequences encoding this enzyme include but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): Q6F8J0, Q8UHP8, Q2IM67, Q5PB05, Q8YS61,
  • Q3M3F2 O67060, O81014, Q5P725, Q81VZ6, Q73FG3, Q81JA2, Q63HI8, Q5LC56, Q64T40, Q9KGK0, Q6HPX2, Q65PH5, Q5WLV8, P37550, Q8AA41, Q6G4E4,
  • Step (v) The conversion CDP-MEP to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) is catalyzed by 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (E.C.
  • sequences encoding this enzyme include but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): Q8UFF4, Q2IQG8, A1USA2, Q6G3Z8, Q6G164, Q89LQ8, ⁇ 5 ⁇ 9, A4YUQ7, Q2YPW1, Q57D18, Q8YHD8, Q8G0H4, A0RN28, Q9PM68, A1W1K9, Q5HSI4, Q9A7I5, Q310X3, Q6ARN9, Q72C30, A1VDX6, Q2NAE1, Q5FQD6, Q0BTD5, Q9ZM19, ⁇ 25664, Q0C0N0, Q28Q60, Q1MR76, Q6ADI0, Q2W4Q8, Q0APQ6, Q11HV9, Q1QM99, Q3SSN8, Q2G708, A1B890, Q4FM31, Q2K8V
  • Step (vi) The conversion of MEcPP to i-hydroxy-2-methyl-2-(E)-butenyl 4- diphosphate (HMB-PP) is catalyzed by i-hydroxy-2-methyl-2-(E)-butenyl-4- diphosphate synthase (E.C.
  • sequences encoding this enzyme include but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): Q82K43, Q9X7W2, Q82ML3, Q9KYR9, Q6FEM3, P58665, Q5PAJ1, P58666, ⁇ 67496, Q5P7B3, Q81LV7, Q730Q8, Q818H8, Q634Q9, Q5L7W2, Q64N34, Q9 KD18, Q6HDN9, Q65HA9, Q5WHB2, P54482, Q8A4T0, Q6G1X4, Q6G104, Q8G7Y6, Q7WHN0, Q7W6P6, Q7VWL0, Q89W9, Q57BA5, Q8YJ17, Q8FYT2, P57374, Q8K9P4, Q62JW4, Q63UT3, Q9PPM1, Q5HV95, Q9A9W0, Q5
  • Step h The conversion of dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) to geranyl diphosphate (GPP) is catalyzed by geranyl pyrophosphate synthase [also known as geranyl diphosphate synthase] (GPPS) (E.C.
  • sequences encoding this enzyme include but are not limited to, one or more genes that encode the following proteins (indicated below as GenPept accession numbers): Q09152, P49351, O24241, Q43315, P49352, ⁇ 24242, P49350, Q8WMY2, P08836, Q92235, O64905, P14324, P49349, P49353, Q920E5, Q92250, P05369, O14230, P08524, P34802, O04046, Q9LUD9, Q9SLG2, O22043, Q758K0, P56966, P80042, Q42698, Q92236, Q94ID7, ⁇ 95749, Q9WTN0, P0A5H9, P0A5H8, P24322, Q6F596, Q9P885, Q43133, Q12051, P39464, P95999, Q58270, O26156, and Q53479-
  • the GPPS is derived from
  • Step i The conversion of geranyl diphosphate (GPP) to geraniol may be catalyzed by geraniol synthase (GES) (E.C. 3.1.7.11), examples of which are found at SEQ ID NO: 1.
  • GES geraniol synthase
  • Other examples of sequences of genes encoding this enzyme include but are not limited to AF529266, AJ457070, and AY362553 (GenBank accession numbers).
  • the GES is derived from Ocimym basilicum .
  • the GES is encoded by the sequence SEQ ID NO: 1 shown in Figure 6.
  • the conversion of geraniol to geraniol acetate may be catalyzed by an alcohol acyltransferase (AAT), examples of which are found at (RhAAT, Rosa hybrida) Accession no. AY850287.1 (SEQ ID NO: 2) (RhAAT, Rosa hybrida).
  • AAT alcohol acyltransferase
  • Other examples of sequences of genes encoding this enzyme include but are not limited to SAAT, Fragaria x ananassa Accession no. AAG13130 and BAAT, Musa acum inata Accession no.
  • the AAT is derived from Rosa hybrida.
  • the AAT is encoded by the sequence, SEQ ID NO: 2, shown in Figure 9.
  • the AAT has the protein sequence, SEQ ID NO: 3, shown in Figure 10. The genes encoding GPPS, GES and AAT will typically be heterologous to the host cell.
  • the at least one microbial organism comprises one, two, three, four or five microbial organisms.
  • the at least one microbial organism is one microbial organism.
  • the invention provides and makes use of at least one microbial organism co-expressing genes encoding the mevalonate pathway and: (i) geranyl pyrophosphate synthase; (ii) a monoterpenoid synthase; (iii) an heterologous alcohol acyltransferase; and optionally (iv) a hydroxylase or a reductase.
  • the microbial organism can be any "host cell” that produces a monoterpenoid ester when transformed with a gene encoding an alcohol acyltransferase.
  • progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • the host cell can be eukaryotic or prokaryotic.
  • the host cells include, but are not limited to, fungi, filamentous fungi, yeast, algae and bacteria.
  • the host cell is a filamentous fungus.
  • the filamentous fungi host cells of the present invention include all filamentous forms of the subdivision
  • Filamentous fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex
  • filamentous fungi host cells of the present invention are morphologically distinct from yeast.
  • Exemplary filamentous fungal cells include, but are not limited to, species of: Achlya, Acrem onium, Aspergillus,
  • the filamentous fungal host cell is a species of: Aspergillus (e.g., A. awam ori, A. fum igatus, A. japonicus, A. nidulans, A. niger, A. aculeatus, A.
  • Aspergillus e.g., A. awam ori, A. fum igatus, A. japonicus, A. nidulans, A. niger, A. aculeatus, A.
  • Fusarium e.g., F. bactridioides, F. cerealis, F. crookw ellense, F. culm orum, F.
  • the host cell is a yeast.
  • the yeast is from one of the genera: Candida, Hansenula, Saccharomy ces, Schizosaccharomy ces, Pichia, Kluyveromyces, and Yarrow ia.
  • the yeast cell is Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodam ae, Pichia m em branaefaciens, Pichia opuntiae, Pichia therm otolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia m ethanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrow ia lipolytica.
  • the host cell is an algal cell such as Chlamydom onas (e.g., C. Reinhardtii) and Phorm idium (P. sp. ATCC29409).
  • algal cell such as Chlamydom onas (e.g., C. Reinhardtii) and Phorm idium (P. sp. ATCC29409).
  • the host cell is a prokaryotic cell.
  • Suitable prokaryotic cells include gram positive, gram negative and gram-variable bacterial cells.
  • Exemplary prokaryotic host cells include, but are not limited to, species of: Agrobacterium , Alicyclobacillus, Anabaena, Anacystis, Acinetobacter Arthrobacter, Azobacter, Bacillus,
  • Camplyobacter Clostridium , Corynebacterium, Chrom atium, Coprococcus,
  • M ethy lobacterium M ethy lobacterium , Mycobacterium, Neisseria, Pantoea,
  • Rhodopseudom onas Roseburia, Rhodospirillum, Rhodococcus, Scenedesm un, Streptomyces, Streptococcus, Synnecoccus, Staphylococcus, Serratia, Salm onella, Shigella, Therm oanaer obacterium, Tropherym a, Tularensis, Tem ecula,
  • the bacterial host cell is non-pathogenic to humans.
  • the bacterial host strain is an industrial strain. Numerous bacterial industrial strains are known and suitable in the present invention.
  • the bacterial host cell is of the Bacillus species, e.g., B.
  • B. thuringiensis B. m egaterium, B. subtilis, B. lentus, B. circulans, B. pum ilus, B. lautus, B. coagulans, B. brevis, B. licheniform is, B. clausii, Geobacillus and B.
  • the bacterial host cell is of the Clostridium species, e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, and C. beijerinckii.
  • the bacterial host cell is of the Corynebacterium species e.g., C. glutam icum and C. acetoacidophilum ln
  • the bacterial host cell is of the Erw inia species, e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata, and Z?. terreus.
  • E. uredovora E. carotovora
  • E. ananas E. herbicola
  • E. punctata E. punctata
  • Z?. terreus e.g., Z?. terreus.
  • the bacterial host cell is of the Pantoea species, e.g., P. citrea and P. agglom erans. In some embodiments, the bacterial host cell is of the Pseudomonas species, e.g., P. pudita, P. m evalonii, and P. sp. D-ol 10. In some embodiments, the bacterial host cell is of the Streptococcus species, e.g., 5. equisim iles, S. pyogenes, and 5. uberis. In some embodiments, the bacterial host cell is of the Streptomyces species, e.g., 5. am bofaciens, S. averm itilis, S. coelicolor, S. aureofaciens, S. aureus, S.
  • the bacterial host cell is of the Zym om onas species, e.g., Z. m obilis and Z. lipolytica.
  • the bacterial host cell is of the Escherichia species, e.g., E. coli.
  • the bacterial host cell may a host cell that is optimised for protein production - such as E. coli BL2l(DE3).
  • the bacterial host cell may a host cell that has increased tolerance of toxic proteins - such as E. coli C43(DE3).
  • E. coli C43(DE3) E. coli C43(DE3).
  • Strains that may serve as suitable host cells including both prokaryotic and eukaryotic strains, are readily accessible to the public from a number of culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
  • ATCC American Type Culture Collection
  • DSM Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH
  • CBS centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • Geranyl pyrophosphate synthase the gene encoding the geranyl pyrophosphate synthase is from one of Abies grandis, Lithosperm um, Mentha x piperita, Mentha spicata, Salvia officinalis, and Vitis vinifera.
  • sequences encoding geranyl pyrophosphate synthase include, but are not limited to, those listed with regard to Step h.
  • the gene encoding the geranyl pyrophosphate synthase is from Abies grandis.
  • the gene encoding the monoterpenoid synthase is from one of
  • Salvia officinalis Rosem arinus officinalis, Pseudotsuga m enzeseii, Abies grandis, Arabidopsis thaliana, Mentha spicata, Artem isia annua, Ocimym basilicum, and Salvia fruticosa.
  • the monoterpenoid synthase is a limonene synthase (LS) such as, but not limited to those from Mentha spicata (LS-MSPI) (e.g., NCBI Accession No.
  • LS-MSPI Mentha spicata
  • the monoterpenoid synthase is a pinene synthase (PS) such as, but not limited to those from A rtem isia annua (PS-AANN) (e.g., NCBI Accession No. AAK58723.1).
  • PS-AANN pinene synthase
  • Such synthases may useful to produce monoterpenoid compounds including, but not limited to, a-pinene and ⁇ -pinene.
  • the monoterpenoid synthase is a fenchol synthase (FS) such as, but not limited to those from Ocimym basilicum (FS-OBAS) (e.g., NCBI Accession No. AAV63790.1).
  • FS-OBAS Ocimym basilicum
  • Such synthases may useful to produce fenchol.
  • the monoterpenoid synthase is a geraniol synthase (GS) such as, but not limited to those from Ocimym basilicum (GS-OBAS). Such synthases may useful to produce geraniol. More suitably, the gene encoding (ii) the monoterpenoid synthase has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1.
  • the monoterpenoid synthase is selected from a geraniol synthase, a terpineol synthase, a linalool synthase, a fenchol synthase and a limonene synthase.
  • the monoterpenoid synthase is selected from a geraniol synthase (EC 3.1.7.11), a (-)-alpha-terpineol synthase (EC 4.2.3.111), a s-linalool synthase (EC 4.2.3.25), a r-linalool synthase (EC 4.2.3.26), a (-)-fenchol synthase (EC 4.2.3.10) and a limonene synthase (EC 4.2.3.20).
  • a geraniol synthase EC 3.1.7.11
  • a (-)-alpha-terpineol synthase EC 4.2.3.111
  • a s-linalool synthase EC 4.2.3.25
  • a r-linalool synthase EC 4.2.3.26
  • a (-)-fenchol synthase (EC 4.2.3.10) and a limonene synthase
  • the monoterpenoid synthase is a geraniol synthase (EC 3.1.7.11) derived from O. basilicum .
  • the heterologous alcohol acyl transferase is an alcohol acetyltransferase.
  • the heterologous alcohol acyl transferase is not a chloramphenicol acetyltransferase.
  • the gene encoding the heterologous alcohol acyl transferase is not an antibiotic resistance gene.
  • the heterologous alcohol acyl transferase is not a protein encoded by an antibiotic resistance gene.
  • the gene encoding the heterologous alcohol acyl transferase is from a eukaryote.
  • the gene encoding the heterologous alcohol acyl transferase is from a plant - such as, but not limited to, lemongrass, rose, or geranium.
  • the gene encoding the heterologous alcohol acyl transferase is from one of A. sativa, A. thaliana, Capsicum annum , C. brew eri, C. m elo, Chrysanthem um x m orifolium , C. roseus, D. caryophyllus, D. variabilis, Fragaria x ananassa, Fragaria vesca, G.
  • sequences of genes encoding heterologous alcohol acyl transferase include but are not limited to BAA74428 AAO12206, AAO38058, AAQ63616, AAQ63615, BAD93691, AAS77402, AAS77404, BAA93475, AAL50566, AAL50565, AAM64817, CAA61258, AAK73661, AAV66311, AAC99311, AAO13736, CAD89104, AAR26385, AAC18062, AAW51126, AAW31948, AAG13130, CAC09062, AAO73071, AAN09797, BAD89275, AAU 14879, AAW22989, AAN09796, AAW51125, AAN09798, AAU06226, CAA94432, AAL77060, CAC09063, AAM75818, AAL92459, AAF27621, Q9FPW3, AAF34254, CAB06430, CAE46932, BAC7863
  • the gene encoding the alcohol acyltransferase is from Rosa hybrid the gene encoding (iii) the heterologous alcohol acyltransferase has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2.
  • the heterologous alcohol acyltransferase has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3.
  • the optional component (iv) is present and is a hydroxylase.
  • the monoterpenoid synthase is a limonene synthase and optional component (iv) is present and is a hydroxylase.
  • the hydroxylase is monoterpenoid hydroxylase that converts a monoterpenoid to a monoterpenoid alcohol.
  • the hydroxylase is a cyctochrome P450.
  • the hydroxylase is a cyctochrome P450 from the CYP153 family.
  • the hydroxylase is a cyctochrome P450 from Mycobacterium . More suitably, the hydroxylase is a cyctochrome P450 from Mycobacterium selected from
  • Mycobacterium sp. strain HXN-1500 and My cobacterium sp. Shizuoka-1 (GenPept accession number WP_099250443).
  • optional component (iv) is absent.
  • the monoterpenoid synthase is selected from a geraniol synthase, a terpineol synthase, a linalool synthase and a fenchol synthase; suitably the optional component (iv) is absent.
  • the optional component (iv) is present and is a reductase.
  • the reductase is monoterpenoid reductase that converts a
  • the reductase is a geraniol reductase (EC 1.6.99.1).
  • reductases include, but are not limited to, old yellow enzyme 2 and 12-oxophytodienoate reductase.
  • Old yellow enzyme 2 (OYE2) is from 5. cerevisiae (Q03558.3) and 12- oxophytodienoate reductase (OPR), is from the plant Hevea brasiliensis, HbOPR Carbon Source
  • the carbon source is selected from carboxylic acids (such as succinic acid, lactic acid, acetic acid), alcohols (e.g., ethanol), sugar alcohols (e.g., glycerol), aldehydes, amino acids, carbohydrates, saturated or unsaturated fatty acids, ketones, peptides, proteins, and mixtures thereof.
  • carboxylic acids such as succinic acid, lactic acid, acetic acid
  • alcohols e.g., ethanol
  • sugar alcohols e.g., glycerol
  • aldehydes amino acids
  • carbohydrates saturated or unsaturated fatty acids
  • ketones e.glycerol
  • the carbon source is a carbohydrate.
  • the carbon source is a carbohydrate selected from monosaccharides, disaccharides, oligosaccharides and polysaccharides.
  • the carbon source is a disaccharide selected from sucrose and lactose.
  • the sugar is an oligosaccharide comprising 3, 4, 5, 6, 7, 8, 9 or 10
  • the carbon source is a polysaccharide selected from starch, cellulose and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt
  • the carbon source is a monosacchride selected from glucose, galactose, xylose, arabinose and fructose.
  • the carbon source is glucose.
  • Two-phase aqueous-organic system Two-phase aqueous-organic system
  • the culturing step is carried out in a two-phase aqueous-organic system.
  • a liquid two-phase aqueous-organic system starting materials, reagents and products will be partitioned depending on their solubility in each layer.
  • the organic layer comprises a solvent that is non-toxic to the microbial organism.
  • the organic phase of the two-phase aqueous-organic system comprises at least one of decane, undecane, dodecane, pentadecane, hexadecane, diisononyl phthalate and diisodecyl phthalate.
  • the organic phase of the two-phase aqueous-organic system comprises an alkane.
  • the organic phase consists essentially of at least one alkane.
  • the organic phase consists of at least one alkane.
  • the organic phase consists of an alkane.
  • the organic phase of the two-phase aqueous-organic system comprises an alkane selected from decane, undecane, dodecane, pentadecane and hexadecane. More suitably, the organic phase of the two-phase aqueous-organic system comprises dodecane. More suitably, the organic phase consists essentially of dodecane. More suitably, the organic phase consists of dodecane. In another aspect, the organic phase of the two-phase aqueous-organic system comprises the monoterpenoid ester. In such aspects, the monterpenoid ester is immiscible with the aqueous phase.
  • the organic phase of the two-phase aqueous-organic system comprises at least one of the monoterpenoid ester, decane, undecane, dodecane, pentadecane, hexadecane, diisononyl phthalate and diisodecyl phthalate.
  • the aqueous layer of the two-phase aqueous-organic system comprises a culture medium.
  • Culture medium in the present invention contains suitable carbon source.
  • culture medium typically contains suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for geraniol production.
  • Suitable growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, Terrific broth (TB) or Yeast medium (YM) broth, or modified versions of such commercially prepared media.
  • Other defined or synthetic growth media may also be used, and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science.
  • Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0. In some embodiments the initial pH is 6.0 to pH 8.0.
  • Microorganism culture may be performed under aerobic, anaerobic, or microaerobic conditions.
  • the aqueous phase of the two-phase aqueous-organic system comprises acetic acid.
  • the aqueous phase of the two-phase aqueous-organic system comprises tryptone, yeast extract and sodium chloride.
  • the aqueous phase of the two-phase aqueous-organic system comprises a carbon source, tryptone, yeast extract and sodium chloride. More suitably, the aqueous phase of the two-phase aqueous-organic system comprises acetic acid, tryptone, glycerol, yeast extract and sodium chloride.
  • the aqueous phase of the two-phase aqueous-organic system comprises acetic acid, tryptone, yeast extract and sodium chloride.
  • the aqueous phase of the two-phase aqueous-organic system comprises a acetic acid, carbon source, tryptone, yeast extract and sodium chloride.
  • the aqueous phase of the two-phase aqueous-organic system comprises acetic acid, tryptone, glycerol, yeast extract and sodium chloride.
  • the culturing step is carried out at a temperature of from 25 0 C to 40 0 C. Such temperatures are suitably for the growth of cells in appropriate medium.
  • the culturing step is carried out at a temperature of from 26 0 C to 35 0 C, more suitably, the culturing step is carried out at a temperature of from 28 0 C to 32 0 C.
  • the culturing step is carried out in the presence of acetic acid.
  • the monterpenoid alcohol is selected from geraniol, terpineol, linalool, fenchol, perillyl alcohol, nerol and citronellol.
  • the monterpenoid alcohol is selected from geraniol, (-)-alpha-terpineol, s- linalool, r-linalool, (-)-fenchol, perillyl alcohol, nerol and citronellol.
  • the monterpenoid alcohol is selected from geraniol, (-)-alpha-terpineol, s-linalool, r-linalool, (-)-fenchol and perillyl alcohol.
  • the monterpenoid alcohol is selected from geraniol, (-)-alpha-terpineol, s-linalool, r-linalool and (-)-fenchol.
  • the monterpenoid alcohol is geraniol.
  • the monoterpenoid ester is selected from an acetate, propanoate, n-butanoate and sec-butanoate.
  • the monterpenoid ester is a monoterpenoid acetate.
  • the monoterpenoid compound produced by the method is a
  • the monoterpenoid ester is selected from geranyl acetate, terpineol acetate, linalyl acetate, fenchyl acetate, perillyl acetate, neryl acetate, citronellyl acetate, geranyl propanoate, terpineol propanoate, linalyl propanoate, fenchyl propanoate, perillyl propanoate, neryl propanoate, citronellyl propanoate, geranyl n-butanoate, terpineol n- butanoate, linalyl n-butanoate, fenchyl n-butanoate, perillyl n-butanoate, neryl n- butanoate, citronellyl n-butanoate, geranyl sec-butanoate, terpineol sec-butanoate, terpine
  • the monoterpenoid ester is selected from geranyl acetate, (-)-alpha- terpineol acetate, s-linalyl acetate, r-linalyl acetate, (-)-fenchyl acetate, perillyl acetate, neryl acetate, citronellyl acetate, geranyl propanoate, (-)-alpha-terpineol propanoate, s- linalyl propanoate, r-linalyl propanoate, (-)-fenchyl propanoate, perillyl propanoate, neryl propanoate, citronellyl propanoate, geranyl n-butanoate, (-)-alpha-terpineol n- butanoate, s-linalyl n-butanoate, r-linalyl n-butanoate, (-)-fenchyl
  • the monoterpenoid ester is selected from geranyl acetate, terpineol acetate, linalyl acetate, fenchyl acetate, perillyl acetate, neryl acetate and citronellyl acetate.
  • the monoterpenoid ester is selected from geranyl acetate, (-)-alpha- terpineol acetate, s-linalyl acetate, r-linalyl acetate, (-)-fenchyl acetate, perillyl acetate, neryl acetate and citronellyl acetate. More suitably, the monoterpenoid ester is selected from geranyl acetate, (-)-alpha- terpineol acetate, s-linalyl acetate, r-linalyl acetate, (-)-fenchyl acetate and perillyl acetate.
  • the monoterpenoid ester is selected from geranyl acetate, (-)-alpha- terpineol acetate, s-linalyl acetate, r-linalyl acetate and (-)-fenchyl acetate. Most suitably, the monoterpenoid ester is selected from geranyl acetate.
  • the method comprises the further step of hydrolysis of the
  • the monoterpenoid compound produced by the method is a monoterpenoid alcohol.
  • the advantage of this approach is that initial production of the monterpenoid ester as described herein enables the efficient and highly selective production of an intermediate that can them be removed from the reaction conditions by removing the organic layer from the two-phase aqueous-organic system.
  • This monoterpenoid ester can then undergo hydrolysis to the monoterpenoid alcohol in good yield without the issues of toxicity and production of side products.
  • the further step of hydrolysis of the monoterpenoid ester comprises a chemical hydrolysis.
  • the hydrolysis step uses base hydrolysis.
  • the base hydrolysis is carried out with sodium hydroxide
  • the hydrolysis step uses acid hydrolysis.
  • the hydrolysis is carried out using an acid in the presence of water.
  • the hydrolysis step uses acid hydrolysis, wherein the acid is selected from hydrochloric and sulfuric acid.
  • Recombinant constructs provided herein can be used to transform cells to express one or more proteins.
  • a recombinant polynucleotide construct can comprise a
  • a polynucleotide encoding one or more polynucleotides as described herein, operably linked to a regulatory region suitable for expressing the polypeptide.
  • a polynucleotide can comprise a coding sequence that encodes the polypeptide as described herein.
  • Recombinant DNA includes DNA which has been genetically engineered and constructed outside of a cell and includes DNA containing naturally occurring DNA or cDNA or synthetic DNA. Vectors containing recombinant polynucleotide constructs such as those described herein are also provided.
  • Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available and well known in the art.
  • the vectors can include, for example, origins of replication, scaffold attachment regions or markers.
  • a marker gene can confer a selectable phenotype on a cell.
  • a marker can confer biocide resistance, such as resistance to an antibiotic.
  • an expression vector can include a tag sequence designed to facilitate manipulation or detection (for example, purification or localization) of the expressed polypeptide.
  • Tag sequences such as luciferase, beta-glucuronidase, green fluorescent protein, glutathione S-transferase, polyhistidine, c-myc or hemagglutinin sequences typically are expressed as a fusion with the encoded polypeptide.
  • Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.
  • the nucleic acid construct comprises genes encoding a mevalonate pathway and: (i) geranyl pyrophosphate synthase; (ii) monoterpenoid synthase; (iii) an alcohol acyltransferase; and optionally (iv) a hydroxylase or a reductase.
  • the present invention provides a combination of two nucleic acid constructs, wherein (a) the first nucleic acid construct comprises genes encoding a metabolic pathway for the production of isopentenyl pyrophosphate and dimethylallyl pyrophosphate and (i) geranyl pyrophosphate synthase and (ii) a monoterpenoid synthase and optionally (iv) a hydroxylase or a reductase; and (b) the second nucleic acid construct comprises (iii) an alcohol acyltransferase; and optionally (iv) a hydroxylase or a reductase.
  • optional component (iv), where present is only present in one of nucleic acid constructs (a) or (b).
  • optional component (iv) is absent from nucleic acid construct (a). More suitably in the combination of two nucleic acid constructs aspects, optional component (iv) is absent from nucleic acid construct (a).
  • optional component (iv) may be absent.
  • a combination of two nucleic acid constructs wherein (a) the first nucleic acid construct comprises genes encoding a mevalonate pathway and (i) geranyl pyrophosphate synthase and (ii) a monoterpenoid synthase; and (b) the second nucleic acid construct comprises (iii) an alcohol acyltransferase; and optionally (iv) a hydroxylase or a reductase.
  • the two nucleic acid constructs can be transformed into the same host cell and co-expressed in the same host cell or the two nucleic acid constructs can be transformed into two different host cells and co-expressed in the two different host cells.
  • the host cell may be the same or a different species of cell.
  • a host cell comprising the nucleic acid construct or a host cell comprising the combination of two nucleic acid constructs or two host cells each comprising one of the nucleic acid constructs is also disclosed.
  • the present invention provides an expression system comprising (a) a first nucleic acid construct or a first microbial organism comprising genes encoding a metabolic pathway for the production of isopentenyl pyrophosphate and dimethylallyl pyrophosphate and (i) geranyl pyrophosphate synthase and (ii) a monoterpenoid synthase; and optionally (iv) a hydroxylase or a reductase; and (b) a second nucleic acid construct or a second microbial organism comprising (iii) a heterologous alcohol acyltransferase; and optionally (iv) a hydroxylase or a reductase.
  • the expression system comprises a first nucleic acid construct.
  • the expression system comprises a second nucleic acid construct.
  • optional component (iv), where present, is only present in one of microbial organism (a) or microbial organism (b).
  • optional component (iv) is absent from microbial organism (a).
  • optional component (iv) is absent from microbial organism (a).
  • optional component (iv) may be absent.
  • the expression system comprises (a) a first nucleic acid construct or a first microbial organism comprising genes encoding a mevalonate pathway and (i) geranyl pyrophosphate synthase and (ii) a monoterpenoid synthase; and (b) a second nucleic acid construct comprising (iii) an alcohol acyltransferase or a second microbial organism comprising a heterologous alcohol acyltransferase; and optionally (iv) a hydroxylase or a reductase.
  • the expression systems described herein can comprise the nucleic acid constructs which may optionally be transformed into a host cell of interest.
  • the expression system may be provided in the form of a kit, optionally comprising instructions.
  • the invention finds application in the highly selective and efficient production of monoterpenoid compounds.
  • the monoterpenoid compounds produced by the culturing step are natural fermentation products. These fermentation monoterpenoid compounds find application in a number of areas.
  • Use of a fermentation monoterpenoid product as described herein as flavouring Use of a fermentation monoterpenoid product as described herein as a fragrence.
  • Use of a fermentation monoterpenoid product as described herein as a cosmetic product Use of a fermentation monoterpenoid product as described herein as a cosmetic ingredient.
  • FIG. 1 Diagram of the two E. coli expression constructs used in this study harbouring (A) the heterologous mevalonate pathway (MEV) leading towards the production of geraniol, and (B ) an alcohol acyltransferase (AAT) capable of esterifying geraniol and acetyl-CoA to produce geranyl acetate.
  • MEV heterologous mevalonate pathway
  • AAT alcohol acyltransferase
  • Plasmid PMIB13 (shown in A) is a modified version of the pJBEI-6410 plasmid originally created by Alonso-Gutierrez et al, (2013) in which the terminal limonene synthase has been exchanged for a geraniol synthase (GES) from O. basilicum . Endogenous cell metabolism converts available sugars into acetyl-CoA, which is then further transformed into geraniol via the expressed mevalonate pathway. Co-transformation with the second construct pET28:AAT (shown in B ) harbouring a codon optimized alcohol acyltransferase from R. hybrida, results in the further conversion geraniol to a geranyl acetate ester in high titres.
  • the origin of all enzymes expressed on each plasmid is color coded according to species of origin.
  • FIG. 1 A Gas chromatographs of the dodecane phase of two-phase cultures of E. coli strains DLG2 (top panel) producing several terpenols, and DLGA3 (bottom panel) producing only geranyl acetate.
  • B The total production (mg/L) of either geraniol or geranyl acetate produced by strain DLG2 and DLGA3, respectively. Cultures were induced with IPTG, followed by the addition of a 10% dodecane top layer for product extraction. Product analysis occurred after 24.I1 of growth at 30°C. Chromatograph peaks for citronellol, nerol, geraniol, farnesol, and geranyl acetate are indicated.
  • FIG. 1 Monoterpene and monoterpene ester production in E. coli under fed-batch fermentation using a two-phase system. A Accumulation of geraniol (purple), nerol (blue), citronellol (red) and total monoterpene (grey) in E. coli strain DLG2. B
  • FIG. 5 Production of geranyl acetate in E. coli strain DLGA3 under fed-batch fermentation using a two-phase system with a semi-defined media. The culture was grown in a fermentation media +2% glucose at 30°C and induced when OD6 00 reached ⁇ 5-5 using ⁇ IPTG, followed by the addition of a 10% dodecane top layer and media supplementation with 20 mM acetic acid.
  • Figure 6 Full length codon optimized sequence of the O. basilicum GES to the genome of E. coli (SEQ ID No. 1).
  • FIG 8 Gas chromatographs showing the monoterpenes formed in E. coli C43 (DE3) after incubation in the presence of either geraniol or nerol.
  • Panels A and C show internal standards of geraniol and nerol, respectively.
  • Panel B and D show the monoterpenes present in the cell pellet of wild type E. coli C43 (DE3) incubated in the presence of either 0.5 mM geraniol or 0.5 mM nerol respectively, for 6 h. The peaks corresponding to citronellol, nerol and geraniol are indicated.
  • Figure 9 shows the RhAAT Rose (R. hybrida) gene sequence (codon optimized to E.coli) expressing the alcohol acyltransferase (SEQ ID No. 2).
  • Figure 10 shows the protein sequence for the alcohol transferase from R. hybrid (SEQ ID No. 3).
  • E. coli strains BlOBlue (Bioline; London, UK) and C43 (DE3) (Lucigen; Middleton, WI) were used for plasmid construction and expression, respectively.
  • Gene sequence optimization and synthesis was performed by GeneArt (Thermo Fisher Scientific; Waltham, MA).
  • Plasmid p JBEI-6410 was obtained from Addgene (Addgene plasmid # 47049, Taek Soon Lee). 1.1 Plasm ids, bacterial strains, and grow th conditions
  • E. coli strain C43 (DE3) was chosen as the host for protein overexpression due to its high tolerance of toxic proteins.
  • Luria Broth (LB) media (10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract) was used for gene cloning and pre-culturing.
  • recombinant strains were cultured in Terrific Broth (TB) media (12 g/L tryptone, 24 g/L yeast extract, 4 ml glycerol, 0.17 M KH 2 P0 4 , and 0.72 M K2HPO 4 ) containing 20 g/L glucose.
  • TB Terrific Broth
  • recombinant strains were cultured in either a modified TB (MTB) media (20 g/L glucose, 12 g/L tryptone, 24 g/L yeast extract, 4 ml glycerol, 5 g/L NaCl), or a semi-defined fermentation (FM) media (20 g/L glucose, 9.8 g/L K2HPO 4 , 5 g/L yeast extract, 0.3 g/L ferric ammonium citrate, 2.1 g/L citric acid monohydrate, 0.06 g/L MgS0 4 and 1 mL of trace element solution which includes 0.37 g/L ( ⁇ 4 ) 6 ⁇ 7 0 24 ⁇ 4 ⁇ 2 0, ⁇ .29 g/L ZnS0 4 -7H 2 0, 2.47 g/L ⁇ 3 ⁇ 0 4 , ⁇ .25 g/L CuS0 4 -5H 2 0 and
  • MTB modified TB
  • FM semi-defined fermentation
  • Rh Rosa hybrida
  • Ec Escherichia coli
  • Sa Staphylococcus aureus
  • Sc Saccharomyces cerevisiae
  • Ab Abies grandis
  • Ob Ocim um basilicum
  • c tr-GPPS truncated sequence of GPPS - The GPPS was truncated to remove the N- terminal targeting sequence ( ⁇ 84aa at N-terminal)
  • Strains DLGA2 and DLGA3 were inoculated into LB broth and grown overnight at 37°C. Cultures were then inoculated at 1% (v/v) into 25ml of TB + 20 g/L glucose medium, and grown at 37°C until an OD 60 o of 1 was reached.
  • Cultures of the DLGA3 strain were induced with 100 ⁇ IPTG and supplemented with either o, 5, 10 or 20 mM acetic acid before a 10% (v/v) dodecane top layer was added to trap geranyl acetate. Cultures were then incubated at 30°C in a rotary shaker (250 rpm) for 24 hours and analysed as described above.
  • Strains DLG2 and DLGA3 were grown overnight at 37 °C in 100 ml of LB medium and used to inoculate a 1.5 L bioreactor (BIOSTAT B plus MO5L, Sartorius, Germany) containing 1.2 L of MTB media supplemented with the relevant antibiotics. Strain DLG2 was induced when OD6 00 reached approximately 20 with 50 ⁇ IPTG, and a 10% dodecane top layer was then fed into the culture. Strain DLGA3 was induced when OD6 00 reached approximately 20 with 125 ⁇ IPTG; 20 mM acetic acid and a 10% dodecane top layer were then fed into the culture.
  • Culture temperature was maintained at 30 °C and pH was maintained at 6.8 by automatic addition of 5 M KOH or 5 M H2SO4 when culture pH deviated by 0.4.
  • Antifoam 204 was used to minimize foam development.
  • Dissolved oxygen was maintained at 20% saturation through combined control of air flow and stir speed. Intermittent feeding of a MTB+65% glucose solution was initiated to maintain culture glucose concentration between 5-10 g/L.
  • Fermentation samples were periodically collected to determine culture OD 60 o, then centrifuged to separate the organic and aqueous layers for further analysis of glucose concentration, and terpene product formation.
  • Strain DLGA3 was also run in fed-batch fermentation using a semi-defined
  • Terpene and ester products were quantified by a model 7890B gas chromatograph and 5977A mass spectrometer (Agilent technologies, Stockport, UK). Samples were separated on a DB-FFAP 30m x 20 ⁇ x 0.25 ⁇ capillary column under the following conditions: 1 ⁇ of sample was injected onto the column which was held at 40°C for 1 minute, the temperature was then ramped at a gradient of 20°C/min to a final temperature of 250°C and held for 8 minutes. Terpene and ester products typically eluted between 7 and 11 minutes and were monitored on both MS and FID detectors. The concentration of product was quantified using calibration curves for each compound analysed.
  • culture glucose was monitored using ion chromatography with a Dionex 5000+ fitted with a 4x250 mm analytical CarboPac PAi column (Thermo Fisher Scientific, USA). Filtered supernatant was injected and run isocratically for 15 minutes at a flow rate of 1.0 ml/min (50 mM NaOH at 30°C). Glucose concentration was quantified using a calibration curve.
  • E. coli harbouring plasmid pET28a: :AAT was inoculated into LB broth supplemented with kanamycin (50 g/ml) and grown overnight at 37°C. Cultures were then inoculated at 1% into 25 ml of TB + 2% glucose medium with kanamycin (50 g/ml), and grown at 37°C until an OD6 00 of 1 was reached. Cultures were then induced with 400 ⁇ IPTG and moved to 20°C and in a rotary shaker (250 rpm) for 2 hours before being fed 10 mM acetic acid and 0.5 mM geraniol. Cultures were then further incubated for 18 hours at 20°C and 250 rpm.
  • the lower temperature was used to minimize terpene volatilization.
  • the OD 60 o was measured and the culture was centrifuged at 13,000 rpm at 4°C to separate the supernatant and pellet fractions.
  • the supernatant was directly extracted into hexane for analysis by gas-chromatography mass-spectometry (GC-MS).
  • GC-MS gas-chromatography mass-spectometry
  • the pellet was resuspended in 0.9% NaCl, sonicated at 12 microns on ice until clear, and then extracted into hexane for analysis by GC-MS.
  • E. coli strain C34(DE3) was inoculated into LB broth and grown overnight at 37°C. The following day, cultures were inoculated at 1% into 5 ml of TB + 2% glucose medium, and grown at 37°C in a rotary shaker (250rpm) until an OD6 00 of 4 was reached.
  • Cultures were then fed either 0.5 mM geraniol or 0.5 mM Nerol and further incubated at 37°C for 6 h before being centrifuged at 13,000 rpm to separate the culture supernatant and pellet. The pellet was resuspended in 0.9% NaCl, sonicated at 12 microns on ice until clear, and then extracted into hexane for analysis of monoterpene conversion GC-MS.
  • E. coli strain BL2i(DE3) New England Biolabs
  • E. coli strain BL2i(DE3) New England Biolabs
  • pET28A::AAT E. coli strain BL2i(DE3) (New England Biolabs) containing the plasmid pET28A::AAT was grown at 37°C in LB broth supplemented with kanamycin (50 g/ ml) overnight.
  • the culture was then inoculated to 1% in 1 L of Terrific Broth (TB) with kanamycin (50 g/ml) and grown at 37°C and 250 rpm until an OD6 00 of 1.0 was reached.
  • TB Terrific Broth
  • kanamycin 50 g/ml
  • the culture was then centrifuged at 4000 rpm for 20 minutes at 4°C and the cell pellet was resuspended in Buffer A (20 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole) and sonicated on ice at 12 microns until clear.
  • Buffer A (20 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole) and sonicated on ice at 12 microns until clear.
  • the soluble lysate fraction was run through a Poly-Prep chromatography column (0.8 x 4 cm, Biorad) with a pre-equilibrated metal affinity resin charged with cobalt (Clontech).
  • the His(6x)-tagged AAT was then eluted into a 2 ml mixture of Buffer A and Buffer B (20 mM Tris pH 8.0, 300 mM NaCl, 1 M imidazole) containing a final imidazole concentration of 500 mM. Protein concentration was determined using a Bradford assay.
  • the malachite green assay was performed in 96-well flat bottom plates in a total volume of 50 ⁇ according to Vardakou et al, (2014).
  • the assay mix contained malachite green assay buffer (25 mM MES, 20 mM CAPS, 50 mM Tris, 2.5 mU of inorganic pyrophosphatase (Saccharomyces cerevisiae), 5 mM MgCl 2 ) pH 7.5, 0.008 ⁇ purified GES enzyme, geranyl pyrophosphate (GPP) ranging in concentration from 10-400 ⁇ , and a fixed concentration of geraniol (either o, 100, 200, or 400 ⁇ ).
  • malachite green assay buffer 25 mM MES, 20 mM CAPS, 50 mM Tris, 2.5 mU of inorganic pyrophosphatase (Saccharomyces cerevisiae), 5 mM MgCl 2 ) pH 7.5,
  • E. coli C43 (DE3) was transformed with plasmid PMIB13 to produce strain DLG2 (Table 1), which when expressed in E. coli produced 35 mg/L of geraniol after 24 hours in a two-phase culture (Fig. 2).
  • this strain produced significant amounts of the monoterpenoids nerol and citronellol, as well as the sesquiterpenoid farnesol.
  • Previous work has determined that the GES enzyme from O.
  • basilicum has strict substrate specificity for synthesizing only geraniol from the geranyl pyrophosphate precursor (Iijima et al, 2004), suggesting that the observation of additional terpene products is the result of modifications made to geraniol by endogenous E. coli enzymes. Fisher et al, (2013) previously demonstrated upon GES expression the resulting terpene profile is heavily dependent on the heterologous host in which it is being expressed. Ultimately, this further endogenous manipulation of geraniol is limiting to industrial level production, and several groups have worked toward minimizing this bioconversion (Zhou et al, 2014). Also hindering industrial production is the high cytotoxicity of geraniol, which is characteristic of monoterpenes.
  • E. coli possesses only restricted ability to utilize acetic acid for biomass growth, instead it is used primarily via the acs pathway (using AMP-forming acetyl-CoA synthase) and the reversible PTA-ACK pathway (using phosphotransacetylase/acetate kinase) for acetyl-CoA formation - indicating it may be used as a strategy to increase intracellular acetyl-CoA (Castano-Cerezo et al, 2009; Krivoruchko et al, 2015; Yang et al, 2016).
  • acetyl-CoA synthase which catalyzes the condensation of acetate and coenzyme A (CoA).
  • ACS acetyl-CoA synthase
  • Streptomyces sp. strain CL190 (responsible for the condensation of two acetyl-CoA's to form acetoactyl-CoA) allows E. coli to make ⁇ -caryophyllene using acetic acid as its sole carbon source.
  • DLG2 and DLGA3 were performed in a 1.5 L batch reactor. Both strains were cultured in a modified TB media (MTB) in which the phosphate buffer salts were replaced with sodium chloride; this substitution was made as the culture pH would be maintained at 6.8 by the addition of acid and base. Cultures were grown at 30°C to minimize product loss through evaporation, and culture OD 60 o and product formation were tracked over the course of 115 hours for both strains. With strain DLG2, geraniol production peaked at 34 hours with 220 mg/L, after this culture geraniol decreased while citronellol and nerol increased (Fig. 4a).
  • MTB modified TB media
  • strain DLGA3 produced exclusively geranyl acetate in a fed-batch bioreactor run with MTB media.
  • the culture was fed 20 mM acetic acid directly post induction, as it was previously shown to increase product titre (Fig. 3).
  • Geranyl acetate peaked at 95 hours with 4.8 g/L (Fig. 4b). This is approximately 2.4 times more geranyl acetate than the previously reported highest titres achieved by Liu et al, (2016), where bioconversion of geraniol to geranyl acetate, via a promiscuous chloramphenicol acetyltransferase (CAT) enzyme, was also used.
  • CAT promiscuous chloramphenicol acetyltransferase
  • strain DLGA3 was cultured in fed-batch using a semi-defined media (FM) that is more typical of industrial production.
  • FM semi-defined media
  • geranyl acetate production peaked at 77 hours with 422 mg/L (Fig. 7), and again was the only product observed over the course of the fermentation. Though these titres are lower than those observed in the rich MTB media (Fig. 4b), further optimization of the fermentation media and run conditions.
  • Reaction Schem e Proposed pathway for the conversion of geraniol to the similar monoterpenoids, nerol and citronellol, by endogenous E. coli enzymes.
  • the GES enzyme from O. basilicum has exclusive substrate specificity for converting GPP to geraniol.
  • the observed accumulation of both nerol and citronellol over time is most likely the result of endogenous enzymes catalysing the isomerization of geraniol to nerol and visa versa, as well as the reduction of both species to citronellol.
  • Escherichia coli Increase of NADH Availability by Overexpressing an NAD+- Dependent Formate Dehydrogenase. Metab. Eng. 4, 217-229.
  • alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli. Bioengineered 6, 288-293.
  • RecA- mediated SOS response provides a geraniol tolerance in Escherichia coli. J.

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Abstract

L'invention concerne un procédé destiné à la production d'un composé monoterpénoïde, d'un organisme microbien et d'un système d'expression. Le procédé comprend une étape de culture d'au moins un organisme microbien co-exprimant des gènes codant pour une voie métabolique, destinée à la production d'isopentényl pyrophosphate et de diméthylallyl pyrophosphate et : (i) d'une géranyle pyrophosphate synthase ; (ii) d'une synthase de monoterpénoïde ; (iii) d'une acyltransférase d'alcool hétérologue ; et éventuellement (iv) d'une hydroxylase ou d'une réductase ; dans des conditions dans lesquelles le constituant (i), le constituant (ii) et le constituant facultatif (iv) exprimés dans le micro-organisme convertissent une source de carbone en vue de produire un monoterpénoïde comprenant un alcool, qui est ensuite converti par le constituant (iii) exprimé dans le micro-organisme en un ester monoterpénoïde, ces conditions comprenant un système aqueux-organique à deux phases.
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