WO2023140793A2 - Bioproduction of natural phenethyl acetate, phenylacetic acid, ethyl phenylacetate, and phenylethyl phenylacetate from renewable feedstock - Google Patents

Bioproduction of natural phenethyl acetate, phenylacetic acid, ethyl phenylacetate, and phenylethyl phenylacetate from renewable feedstock Download PDF

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WO2023140793A2
WO2023140793A2 PCT/SG2023/050036 SG2023050036W WO2023140793A2 WO 2023140793 A2 WO2023140793 A2 WO 2023140793A2 SG 2023050036 W SG2023050036 W SG 2023050036W WO 2023140793 A2 WO2023140793 A2 WO 2023140793A2
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derivative
enzymes
styrene
cells
reaction
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WO2023140793A3 (en
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Zhi Li
Balaji SUNDARA SEKAR
Xirui LI
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National University Of Singapore
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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
    • C12P7/62—Carboxylic acid esters
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • 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
    • C12P5/002—Preparation of hydrocarbons or halogenated hydrocarbons cyclic
    • C12P5/005—Preparation of hydrocarbons or halogenated hydrocarbons cyclic aromatic
    • 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
    • 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
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • 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
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids

Definitions

  • This invention relates to an artificial enzyme cascade for the efficient production of natural phenethyl acetate, ethyl phenyl acetate and phenylethyl phenylacetate from a feedstock (e.g. styrene, L-phenylalanine or glucose and glycerol via combination of a natural biosynthesis pathway for glucose/glycerol with an artificial enzyme cascade.
  • a feedstock e.g. styrene, L-phenylalanine or glucose and glycerol
  • This provides a simple and high-yielding process (e.g. 13.6 g/L titer; >80% yield) which may be attractive to industry.
  • This invention is also green, sustainable and uses low-cost renewable feedstocks for the efficient production of natural aroma chemicals phenethyl acetate, ethyl phenyl acetate and phenylethyl phenylacetate.
  • Aroma chemicals such as phenethyl acetate (PEA; FEMA number: 2857; Flavis number: 9.031 ; Flavour profile: flower; honey; and rose), phenylacetic acid (PAA; FEMA number: 2878; Flavis number: 8.038; Flavour profile: honey), ethyl phenylacetate (Et-PA; FEMA number: 2452; Flavis number: 9.784; Flavour profile: floral; fruit; honey; and rose) and phenylethyl phenylacetate (PE-PA; FEMA number: 2866; Flavis number: 9.707; Flavour profile: floral) are high-value chemicals used as food and flavour ingredients. These compounds can be extracted from natural sources, but with limited availability and high price.
  • the price of natural PEA and Et-PEA are 1 ,760-2,200 LISD/Kg and the market of PEA is -2000 tons/year.
  • Several chemical methods were established for the synthesis of PEA, PAA, Et-PA, and PE-PA. However, these chemical syntheses start from petroleum derived chemicals, require the use of toxic reagents, and generate by-products and large amount of waste. In addition, the generated by-products affect the aroma and organoleptic profiles for flavor applications. Moreover, chemically synthesized flavor compounds are less preferred in food and cosmetics industry and have restricted use as flavoring agent in Europe and U.S.
  • Yeast is a natural producer of these chemicals during fermentation such as wine making process.
  • L-phenylalanine L-Phe
  • ATFs alcohol acetyl transferases
  • a method for producing phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof using four or more enzymes comprises subjecting styrene or a derivative thereof to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.
  • the epoxidase is a styrene monooxygenase, optionally wherein the styrene monooxygenase is from Pseudomonas sp. VLB120, or its mutants or similar enzymes with more than 50% identity; and/or
  • the isomerase is a styrene oxide isomerase, optionally wherein the styrene oxide isomerase is from Pseudomonas sp. VLB 120, or its mutants or similar enzymes with more than 50% identity; and/or
  • the reductase is a phenylacetaldehyde reductase, optionally wherein the phenylacetaldehyde reductase is from Solanum lypersicum, or its mutants or similar enzymes with more than 50% identity; and/or
  • the transferase is an alcohol acetyl transferase, optionally wherein the alcohol acetyl transferase is from Saccharomyces cerevisiae, or its mutants or similar enzymes with more than 50% identity.
  • the epoxidase is a styrene monooxygenase, optionally wherein the styrene monooxygenase is from Pseudomonas sp. VLB120, or its mutants or similar enzymes with more than 50% identity; and/or
  • the isomerase is a styrene oxide isomerase, optionally wherein the styrene oxide isomerase is from Pseudomonas sp. VLB 120, or its mutants or similar enzymes with more than 50% identity; and/or
  • the oxidase or aldehyde dehydrogenase is aldehyde dehydrogenase (EcALDH) from Escherichia coli K12, or its mutants or similar enzymes with more than 50% identity; and/or
  • the lipase is Novozyme 435 from Candida Antarctica or TL lipozyme from Thermomyces lanuginosus, or their mutants or similar enzymes with more than 50% identity.
  • the method further comprises providing styrene or a derivative thereof by generating trans-cinnamic acid or a derivative thereof from L-phenylalanine or a derivative thereof by a deamination reaction catalyzed by an ammonia lyase and generating styrene or a derivative thereof from the transcinnamic acid or a derivative thereof in a decarboxylation reaction catalyzed by a decarboxylase.
  • the decarboxylase is phenylacrylic acid decarboxylase from Aspergillus niger, or its mutants or similar enzymes with more than 50% identity.
  • the microbial cells producing L- phenylalanine from glucose or glycerol overexpress at least one enzyme is selected from one or more of the group consisting of DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), or their mutants or similar enzymes with more than 50% identity, optionally wherein the microbial cells producing L-phenylalanine from glucose or glycerol overexpress at least one enzyme is:
  • the one-pot reaction system comprises an aqueous medium.
  • the one-pot reaction system comprises use of a bi-phasic medium, optionally wherein the bi-phasic medium consists of an aqueous and solid resin medium, or an aqueous and organic solvent medium (e.g. the organic solvent medium is selected from one or more alkanes solvents and/or one or more ester solvents).
  • the one-pot reaction system comprises use of absorbent resins or nanomaterials for in-situ removal of product.
  • One or more recombinant prokaryotic or eukaryotic cells selected from the group comprising bacterial cells, yeast cells, mammalian cells and insect cells, wherein said cells comprise at least one expression construct as described in Clause 22.
  • a method for producing phenylacetic acid (PAA) or a derivative thereof using five or more enzymes comprises subjecting L-phenylalanine or a derivative thereof to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.
  • the decarboxylase is phenylacrylic acid decarboxylase from Aspergillus niger, or its mutants or similar enzymes with more than 50% identity; and/or
  • the epoxidase is a styrene monooxygenase, optionally wherein the styrene monooxygenase is from Pseudomonas sp. VLB120, or its mutants or similar enzymes with more than 50% identity; and/or
  • the isomerase is a styrene oxide isomerase, optionally wherein the styrene oxide isomerase is from Pseudomonas sp. VLB 120, or its mutants or similar enzymes with more than 50% identity; and/or
  • the oxidase or aldehyde dehydrogenase is aldehyde dehydrogenase (EcALDH).
  • the microbial cells producing L- phenylalanine from glucose or glycerol overexpress at least one enzyme is selected from one or more of the group consisting of DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), or their mutants or similar enzymes with more than 50% identity, optionally wherein the microbial cells producing L-phenylalanine from glucose or glycerol overexpress at least one enzyme is:
  • the one-pot reaction system comprises use of a bi-phasic medium, optionally wherein the bi-phasic medium consists of an aqueous and solid resin medium, or an aqueous and organic solvent medium (e.g. the organic solvent medium is selected from one or more alkanes solvents and/or one or more ester solvents).
  • the bi-phasic medium consists of an aqueous and solid resin medium, or an aqueous and organic solvent medium (e.g. the organic solvent medium is selected from one or more alkanes solvents and/or one or more ester solvents).
  • One or more recombinant prokaryotic or eukaryotic cells selected from the group comprising bacterial cells, yeast cells, mammalian cells and insect cells, wherein said cells comprise at least one expression construct as described in Clause 41.
  • a kit comprising at least one isolated nucleic acid according to any one of Clauses 38 to 40.
  • FIG. 1 depicts the schematic representation of the sustainable bioproduction of natural PEA, PAA, Et-PA and PE-PA from renewable feedstock L-phenylalanine (L-Phe), glucose, and glycerol.
  • FIG. 2 depicts the synthesis of natural PEA and PAA from bio-based L-Phe and renewable feedstock glucose and glycerol, a) i) The conversion of L-Phe to PEA via one-pot cascade deamination-decarboxylation-epoxidation-isomerization-reduction-transferase reaction.
  • Enzymes involved are phenylalanine ammonia lyase (PAL), phenylacrylic acid decarboxylase (PAD; consisting of PAD and FDC), styrene monooxygenase (SMO, consisting of StyA and StyB), styrene oxide isomerase (SOI), phenylacetaldehyde reductase (PAR), and alcohol acetyl transferase (ATF).
  • PAL phenylalanine ammonia lyase
  • PAD phenylacrylic acid decarboxylase
  • SMO styrene monooxygenase
  • SOI styrene oxide isomerase
  • PAR phenylacetaldehyde reductase
  • ATF alcohol acetyl transferase
  • the enzymes involved are PAL, PAD, SMO, SOI, and aldehyde dehydrogenase (EcALDH).
  • PAL PAL
  • PAD synthesis of L-Phe
  • SMO sulfur dioxide
  • SOI sulfur dioxide
  • EcALDH aldehyde dehydrogenase
  • FIG. 3 depicts a) schematic representation of the plasmid expressing ATF and codon- optimized ATF (Opt-ATF). b) Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the soluble and particulate fractions of E. co//-pCDFDuet (Lane 1), E. co//-pCDFDuet-ATF (Lane 2) and E. co//-pCDFDuet-Opt-ATF (Lane 3).
  • SDS-PAGE Sodium dodecyl-sulfate polyacrylamide gel electrophoresis
  • ATF is marked with an arrow, c) Time course of biotransformation of 45 mM 2-phenylethanol (2- PE) to PEA with 10 g cdw/L cells of E. coli Opt-ATF. Reaction conditions: two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose) and n-hexadecane (1 :1 v/v); 30 °C; 220 rpm; and 4 h. Concentrations are normalized to aqueous phase volume. Experiments were performed in duplicates with error bars showing ⁇ s.d.
  • FIG. 4 depicts the high-performance liquid chromatography (HPLC) chromatograms showing the consumption of 2-PE during the biotransformation of 2-PE to PEA by 10 g cdw/L of a) E. coli T7 express (negative control) and b) E. co//-Opt-ATF.
  • the samples at 0 and 2 h were analysed.
  • Reaction conditions two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose, 45 mM 2-PE) and n-hexadecane (1 :1 v/v); 30 °C; 220 rpm; and 2 h.
  • FIG. 5 depicts gas chromatography-mass spectrometry (GC-MS) analysis, a) GC chromatogram showing the production of PEA in biotransformation of 2-PE to PEA with 10 g cdw/L of E. coli Opt-ATF. b) MS profile of the 2-PEA peak eluting at 6.886 min. The sample at 2 h was measured. Reaction conditions: two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose, 45 mM 2-PE) and n-hexadecane (1 :1 v/v); 30 °C; 220 rpm; and 2 h.
  • phosphate buffer 200 mM, pH 8.0, 2% glucose, 45 mM 2-PE
  • n-hexadecane (1 :1 v/v
  • FIG. 6 depicts a) E. co//-Sty-PEA harboring three plasmids for the expression of PAL, PAD, SMO, SOI, PAR and ATF. b) SDS-PAGE analysis of soluble fractions of E. co//-Sty-PEA. Lane M, Protein ladder; Lane 1 , E. co//-pCDFDuet (negative control); Lane 2, E. co//-Sty-2-PE (negative control); and Lane 3, E. co//-Sty-PEA. c) Concentration of the accumulated 2-PE and produced PEA at 24 h in the biotransformation of 50, 80 and 100 mM L-Phe, respectively, with 10 g cdw/L cells of E.
  • reaction conditions two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose) and n-hexadecane (1 :1 v/v); 30 °C; and 24 h.
  • d Optimization of reaction conditions by modifying the organic phase in the biotransformation of 100 mM L-Phe to PEA with 15 g cdw/L cells of E. co//-Sty-PEA.
  • Reaction conditions two- phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose; Aq) and n-hexadecane (Hx) or ethyl oleate (EO) in 1 :1 or 1 :0.5 v/v; 30 °C; and 24 h.
  • e, f Time courses of the cascade conversion of 100 mM L-Phe to PEA with optimized reaction conditions using 15 g cdw/L E. co//-Sty-PEA with (e) ethyl oleate and (f) biodiesel as organic phases.
  • FIG. 7 depicts the time courses of the accumulated 2-PE and produced PEA at 24 h in the biotransformation of a) 50 mM, b) 80 mM and c) 100 mM L-Phe to PEA, respectively, with 10 g cdw/L cells of E. co//-Sty-ATF.
  • Reaction conditions two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose) and n-hexadecane (1 :1 v/v); 30 °C; and 24 h.
  • FIG. 8 depicts the optimization of reaction conditions for the cascade conversion of L-Phe to PEA. Concentration of the synthesized PEA in the biotransformation with varying a) amount of whole cells and b) glucose addition. Reaction conditions: two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose) and n-hexadecane (1 :1 v/v); 30 °C; and 24 h.
  • FIG. 9 depicts a) E. co//-Sty-PAA harboring two plasmids for the expression of PAL, PAD, SMO, SOI and EcALDH. b) SDS-PAGE analysis of soluble fractions of E. co//-Sty-PAA. Lane M, Protein ladder; Lane 1 , E. co//-pCDFDuet (negative control); and Lane 2, E. co//-Sty-PAA. c) Time course of the cascade conversion of 100 mM of L-Phe to PAA with 15 g cdw/L cells of E. co//-Sty-PAA.
  • FIG. 10 depicts a) coupled fermentation and biotransformation approach for the production of PEA and PAA from renewable feedstock glucose and glycerol, respectively. Fermentation of glucose and glycerol to L-Phe was performed with E. coli NST74-Phe, followed by biotransformation of 67-78 mM biosynthesized L-Phe to PEA and PAA with 15 g cdw/L E. coli- Sty-PEA and E. co//-Sty-PAA, respectively, in a two-phase system with fermented media containing phosphate buffer and ethyl oleate (1 :0.5, v/v).
  • FIG. 11 depicts the screening of lipases for the conversion of PAA to Et-PA and PE-PA, respectively.
  • FIG. 12 depicts the a) production of natural Et-PA and PE-PA from bio-based L-Phe and renewable feedstock glucose, i) Bioproduction of PAA from L-Phe or glucose, followed by lipase-catalyzed esterification of PAA to Et-PA. ii) Bioproduction of PAA and 2-PE from L-Phe or glucose, followed by lipase-catalyzed esterification of PAA and 2-PE to PE-PA.
  • a method for producing phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof using one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes which method comprises subjecting styrene or a derivative thereof to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.
  • the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features.
  • the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention.
  • the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.
  • the phrase, “consists essentially of” and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present.
  • the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.
  • the styrene itself may be produced from L-phenylalanine by a further enzymatic cascade, with the L-phenylalanine being obtained either from the cell’s natural metabolic processes or an enhanced enzymatic pathway that provides an up-regulated production of L-phenylalanine.
  • These multiple reaction steps may be performed simultaneously or sequentially in one reaction vessel, to allow for the green, efficient, and economical production of the desired products directly from styrene or a derivative thereof, phenylalanine or a derivative thereof, glucose or glycerol.
  • one-pot cascade reactions may avoid the expensive and energy-consuming isolation and purification of intermediates, minimize waste generation, and overcome the possible thermodynamic hurdles normally encountered in traditional multi-step synthesis.
  • multiple enzymes may be coexpressed inside one recombinant microbe strain, and the whole cells of the strain may be directly applied as catalysts for a series of cascade reactions in one pot.
  • the enzymes could be separately expressed in cells of different strains, purified individually, or immobilized (the purified enzymes or cells containing all or some of said enzymes).
  • the biocatalysts enzymes, cells, immobilized enzymes, and immobilized cells
  • a method for producing an phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof comprises subjecting styrene or a derivative thereof (or other suitable starting materials mentioned herein) to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.
  • Styrene and derivatives thereof can be manufactured on very large scale in the petrochemical industry (e.g. by hydrocarbon cracking), and so form easily available and cheap starting materials for organic synthesis of the type discussed herein. For example, many aromatic and aliphatic alkenes are produced in very large amounts and at very low price. As discussed hereinbelow, styrenes and substituted styrenes are very useful substrates to produce various desirable products.
  • the current invention also allows for the conversion of L-phenylalanine and derivatives thereof into styrene and derivatives thereof by way of further enzyme-catalyzed transformations which will be discussed hereinbelow. This may enable access to styrene derivatives that may otherwise be difficult to obtain access to and provide a greater pool of possible styrene substrates for use in the enzyme-catalyzed transformations described herein.
  • the term “derivative thereof” as applied to styrene and L-phenylalanine relates to a compound where the benzene ring contains one or more substituents (e.g. 1 , 2, 3, 4or 5, such as 1 to 3, such as 1 or 2 substituents) that are not H.
  • substituents may be halo, alkyl, cycloalkyl, aryl, heterocyclic, OH, NH 2 , SH and combinations thereof (e.g. alkyl aryl, Oalkyl, N(alkyl)H, N(alkyl) 2 , N(alkyl)(aryl) etc).
  • alkyl refers to an unbranched or branched, saturated or unsaturated hydrocarbyl radical (so forming, for example, an alkenyl or alkynyl), which may be substituted or unsubstituted (with, for example, one or more halogen atoms).
  • the alkyl group may be Ci-w alkyl and, more preferably, Ci-e alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl).
  • alkenyl and alkynyl are to be interpreted accordingly.
  • cycloalkyl refers to an unbranched or branched, saturated or unsaturated hydrocarbyl radical (so forming, for example, a cycloalkenyl group) that may be substituted or unsubstituted.
  • the cycloalkyl group may be C3-12 cycloalkyl and, more preferably, C5-10 (e.g. C5-7) cycloalkyl.
  • cycloalkenyl is to be interpreted accordingly.
  • halogen when used herein, includes fluorine, chlorine, bromine and iodine.
  • aryl when used herein includes Ce-i4 (such as Ce-i3 (e.g. Ce- )) aryl groups that may be substituted or unsubstituted. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 14 ring carbon atoms, in which at least one ring is aromatic. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an aromatic ring.
  • Ce-14 aryl groups include phenyl, naphthyl and the like, such as 1 ,2,3,4-tetrahydronaphthyl, indanyl, indenyl and fluorenyl. Most preferred aryl groups include phenyl.
  • aryl alkyl is to be interpreted in line with the definitions provided hereinbefore for “alkyl” and “aryl”, where the point of attachment of the group to the rest of the compound of formula (I) or (II) is through the alkyl portion of the aryl alkyl group.
  • heterocyclic refers to a fully saturated, partly unsaturated, wholly aromatic or partly aromatic ring system in which one or more (e.g. one to four) of the atoms in the ring system is other than carbon (i.e. a heteroatom, which heteroatom is preferably selected from N, O and S), and in which the total number of atoms in the ring system is between three and twelve (e.g. between five and ten).
  • the heterocyclic groups may be substituted or unsubstituted.
  • Heterocyclic groups that may be mentioned include 7- azabicyclo[2.2.1]heptanyl, 6-azabicyclo[3.1.1]heptanyl, 6-azabicyclo[3.2.1]octanyl, 8- azabicyclo[3.2.1]octanyl, aziridinyl, azetidinyl, dihydropyranyl, dihydropyridyl, dihydropyrrolyl (including 2,5-dihydropyrrolyl), dioxolanyl (including 1 ,3-dioxolanyl), dioxanyl (including 1 ,3- dioxanyl and 1 ,4-dioxanyl), dithianyl (including 1 ,4-dithianyl), dithiolanyl (including 1 ,3- dithiolanyl), imidazolidinyl, imidazolinyl, morpholinyl, 7-oxabicyclo[2.2.1]heptanyl, 6-
  • Substituents on heterocyclic groups may, where appropriate, be located on any atom in the ring system including a heteroatom.
  • heterocyclic groups may be via any atom in the ring system including (where appropriate) a heteroatom (such as a nitrogen atom), or an atom on any fused carbocyclic ring that may be present as part of the ring system.
  • Heterocyclic groups may also be in the N- or S- oxidised form.
  • Heterocyclic groups that may be mentioned herein include cyclic amino groups such as pyrrolidinyl, piperidyl, piperazinyl, morpholinyl or a cyclic ether such as tetrahydrofuranyl.
  • heterocyclic alkyl is to be interpreted in line with the definitions provided hereinbefore for “alkyl” and “heterocyclic”, where the point of attachment of the group to the rest of the compound of formula (I) or (II) is through the alkyl portion of the heterocyclic alkyl group.
  • styrene and L-phenylalanine derivatives that may be mentioned herein include those in which the phenyl ring of styrene and/or phenylalanine is mono- or disubstituted by (a) substituent(s) selected from F, Cl, Br, CH3 or OCH3.
  • the phenyl ring of styrene and/or phenylalanine may be monosubstituted by o-F, m-F, p-F, m-CI, p-CI, m-Br, p-Br, m- CH 3 , p-CH 3 , or p-OCHs.
  • the styrene derivative may be indene which is either unsubstituted or substituted as described for styrene above.
  • amino acid or “amino acid sequence,” as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • isolated is herein defined as a biological component (such as a nucleic acid, peptide or protein) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins.
  • Nucleic acids, peptides and proteins which have been isolated thus include nucleic acids and proteins purified by standard purification methods.
  • the term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • nucleic acid or “nucleic acid sequence,” as used herein, refer to an oligonucleotide, nucleotide, polynucleotide, or any fragment thereof, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • fragments refers to those nucleic acid sequences which are greater than about 60 nucleotides in length, and most preferably are at least about 100 nucleotides, at least about 1000 nucleotides, or at least about 10,000 nucleotides in length which are not full-length native sequence but retain catalytic enzyme activity.
  • oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray.
  • oligonucleotide is substantially equivalent to the terms “amplimers,” “primers,” “oligomers,” and “probes,” as these terms are commonly defined in the art.
  • the terms ‘variant’ and ‘mutant’ are used interchangeably herein.
  • the at least one nucleic acids encoding at least one catalytic enzyme may encode a variant or mutant of the exemplified catalytic enzyme which retains activity.
  • a "variant" of a catalytic enzyme refers to an amino acid sequence that is altered by one or more amino acids.
  • the variant may have "conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan).
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • variant enzymes are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, homologous or identical at the amino acid level to an exemplary amino acid sequence described herein (e.g., catalase, alcohol dehydrogenase, a-transaminase) or a functional fragment thereof — e.g., over a length of about: 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the length of the mature reference sequence.
  • an exemplary amino acid sequence described herein e.g., catalase, alcohol dehydrogenase, a-transaminase
  • a functional fragment thereof e.g., over a length of about: 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the length of the mature reference sequence.
  • PAR phenylacetaldehyde reductase
  • ADH alcohol dehydrogenase
  • a vector can include one or more catalytic enzyme nucleic acid(s) in a form suitable for expression of the nucleic acid(s) in a host cell.
  • the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence(s) to be expressed.
  • the term "regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences such as the T7 IPTG-inducible promoters disclosed in the Examples herein.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., catalytic enzyme proteins, fusion proteins, and the like).
  • the recombinant expression vectors of the invention can be designed for expression of catalytic enzyme proteins in prokaryotic or eukaryotic cells.
  • polypeptides of the invention can be expressed in bacteria (e.g., E. coli), insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif.
  • the recombinant expression vector(s) can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S.
  • GST glutathione S-transferase
  • E. coli To maximize recombinant protein expression in E. coli is to express the protein in a host bacterium with an impaired capacity to proteolytical ly cleave the recombinant protein (Gottesman, S. Gene Expression Technology: Methods in Enzymology 1990 185, Academic Press, San Diego, Calif. 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., Nucleic Acids Res. 199220: 2111-2118).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques and is described in the Examples.
  • the catalytic enzyme expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector, a vector for expression in bacterial cells, e.g. a plasmid vector, or a vector suitable for expression in mammalian cells.
  • the expression vector's control functions can be provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g., by means of a signal transduction pathway), by a chemical (e.g., Isopropyl p-D-1 -thiogalactopyranoside (IPTG)) or by a heterologous polypeptide.
  • a promoter regulated by a steroid hormone e.g., by a polypeptide hormone (e.g., by means of a signal transduction pathway)
  • a chemical e.g., Isopropyl p-D-1 -thiogalactopyranoside (IPTG)
  • IPTG Isopropyl p-D-1 -thiogalactopyranoside
  • the methods described herein make use of enzymes to catalyse a sequence of reactions. While these reactions may be performed individually or, more particularly, two or more of them in combination, all of the reactions may be combined into a cascade reaction sequence that provides the product from the initial starting material in one pot, thereby eliminating the need for isolation of the intermediates and, potentially, increasing the overall yield of the reaction sequence.
  • These cascade reactions may involve the use of one or more reactive components selected from the group consisting of cells, immobilized cells, cell extracts, isolated enzymes and immobilized enzymes in said reaction vessel.
  • the invention provides at least one expression construct comprising at least one nucleic acid sequence that is heterologous according to any aspect of the invention.
  • the at least one construct comprises a plasmid suitable for expression of at least one catalytic enzyme in a bacterium.
  • a host cell which includes at least one nucleic acid molecule described herein, e.g., at least one catalytic enzyme nucleic acid molecule within a recombinant expression vector or a catalytic enzyme nucleic acid molecule containing sequences which allow homologous recombination into a specific site of the host cell's genome.
  • the terms "host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • at least one catalytic enzyme protein can be expressed in bacterial cells (such as E. coli), insect cells (such as Spodoptera frugiperda Sf9 cells), yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman Cell 1981 , I23: 175-182)).
  • bacterial cells such as E. coli
  • insect cells such as Spodoptera frugiperda Sf9 cells
  • yeast or mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman Cell 1981 , I23: 175-182)
  • Other suitable host cells are known to those skilled in the art.
  • One or more vector DNAs can be introduced into host cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • a host cell may comprise one, two, three, four or more plasmids, each of which may express at least one catalytic enzyme directed to a chemical transformation in the pathway from an alkene starting material to the desirable products mentioned herein.
  • the host cell may also include a vector to express catalytic enzymes for providing the alkene by, for example, generating a vinyl carboxylic acid from an a-amino acid by a deamination reaction catalyzed by an ammonia lyase and generating the alkene from the vinyl carboxylic acid in a decarboxylation reaction catalyzed by a decarboxylase.
  • catalytic enzymes for providing the alkene by, for example, generating a vinyl carboxylic acid from an a-amino acid by a deamination reaction catalyzed by an ammonia lyase and generating the alkene from the vinyl carboxylic acid in a decarboxylation reaction catalyzed by a decarboxylase.
  • the catalytic enzymes required to transform an alkene starting material to phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof are arranged on expression vectors as modules, wherein each module comprises a combination of catalytic enzymes to perform specific reactions within the overall system.
  • a host cell comprising the said first and third modules, on the same or separate plasmids, is capable of catalyzing the conversion of a terminal alkene to phenethyl acetate.
  • a host cell comprising said first, second and third module is capable of catalyzing the conversion of a terminal alkene to phenethyl acetate, or a derivative thereof. If module 4 is co-expressed in the host cell, an a-amino acid feed stock can be provided for the cell to generate its own terminal alkene for the cascade reactions.
  • one or more recombinant prokaryotic or eukaryotic cells selected from the group comprising bacterial cells, yeast cells, mammalian cells and insect cells, wherein said cells comprise at least one expression construct and/or heterologous nucleic acid molecule that encodes at least one catalytic enzyme required in the pathway from alkene to phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof.
  • PDA phenethyl acetate
  • Et-PA ethyl phenylacetate
  • PE-PA phenylethyl phenylacetate
  • the cells may contain a single expression vector or construct, such as a plasmid, which expresses a single catalytic enzyme or co-expresses a plurality of catalytic enzymes as described herein under the control of at least one regulatory element.
  • the catalytic enzymes may be arranged in the plasmid as an individual artificial operon under the control of a promoter with one ribosome-binding site before every gene, or arranged with individual promoters. Accordingly, a one-pot synthesis cascade may be achieved with cells expressing all required catalytic enzymes on one or several plasmids, or with different recombinant cells which each express a specific repertoire of catalytic enzymes, providing the necessary cells are included for a particular chemical transformation.
  • said catalytic enzymes for use in the invention have at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, homology or amino acid identity with at least one enzyme mentioned herein.
  • a host cell of the invention can be used to produce (i.e. , express) one or more catalytic enzyme proteins. Accordingly, the invention further provides methods for producing one or more catalytic enzyme proteins, e.g., one or more catalytic enzyme proteins described herein, using the host cells of the invention.
  • the method includes culturing the host cell of the invention (into which one or more recombinant expression vector(s) encoding one or more catalytic enzyme proteins has/have been introduced) in a suitable medium such that one or more catalytic enzyme proteins is/are produced.
  • the method further includes isolating one or more catalytic enzyme proteins from the medium or the host cell.
  • kits comprising at least one recombinant cell, expression construct or isolated nucleic acid according to any aspect of the invention.
  • the kit can be used to provide one or more components for a one-pot system to enzyme-catalyze the production of phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof from a suitable starting material.
  • PEA phenethyl acetate
  • Et-PA ethyl phenylacetate
  • PE-PA phenylethyl phenylacetate
  • Further aspects of the invention may relate to a method for producing phenylacetic acid (PAA) or a derivative thereof using five or more enzymes, which method comprises subjecting L- phenylalanine or a derivative thereof to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system. This may be achieved using broadly the same technology as discussed hereinbefore and/or throughout the specification.
  • PAA phenylacetic acid
  • the lipase may form part of the enzymatic cascade within one or more recombinant cells, or it may be provided as an immobilised enzyme.
  • the immobilised lipase may be provided on a support - such as described in Facin, etal., Ind. Eng. Chem. Res. 2019, 58, 14, 5358-5378.
  • chemical-based methods are low-yielding, and suffer from by-product accumulation. These by-products can affect the aroma and organoleptic profile of the product.
  • the currently disclosed enzyme cascade avoids the accumulation of by-products, thereby overcoming this issue.
  • aroma chemicals produced from chemical-based methods are not preferred in the food industry.
  • the disclosed enzyme cascade produces ‘natural’ products with a higher market value and will be more readily accepted for use in the food industry.
  • Chemical-based methods are also multi-step in nature and involve purification of intermediates at each step, which is an energy-intensive process.
  • the current invention is performed in one-pot, making it much more energy- and material-efficient and easy to scale up.
  • the currently disclosed method to make the above-mentioned materials use chemicals and acidic or basic catalysis at high temperatures. This is avoided by the current invention, which uses mild reaction conditions only.
  • current synthetic methods use petroleum- dervied chemicals which are unsustainable.
  • the current invention uses renewable feedstocks such as styrene (which can be derived from L-phenylalanine), L-phenylalanine, glucose or glycerol and “greener” solvents, thereby minimising environmental impact.
  • the current invention provides a method that is efficient and can provide a high product titer (e.g. 13.6 g/L) and yield (e.g. >80% conversion).
  • Isopropyl p-D-1 -thiogalactopyranoside (IPTG, 99%) was purchased from 1 st base Singapore.
  • Biodiesel (B100) was purchased from Alpha Biofuels Singapore.
  • Ethyl acetate (EA, HPLC grade) and acetonitrile (HPLC grade) were obtained from Tedia.
  • Luria broth (LB, Miller) powder and BactoTM yeast extract were purchased from Becton Dickinson Germany. 5x SDS sample loading dye was purchased from GenScript (USA).
  • Plasmid isolation kit and gel extraction kit were purchased from Qiagen USA. All DNA-modifying enzymes including DNA polymerases and restriction digestion enzymes required in gene cloning were purchased from Thermo Fisher Scientific USA.
  • Seed medium for the production of L-Phe in shaking flask’ glucose/glycerol 20 g/L, (NH4)2SO4 10 g/L, KH 2 PO4 1.5 g/L, MgSO 4 5 g/L, yeast extract 4 g/L, FeSO4 15 mg/L, sodium citrate 0.5 g/L and thiamine HCI 0.1 g/L.
  • the pH was adjusted to 7.0 with NH4OH.
  • Fermentation medium for the production of L-Phe in bioreactor glucose/glycerol 10 g/L, (NH4)2SO4 10 g/L, KH2PO4 5 g/L, MgSC 5 g/L, yeast extract 5 g/L, FeSC 15 mg/L, MnSC 15 mg/L, and betaine 1 g/L.
  • the pH was maintained at ⁇ 6.8 with NH4OH.
  • Modified M9 medium glucose 20 g/L, yeast extract 6 g/L, Na2HPO4 6 g/L, KH2PO4 3 g/L, NH4CI 1 g/L, NaCI 0.5 g/L, MgSC 1 mM, CaCh 0.1 mM, and trace metal solution 1 mL/L.
  • the composition of trace metal solution (per litre): 0.1 M HCI solution, 8.3 g FeCL, 0.84 g ZnCh, 0.13 g CUCI 2 ’2H 2 O, 0.1 g CoCI 2 ’2H 2 O, 0.1 g H3BO3, 0.1 g Na 2 MoO 4 and 0.016 g MnCI 2 .
  • L-Phe, 2-PE and PAA in the aqueous phase were measured by HPLC (LC-20AD, Prominence, Shimadzu Corporation). Detector, Photodiode array (DAD); Column, Agilent Poroshell 120 SB-C18 (150 x 4.6 mm, 2.7 pm); Eluent, 30% acetonitrile and 70% ultrapure water containing 0.1 % TFA; Flow rate, 0.5 mL/min; Column temperature, 30 °C. Acetophenone was used as an internal standard. Retention time: L-Phe, 3.5 min; 2-PE, 9.6 min; PAA, 9.2 min; and acetophenone, 15.9 min.
  • PEA, Et-PA and PE-PA in organic phases were analyzed by GC (Agilent 7890A). Detector, FID; Column, Agilent HP-5 column (30 m x 0.32 mm x 0.25 mm); Temperature programme: 70 °C, hold for 1 min; increase 25 °C/min to 200 °C, hold for 1 min; increase 50 °C/min to 280 °C, hold for 1 min. Benzyl alcohol was used as an internal standard. Retention time: PEA, 8.15 min; 8.06 Et-PA, min; 11.09 PE-PA, min; and benzyl alcohol, 5.9 min.
  • the organic phase containing PEA, Et-PA and PE-PA were subjected to GC/MS analysis using a 7890B GC system with an Agilent 5977A MSD and an HP-5MS column (30 m x 0.32 mm x 0.25 mm); Temperature programme: 70 °C, hold for 1 min; increase 25 °C/min to 200 °C, hold for 1 min; and increase 50 °C/min to 280 °C, hold for 1 min.
  • Glucose, glycerol, and acetate levels during fed-batch fermentation was monitored by HPLC (LC-20AD, Prominence, Shimadzu Corporation). Detector, refractive index detector; Column, Aminex-HPX87H column (Biorad, USA); Eluent, 5 mM sulfuric acid in ultrapure water; Flow rate, 0.5 mL/min; Column temperature, 30 °C. Retention time: glucose, 10.1 min; glycerol, 15.2 min; and acetate, 17.4 min.
  • the cell pellets were normalized to 10 ODeoo by resuspending in 20 mM potassium phosphate buffer (pH 8.0). 1mL of resuspended cells were taken in 2 mL lysis tube followed by the addition of 1 g of 0.1 mm glass beads. The cells were broken using FastPrep-24 homogenizer for 20 s and 6 cycles. The broken cells were centrifuged at 4,000 x g for 5 min to remove unbroken cells and glass beads. 500 pL of supernatant was further centrifuged at 13,000 x g for 30 min to separate soluble and particulate fractions. The soluble fractions (supernatant) were mixed with 5x SDS sample loading dye, heated at 95 °C for 15 min, and resolved in 12% SDS-PAGE gel.
  • a six-enzyme artificial cascade was designed for the conversion of L-Phe to PEA (FIG. 2ai).
  • the cascade consists enzymes for the following reactions: deamination of L-Phe to transcinnamic acid using PAL; decarboxylation of trans-cinnamic acid to styrene using PAD; epoxidation of styrene to styrene oxide using SMO; isomerization of styrene oxide to phenylacetaldehyde using SOI; reduction of phenylacetaldehyde to 2-PE using PAR; and transfer reaction of 2-PE to PEA using ATF.
  • PAL of Arabidopsis thaliana, PAD of Aspergillus niger, SMO and SOI of Pseudomonas sp. VLB120 and PAR of Solanum lypersicum were selected for establishing the newly proposed cascade to convert L-Phe to PEA.
  • ATF of Saccharomyces cerevisiae S. cerevisiae was reported to synthesize various esters using alcohols and acyl-CoAs (A. B. Mason & J. P. Dufour, Yeast 2000, 16, 1287-1298; and W. Li et a!., Eur. Food Res. Technol. 2018, 244, 555- 564), it was thus examined for the production of PEA from 2-PE.
  • the gene coding for ATF was amplified from the genome of S. cerevisiae using PCR and cloned into pCDFDuet plasmid (pCDF-ATF) for expression in E. coli.
  • pCDF-ATF pCDFDuet plasmid
  • ATF gene was also codon-optimized (Opt-ATF), synthesized, and cloned into pCDFDuet plasmid (pCDF-Opt-ATF).
  • the recombinant plasmids pCDF-ATF and pCDF-Opt-ATF were transformed into individual E. coli T7 express strains to construct E. coli ATF and E.
  • the glycerol stock of recombinant E. coli strain was inoculated in 2 mL LB medium with appropriate antibiotics and grown at 37 °C and 220 rpm for 6-8 h. 1 mL of the culture was added to 50 mL of modified M9 medium with appropriate antibiotics (50 pg/mL streptomycin) in a 250 mL flask and grown at 37 °C and 220 rpm. At 2 h, 0.5 mM IPTG was added and the growth temperature was set at 22 °C. After 16 h of growth at 22 °C, the cells were harvested by centrifugation at 2,000 x g for 10 min. The harvested cells were used for SDS-PAGE analysis and biotransformation of 2-PE to PEA.
  • the genes coding for PAL and PAD were amplified using PCR and cloned in pRSFDuet plasmid.
  • the genes coding for SMO, SOI and PAR were amplified using PCR and cloned in pETDuet plasmid.
  • the genes coding for ATF was amplified from S. cerevisiae.
  • the codon- optimized ATF was synthesized by Genscript and cloned in pCDFDuet plamid.
  • the presence of recombinant genes in the engineered strains were confirmed using colony PCR and the expression of recombinant genes were confirmed using SDS-PAGE analysis.
  • the confirmed strains were grown in 2 mL LB medium with respective antibiotics at 37 °C for 12 h. Glycerol stocks were made for all recombinant strains and stored at -80 °C until further usage.
  • phosphate buffer 200 mM, pH 8 containing 15 g cdw/L of E. coli-Sty-PEA, 100 mM L- Phe, and 2% glucose were mixed with 50 mL ethyl oleate.
  • the reaction was performed at 30 °C and 220 rpm. 2% of glucose was added to the reaction at 6 h.
  • the reaction mixtures were centrifuged at 10,000 x g for 10 min to separate the aqueous and ethyl oleate phases.
  • the ethyl oleate phase containing PEA was dried over Na2SO4, filtered, and loaded onto a silica column.
  • S. cerevisiae is known to produce various phenylesters during the fermentation process to produce wines. Therefore, alcohol acetyl transferases from S. cerevisiae were analyzed for the production of PEA.
  • ATF was reported to be highly active in S. cerevisiae. ATF was amplified from S. cerevisiae genome and cloned under the control of T7 promoter into pCDFDuet plasmid.
  • the recombinant E. coli was grown and the expression of ATF was studied (FIG. 3b).
  • the expression of ATF was checked in E. coli in soluble and particulate fractions. It was identified that most of the expression was present in the particulate fractions, which denotes the production of enzymes as inactive inclusion bodies.
  • the expression of ATF in soluble fraction was weak, therefore, the ATF gene was codon-optimized for efficient expression in E. coli, synthesized, cloned into pCDFDuet plasmid, and expressed in the recombinant strain E. coli Opt-ATF.
  • the soluble expression of ATF significantly improved in E. coli Opt-ATF, as shown in SDS-PAGE (FIG. 3b).
  • Example 3 Engineering of E. coli expressing six-enzyme cascade and one-pot biotransformation of L-Phe to PEA
  • the plasmid expressing optimized ATF was transformed into E. coli which contained recombinant plasmids expressing PAL, PAD, StyA, StyB, StyC and PAR and named as E. co//-Sty-PEA (FIG. 6a).
  • pRSF-PE-l pRSFDuet-padl-fcfcl-pa/
  • pRSF-PE-ll pETDuet-styA-styB-styC-par plasmids were transformed into competent cells of E. coli Opt-ATF to construct E. coli-Sty- PEA, a recombinant strain harboring three recombinant plasmids expressing a six-enzyme cascade.
  • E. co//-Sty-PEA was inoculated in 2 mL LB medium with appropriate antibiotics (25 pg/mL streptomycin, 25 pg/mL kanamycin or 50 pg/mL ampicillin) and grown at 37 °C and 220 rpm for 6-8 h.
  • 1 mL of the culture was added to 50 mL modified M9 medium with appropriate antibiotics (25 pg/mL streptomycin, 25 pg/mL kanamycin or 50 pg/mL ampicillin) in a 250 mL flask and grown at 37 °C and 220 rpm.
  • 0.5 mM IPTG was added and the growth temperature was set at 22 °C.
  • the cells were harvested by centrifugation at 2000 x g for 10 min and used for SDS-PAGE analysis and biotransformation of L-Phe to PEA.
  • E. coli-ATF The resting cells of E. coli-ATF was resuspended in phosphate buffer containing 50 mM 2-PE and biotransformation was performed in a two-phase system (phosphate buffer and n- hexadecane in 1 :1 v/v) for 6 h at 30 °C.
  • the biotransformation of L-Phe to PEA was performed with the resting cells of E. co//-Sty-PEA in phosphate buffer containing 50, 80 or 100 mM L-Phe and n-hexadecane (1:1 v/v) for 24 h at 30 °C. Samples were analyzed at various time points.
  • FIGS. 6b The expression of all the seven recombinant proteins were confirmed by SDS-PAGE analysis (FIG. 6b).
  • the recombinant strain was used for the biotransformation of L-Phe to PEA.
  • Samples were analyzed at various time points and time-course of the biotransformation is shown in FIGS. 6c and 7.
  • 36, 49.3, and 52.8 mM PEA were produced and 10, 15.6, and 16.1 mM 2-PE were accumulated from 50, 80, and 100 mM L-Phe, respectively.
  • FIGS. 6c and 7 significant amount of 2-PE was accumulated thus reducing the conversion efficiency of L-Phe to PEA.
  • the reason for the accumulation of 2-PE in the biotransformation of L-Phe to PEA could be due to the low availability of acetyl-CoA which is the cofactor for the final step of the cascade.
  • the reactions were investigated by modifying the amount of cells used and amount of glucose added to the reaction (FIG. 8). Increasing the amount of whole cells from 10 to 15 g cdw/L increased the PEA production from 52.8 to 68.4 mM. However, further increase to 20 g cdw/L reduced the production to 64.7 mM. Since glucose could be used for acetyl-CoA biosynthesis and the initial 2% glucose added could not be sufficient, additional glucose (2%) was added to the reaction at 6 h. Upon this addition, L-Phe to PEA biotransformation with 15 g cdw/L E. co//-Sty-PEA increased PEA production from 68.4 to 79.3 mM giving rise to 79.3% conversion.
  • n-hexadecane as an organic phase can give higher titer in the biotransformation of L-Phe to PEA, the process is not sustainable as n-hexadecane is a petrochemical.
  • replacing n-hexadecane with greener ethyl oleate with a reduced amount of organic phase was attempted (FIG. 6d).
  • ATF was proven to be a good catalyst for the conversion of 2-PE to PEA, a key reaction in the six-enzyme cascade.
  • the cascade reactions were optimized by the addition of glucose to generate acetyl-CoA and the use of aqueous-organic two phase system (1 :0.5 v/v) to minimize the toxicity of intermediates and product.
  • the green solvents (ethyl oleate and biodiesel) were proven to be better than n-hexadecane for the same biotransformation, resulting in high-yielding production of 83.1 mM (13.6 g/L) of PEA from L- Phe with 83.1% conversion. Therefore, the present disclosure is a simple and high-yielding process (13.6 g/L titer; >80% yield) and do not use any toxic materials or petroleum-derived substrates, which is attractive for industries.
  • Example 5 Design of five-enzyme cascade, engineering of E. coli expressing the cascade, and one-pot biotransformation of L-Phe to PAA
  • a five-enzyme artificial cascade was designed for the conversion of L-Phe to PAA by overexpressing enzymes for the following reactions: deamination of L-Phe to trans-cinnamic acid using PAL; decarboxylation of trans-cinnamic acid to styrene using PAD; epoxidation of styrene to styrene oxide using SMO; isomerization of styrene oxide to phenylacetaldehyde using SOI; and oxidation of phenylacetaldehyde to PAA using EcALDH (FIG. 2aii). SMO and SOI of Pseudomonas sp.
  • VLB120 and ALDH of Escherichia coli enzymes were used for establishing the proposed cascades.
  • PAL of Arabidopsis thaliana
  • PAD of Aspergillus niger
  • the recombinant strain expressing PAL, PAD, SMO, SOI, and EcALDH from two plasmids was constructed as E. coli- Sty-PAA for the conversion of L-Phe to PAA (FIG. 9a).
  • the genes coding for PAL and PAD were amplified using PCR and cloned in pACYCDuet plasmid.
  • the genes coding for SMO, SOI and EcALDH were amplified using PCR and cloned in pRSFDuet plasmid.
  • the presence of recombinant genes in the engineered strains were confirmed using colony PCR and the expression of recombinant genes were confirmed using SDS-PAGE analysis.
  • the confirmed strains were grown in 2 mL LB medium with respective antibiotics at 37 °C for 12 h. Glycerol stocks were made for all recombinant strains and stored at -80 °C until further usage.
  • PAL PAL
  • PAD FDC
  • StyC styrene oxide isomerase
  • ALDH aldehyde dehydrogenase
  • pACYCDuet expressing pad1, fdc1, and pal and pRSFDuet expressing styA, styB, styC and EcALDH were transformed into E. coli T7 express and the recombinant strain was named as E. co//-Sty-PAA.
  • E. co//-Sty-PAA was inoculated in 2 mL LB medium with appropriate antibiotics (50 pg/mL kanamycin or 25 pg/mL chloramphenicol) and grown at 37 °C and 220 rpm for 8 h.
  • 1 mL of the culture was added to 50 mL modified M9 medium with appropriate antibiotics (50 pg/mL kanamycin or 25 pg/mL chloramphenicol) in a 250 mL flask and grown at 37 °C and 220 rpm.
  • 0.5 mM IPTG was added and the growth temperature was set at 22 °C.
  • the cells were harvested by centrifugation at 2000 x g for 10 min and used for SDS- PAGE analysis and biotransformation of L-Phe to PAA.
  • L-Phe to PAA biotransformation was performed in a two-phase reaction with 5 mL ethyl oleate and 10 mL phosphate buffer (200 mM, pH 8.0) containing 15 g cdw/L of E. co//-Sty-PAA cells, 100 mM L-Phe, and 2% glucose.
  • the reaction was performed at 30 °C and 220 rpm for 10 h. 200 pL of the reaction mixture was collected at various time points of the reaction and centrifgued at 10,000 x g for 10 min and the aqueous phase was analyzed using reverse phase HPLC to measure the concentration of L-Phe and PAA.
  • Example 6 Production of natural PEA and PAA from renewable feedstock glucose and glycerol by coupling L-Phe biosynthesis pathway and artificial enzyme cascades
  • L-Phe is produced naturally by E. coli via growing cell fermentation of sugars such as glucose and glycerol through shikimate pathway.
  • sugars such as glucose and glycerol
  • our artificial enzyme cascades could convert L-Phe to PEA and PAA, it is desirable to combine it with the natural L-Phe biosynthesis pathway (shikimate pathway) to produce PEA and PAA directly from glucose or glycerol (FIG. 2b).
  • the fermentative production of L-Phe from glucose or glycerol (renewable feedstocks) was thus coupled with the artificial cascades biotransformation of L-Phe to PEA and PAA, respectively (FIG. 10a).
  • E. coli NST74-Phe engineered E. coli
  • the biosynthesized L-Phe will be transformed into PEA by performing whole-cells catalyzed biotransformation using fermented media and recombinant E. coli expressing artificial enzyme cascades.
  • co//-Sty-PEA 0.378 g of phosphate powder (0.36 g of K2HPO4 and 18.2 mg KH2PO4), and 0.2 g glucose were added to 10 mL of fermentation broth (containing 67 or 78 mM L-Phe) to form 11 mL of aqueous system containing 15 g cdw/L of cells, 61 or 71 mM L-Phe, 2% glucose and 200 mM phosphate buffer. 5 mL ethyl oleate was added, and biotransformation was performed at 30 °C and 220 rpm. Additional glucose (2%) was added at 6 h. At 24 h, the organic phase was collected to quantify the concentration of PEA by GC.
  • Fermentative production of L-Phe from glucose and glycerol was performed using E. coli NST74-Phe to achieve 67-80 mM L-Phe and the fermentation culture containing biosynthesized L-Phe and E. coli NST74-Phe cells was stored at 4 °C or used directly in biotransformation.
  • E. co//-Sty-PAA was grown as described above in Example 5 to prepare the cells for biotransformation. 0.6 g of wet cells of E.
  • co//-Sty-PAA 0.378 g of phosphate powder (0.36 g of K2HPO4 and 18.2 mg KH2PO4), and 0.2 g glucose were added to 10 mL of fermentation broth (containing 67 or 78 mM L-Phe) to form 11 mL of aqueous system containing 15 g cdw/L of cells, 61 or 71 mM L-Phe, 2% glucose and 200 mM phosphate buffer. 5 mL ethyl oleate was added, and biotransformation was performed at 30 °C and 220 rpm. At 12 h, the reaction mixture was centrifuged at 10,000 x g for 10 min and the aqueous phase was collected to quantify the concentration of PAA by reverse phase HPLC.
  • the titer of PEA and PAA achieved in our processes from glucose or glycerol is 13-fold (D. Guo et al., J. Agric. Food Chem. 2018, 66, 5886-5891) and 158-fold (L. Zhang et al., Amb Express 2017, 7, 1-7) higher than those from the reported productions from glucose (no report from glycerol), respectively. Green, sustainable, and efficient bioproduction of natural PEA and PAA from renewable feedstock glucose and glycerol were thus established.
  • PEA and PAA from low-cost renewable feedstocks such as glucose and glycerol were successfully demonstrated by coupling of artificial enzyme cascade and L-Phe biosynthesis pathway.
  • 63.1 mM (10.4 g/L) of PEA and 67.8 mM (9.2 g/L) PAA were produced from glucose or glycerol, respectively.
  • the high-yielding one-pot syntheses of PEA and PAA from L-Phe, glucose, or glycerol using novel enzyme cascades, greener solvents, and renewable resources provide green, sustainable and efficient production of PEA and PAA as high-value natural aroma compounds.
  • a six-enzyme artificial cascade was designed for the conversion of L-Phe to Et-PA: deamination of L-Phe to trans-cinnamic acid using PAL; decarboxylation of trans-cinnamic acid to styrene using PAD; epoxidation of styrene to styrene oxide using SMO; isomerization of styrene oxide to phenylacetaldehyde using SOI; oxidation of phenylacetaldehyde to PAA using EcALDH; and esterification of PAA to Et-PA using lipase.
  • Two commercially available immobilized lipases such as Novozyme 435 (Candida Antarctica lipase B) (B.
  • E. coli expressing PAL, PAD, SMO, SOI, EcALDH was used for the biotransformation of L-Phe to PAA.
  • the biotransformation was performed at 30 °C, 220 rpm in a two-phase system (phosphate buffer and ethyl oleate in 1 :0.5 v/v).
  • the biosynthesized PAA was isolated from the biotransformation using silica column chromatography by following the preparation of natural PAA by biotransformation protocol in Example 5, and used for the production of Et-PA.
  • the biotransformation protocol was optimized by increasing the stirrer speed.
  • Natural PAA was prepared from glucose using the coupled fermentation-biotransformation approach, isolated and used in this esterification reaction to produce natural Et-PA (FIGS. 12ai and b). Both Novozyme 435 and TL lipozyme were able to convert PAA to Et-PA. For the esterification of 20 mM natural PAA and 40 mM ethanol, Novozyme 435 produced 16.6 mM (2.7 g/L) Et-PA with a conversion of 83.1 % while TL lipozyme gave 11.1 mM (1.8 g/L) Et-PA with the conversion of 55.5%. Accordingly, Novozyme 435 is a better catalyst for the production of Et-PA from PAA which was derived from glucose. Therefore, Novozyme 435 was chosen for the production of Et-PA. Thus, the novel bioproduction of Et-PA directly from glucose was achieved, demonstrating the first example of sugar-derived Et-PA production.
  • the lipases Novozyme 435 and TL lipozyme were also examined for the esterification of natural PAA to natural PE-PA.
  • a six-enzyme artificial cascade was designed for the conversion of L-Phe to PE-PA: deamination of L-Phe to trans-cinnamic acid using PAL; decarboxylation of trans-cinnamic acid to styrene using PAD; epoxidation of styrene to styrene oxide using SMO; isomerization of styrene oxide to phenylacetaldehyde using SOI; oxidation of phenylacetaldehyde to PAA using EcALDH; and esterification of PAA to PE-PA using lipase.
  • E. coli expressing PAL, PAD, SMO, SOI, EcALDH was used for the biotransformation of L-Phe to PAA.
  • the biotransformation was performed at 30 °C and 220 rpm in a two-phase system (phosphate buffer and ethyl oleate in 1 :0.5 v/v).
  • the biosynthesized phenylacetic acid was isolated from the biotransformation using silica column chromatography by following the preparation of natural PAA by biotransformation protocol in Example 5, and used for the production of PE-PA.
  • the biotransformation protocol was optimized by increasing the stirrer speed and reducing the water content from 2% to 1%.
  • Novozyme 435 was screened two commercially available immobilized lipases such as Novozyme 435 and TL lipozyme. Both Novozyme 435 and TL lipozyme were able to convert PAA to PE-PA in the presence of 2-PE. Novozyme 435 produced PE-PA with the conversion of 51.7% and TL lipozyme produced PE-PA with the conversion of 77.3%.
  • PAA and 2-PE (B. S. Sekar, B. R. Lukito & Z. Li, ACS Sustain. Chem. Eng. 2019, 7, 12231- 12239) were first prepared from glucose using the coupled fermentation-biotransformation approach, isolated, and then used for the esterification reaction to produce natural PE-PA (FIGS. 12aii and c). Both Novozyme 435 and TL lipozyme were able to convert PAA to PEPA. Esterification of 20 mM biosynthesized PAA with 40 mM biosynthesized 2-PE by using Novozyme 435 produced 11.3 mM (2.7 g/L) PE-PA with a conversion of 56.5%.
  • TL lipozyme is much more suitable lipase for the production of PE-PA from PAA and 2-PE which were derived from glucose. Therefore, TL lipozyme was chosen for the production of PE-PA.
  • TL lipozyme was chosen for the production of PE-PA.
  • this work discloses the design and development of an artificial enzyme cascade for the efficient production of natural PEA, Et-PA and PE-PA from biobased L-Phe via artificial enzyme cascade and from renewable feedstocks such as glucose and glycerol via the combination of natural biosynthesis pathway and artificial enzyme cascade.
  • This work is also simple, green, sustainable and uses low-cost renewable feedstocks for the efficient production of natural aroma chemicals (PEA, Et-PA and PE-PA), thus providing an economical and sustainable approach for the production of these high value natural aroma chemicals, which is in agreement with the current market and societal needs. Therefore, our work will attract great interest from industries to develop commercial production of natural PEA, Et-PA and PEPA.
  • the strain for the conversion of L-Phe to PAA was constructed by transforming the plasmid overexpressing PAL and PAD E. co//-StyABC-EcALDH.
  • Recombinant E. coli expressing PAL, PAD, StyA, StyB, StyC and EcALDH was inoculated in 2 mL LB medium containing appropriate antibiotics and grown at 37 °C, 220 rpm for 6-8 h. 1 mL of the culture was added to 50 mL of modified M9 medium with appropriate antibiotics in a 250 mL flask. After 2 h of growth at 37 °C, 0.1 mM IPTG and the growth temperature was set at 22 °C.
  • the cells were harvested by centrifugation at 4000 rpm for 10 min. To 5 mL of fermentation broth containing 70-80 mM biosynthesized L-Phe, phosphate buffer (200 mM, pH 8), 2% glucose, and 1 mL of ethyl oleate were added. Harvested cells of E. coli (15 g cdw/L) were added and biotransformation was performed at 30 °C, 220 rpm in a two-phase system (phosphate buffer and ethyl oleate in 1 :0.5 v/v).
  • the produced PAA was extracted from aqueous phase using silica gel column chromatography by following the preparation of natural PAA by biotransformation protocol in Example 5 and used in the biotransformation of PAA to Et-PA and PE-PA using Novozyme 435 and TL lipozyme, respectively.

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Abstract

Disclosed herein is a method for producing phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof using four or more enzymes, which method comprises subjecting styrene or a derivative thereof to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.

Description

Bioproduction of natural phenethyl acetate, phenylacetic acid, ethyl phenylacetate, and phenylethyl phenylacetate from renewable feedstock
Field of Invention
This invention relates to an artificial enzyme cascade for the efficient production of natural phenethyl acetate, ethyl phenyl acetate and phenylethyl phenylacetate from a feedstock (e.g. styrene, L-phenylalanine or glucose and glycerol via combination of a natural biosynthesis pathway for glucose/glycerol with an artificial enzyme cascade. This provides a simple and high-yielding process (e.g. 13.6 g/L titer; >80% yield) which may be attractive to industry. This invention is also green, sustainable and uses low-cost renewable feedstocks for the efficient production of natural aroma chemicals phenethyl acetate, ethyl phenyl acetate and phenylethyl phenylacetate.
Background
The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
Aroma chemicals such as phenethyl acetate (PEA; FEMA number: 2857; Flavis number: 9.031 ; Flavour profile: flower; honey; and rose), phenylacetic acid (PAA; FEMA number: 2878; Flavis number: 8.038; Flavour profile: honey), ethyl phenylacetate (Et-PA; FEMA number: 2452; Flavis number: 9.784; Flavour profile: floral; fruit; honey; and rose) and phenylethyl phenylacetate (PE-PA; FEMA number: 2866; Flavis number: 9.707; Flavour profile: floral) are high-value chemicals used as food and flavour ingredients. These compounds can be extracted from natural sources, but with limited availability and high price. For instance, the price of natural PEA and Et-PEA are 1 ,760-2,200 LISD/Kg and the market of PEA is -2000 tons/year. Several chemical methods were established for the synthesis of PEA, PAA, Et-PA, and PE-PA. However, these chemical syntheses start from petroleum derived chemicals, require the use of toxic reagents, and generate by-products and large amount of waste. In addition, the generated by-products affect the aroma and organoleptic profiles for flavor applications. Moreover, chemically synthesized flavor compounds are less preferred in food and cosmetics industry and have restricted use as flavoring agent in Europe and U.S. Therefore, natural PEA, PAA, Et-PA and PE-PA are highly demanded in the food and flavour industries and methods for producing these natural chemicals are highly wanted. The use of microbial fermentation and enzyme-based technologies to convert natural resources to these compounds are considered natural, giving rise to natural products. Therefore, several researches were focussed on the production of these aroma chemicals using microbes. Yeast is a natural producer of these chemicals during fermentation such as wine making process. In yeast, L-phenylalanine (L-Phe) is converted to 2-PE via the Ehrlich pathway, which will be further converted to esters using lipases or alcohol acetyl transferases (ATFs). However, the Ehrlich pathway is inefficient that leads to low product titers (100-300 mg/L) and the fermentation process takes 10-14 days (A. G. Cordente et al., Appl. Microbiol. Biotechnol. 2018, 102, 5977-5988; and B. Zhang et al., Process Biochem. 2020, 90, 44-49), thus it is not suitable for commercial level production of PEA, Et-PA and PE-PA. Engineering of Ehrlich pathway was also attempted in Escherichia coli for the production of natural PEA from glucose (D. Guo et al., MicrobiologyOpen 2017, 6, e00486; and D. Guo et al., J. Agric. Food Chem. 2018, 66, 5886-5891). However, only 687 mg/L of phenethyl acetate could be achieved (D. Guo et al., J. Agric. Food Chem. 2018, 66, 5886-5891).
Therefore, more efficient biosynthesis pathway and high yielding bioprocess needs to be developed for the production of these high-value natural aroma chemicals.
Summary of Invention
Aspects and embodiments of the invention will now be discussed by the following numbered clauses.
1. A method for producing phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof using four or more enzymes, which method comprises subjecting styrene or a derivative thereof to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.
2. The method of Clause 1 , wherein the method produces producing phenethyl acetate or a derivative thereof, comprising the steps of:
(a) generating styrene oxide or a derivative thereof from styrene or a derivative thereof by conducting an epoxidation reaction catalyzed by an epoxidase to form styrene oxide or a derivative thereof;
(b) generating phenylacetaldehyde or a derivative thereof from styrene oxide or a derivative thereof by conducting an isomerization reaction catalysed by an isomerase to form phenylacetaldehyde or a derivative thereof; (c) generating 2-phenethylethanol or a derivative thereof from phenylacetaldehyde or a derivative thereof by conducting a reduction reaction catalyzed by a reductase to form 2- phenethylethanol or a derivative thereof; and
(d) generating phenethyl acetate or a derivative thereof from 2-phenethylethanol or a derivative thereof by conducting an esterification reaction catalysed by a transferase to form phenethyl acetate or a derivative thereof.
3. The method according to Clause 2, wherein:
(a) the epoxidase is a styrene monooxygenase, optionally wherein the styrene monooxygenase is from Pseudomonas sp. VLB120, or its mutants or similar enzymes with more than 50% identity; and/or
(b) the isomerase is a styrene oxide isomerase, optionally wherein the styrene oxide isomerase is from Pseudomonas sp. VLB 120, or its mutants or similar enzymes with more than 50% identity; and/or
(c) the reductase is a phenylacetaldehyde reductase, optionally wherein the phenylacetaldehyde reductase is from Solanum lypersicum, or its mutants or similar enzymes with more than 50% identity; and/or
(d) the transferase is an alcohol acetyl transferase, optionally wherein the alcohol acetyl transferase is from Saccharomyces cerevisiae, or its mutants or similar enzymes with more than 50% identity.
4. The method according to Clause 3, wherein the method is conducted in one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes), where the recombinant microbial cell is E. coli pETDuet-StyAB-StyC-PAR, pCDFDuet-Opt-ATF.
5. The method of Clause 1 , wherein the method produces producing ethyl phenylacetate or a derivative thereof or phenylethyl phenylacetate or a derivative thereof, comprising the steps of:
(a) generating styrene oxide or a derivative thereof from styrene or a derivative thereof by conducting an epoxidation reaction catalyzed by an epoxidase to form styrene oxide or a derivative thereof;
(b) generating phenylacetaldehyde or a derivative thereof from styrene oxide or a derivative thereof by conducting an isomerization reaction catalysed by an isomerase to form phenylacetaldehyde or a derivative thereof; (c) generating phenylacetic acid or a derivative thereof from phenylacetaldehyde or a derivative thereof by conducting an oxidation reaction catalyzed by an oxidase or an aldehyde dehydrogenase to form phenylacetic acid or a derivative thereof; and
(d) generating ethyl phenylacetate or a derivative thereof from phenylacetic acid or a derivative thereof by conducting an esterification reaction using a lipase in a reaction medium comprising ethanol to form ethyl phenylacetate or a derivative thereof; or
(e) generating phenylethyl phenylacetate or a derivative thereof from phenylacetic acid or a derivative thereof by conducting an esterification reaction using a lipase in a reaction medium comprising 2-phenylethanol to form ethyl phenylacetate or a derivative thereof.
6. The method according to Clause 5, wherein:
(a) the epoxidase is a styrene monooxygenase, optionally wherein the styrene monooxygenase is from Pseudomonas sp. VLB120, or its mutants or similar enzymes with more than 50% identity; and/or
(b) the isomerase is a styrene oxide isomerase, optionally wherein the styrene oxide isomerase is from Pseudomonas sp. VLB 120, or its mutants or similar enzymes with more than 50% identity; and/or
(c) the oxidase or aldehyde dehydrogenase is aldehyde dehydrogenase (EcALDH) from Escherichia coli K12, or its mutants or similar enzymes with more than 50% identity; and/or
(d) the lipase is Novozyme 435 from Candida Antarctica or TL lipozyme from Thermomyces lanuginosus, or their mutants or similar enzymes with more than 50% identity.
7. The method according to Clause 6, wherein the method is conducted in one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes), where the recombinant microbial cell is E. coli pRSFDuet-StyABC-EcALDH in the presence of an immobilised lipase.
8. The method according to any one of the preceding clauses, wherein the method further comprises providing styrene or a derivative thereof by generating trans-cinnamic acid or a derivative thereof from L-phenylalanine or a derivative thereof by a deamination reaction catalyzed by an ammonia lyase and generating styrene or a derivative thereof from the transcinnamic acid or a derivative thereof in a decarboxylation reaction catalyzed by a decarboxylase.
9. The method according to Clause 8, wherein: (a) the ammonia lyase is phenylalanine ammonia lyase from Arabidopsis thaliana, or its mutants or similar enzymes with more than 50% identity; and/or
(b) the decarboxylase is phenylacrylic acid decarboxylase from Aspergillus niger, or its mutants or similar enzymes with more than 50% identity.
10. The method according to any one of Clauses 2 to 4, wherein the method is conducted in one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes), where the recombinant microbial cell is E. coli pRSFDuet- PAL-PAD, pETDuet-StyAB-StyC-PAR, pCDFDuet-Opt-ATF.
11 . The method according to any one of Clauses 8 to 10, wherein the method is conducted in one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes), where the recombinant microbial cell is E. coli pACYCDuet- PAL-PAD, pRSFDuet-StyABC-EcALDH in the presence of an immobilised lipase.
12. The method according to any one of Clauses 8 to 11 , wherein the method further comprises cells that overexpress the natural L-phenylalanine biosynthetic pathway, which cells convert glucose or glycerol to L-phenylalanine.
13. The method according to Clause 12 wherein the microbial cells producing L- phenylalanine from glucose or glycerol overexpress at least one enzyme is selected from one or more of the group consisting of DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), or their mutants or similar enzymes with more than 50% identity, optionally wherein the microbial cells producing L-phenylalanine from glucose or glycerol overexpress at least one enzyme is:
(a) a feedback inhibition resistant mutant of AroG;
(b) a feedback inhibition resistant mutant of PheA.
14. The method according to Clause 13, wherein the microbial cells producing L- phenylalanine from glucose or glycerol that overexpress at least one enzyme is one in which the microbial cells are mutated for deletion or inactivation of err and/or tyrA genes.
15. The method according to any one of the preceding clauses, wherein the one-pot reaction system comprises an aqueous medium. 16. The method according to any one of Clauses 1 to 15, wherein the one-pot reaction system comprises use of a bi-phasic medium, optionally wherein the bi-phasic medium consists of an aqueous and solid resin medium, or an aqueous and organic solvent medium (e.g. the organic solvent medium is selected from one or more alkanes solvents and/or one or more ester solvents).
17. The method according to any one of the preceding clauses, wherein the one-pot reaction system comprises use of absorbent resins or nanomaterials for in-situ removal of product.
18. An isolated nucleic acid molecule encoding at least one heterologous catalytic enzyme selected from the group comprising:
(a) an epoxidase for conducting an epoxidation reaction to form styrene oxide or a derivative thereof from styrene or a derivative thereof;
(b) an isomerase for generating phenylacetaldehyde or a derivative thereof from styrene oxide by an isomerization reaction; and
(c) a reductase for generating 2-phenethylethanol or a derivative thereof by a reduction reaction from phenylacetaldehyde or a derivative thereof;
(d) a transferase for generating phenethyl acetate or a derivative thereof by an esterification reaction from 2-phenethylethanol or a derivative thereof;
(e) an ammonia lyase for generating trans-cinnamic acid or a derivative thereof from L-phenylalanine or a derivative thereof by a deamination reaction;
(f) a decarboxylase for generating styrene or a derivative thereof from trans- cinnamic acid or a derivative thereof in a decarboxylation reaction;
(g) an oxidase or an aldehyde dehydrogenase for generating phenylacetic acid or a derivative thereof from phenylacetaldehyde by an oxidation reaction; and
(h) a lipase for generating ethyl phenylacetate or phenylethyl phenylacetate, or derivatives thereof, from phenylacetic acid or a derivative thereof in an esterification reaction.
19. The isolated nucleic acid of Clause 18, encoding a plurality of said catalytic enzymes.
20. The isolated nucleic acid molecule of Clause 19, wherein said plurality of catalytic enzymes is arranged as at least one module selected from the group comprising: i) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms styrene or a derivative thereof to phenylacetaldehyde or a derivative thereof; ii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms phenylacetaldehyde or a derivative thereof to 2-phenylethanol or a derivative thereof; iii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms 2-phenylethanol or a derivative thereof to phenethyl acetate or a derivative thereof; and iv) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms an L-phenylalanine or a derivative thereof to styrene or a derivative thereof, or any combination thereof.
21. The isolated nucleic acid molecule of Clause 19, wherein said plurality of catalytic enzymes is arranged as at least one module selected from the group comprising: i) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms styrene or a derivative thereof to phenylacetaldehyde or a derivative thereof; ii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms phenylacetaldehyde or a derivative thereof to 2-phenylacetic acid or a derivative thereof; iii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms 2-phenylacetic acid or a derivative thereof to phenylethyl phenylacetate or a derivative thereof or phenethyl acetate; and iv) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms an L-phenylalanine or a derivative thereof to styrene or a derivative thereof, or any combination thereof.
22. An expression construct comprising at least one nucleic acid molecule as described in any one of Clauses 19 to 21.
23. One or more recombinant prokaryotic or eukaryotic cells selected from the group comprising bacterial cells, yeast cells, mammalian cells and insect cells, wherein said cells comprise at least one expression construct as described in Clause 22.
24. The one or more recombinant prokaryotic or eukaryotic cells according to Clause 23, wherein said cells are recombinant bacterial cells. 25. A kit comprising at least one isolated nucleic acid according to any one of Clauses 19 to 21.
26. The method according to Clause 4, wherein the method further comprises acetyl CoA.
27. The method according to any one of Clauses 1 to 17 and 27, wherein the at least four enzymes used in the method are provided:
(a) in whole cells genetically engineered to overexpress the at least four enzymes, optionally wherein the at least four overexpressed enzymes are located on one or more plasmids or integrated in the chromosome of each of the one or more recombinant microbial cells;
(b) in a cell-free extract;
(c) as purified enzymes; or
(d) as immobilized enzymes.
28. A method for producing phenylacetic acid (PAA) or a derivative thereof using five or more enzymes, which method comprises subjecting L-phenylalanine or a derivative thereof to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.
29. The method of Clause 28, wherein the method produces producing phenylacetic acid or a derivative thereof, comprising the steps of:
(a) generating trans-cinnamic acid or a derivative thereof from L-phenylalanine or a derivative thereof by a deamination reaction catalyzed by an ammonia lyase;
(b) generating styrene or a derivative thereof from the trans-cinnamic acid or a derivative thereof in a decarboxylation reaction catalyzed by a decarboxylase;
(c) generating styrene oxide or a derivative thereof from styrene or a derivative thereof by conducting an epoxidation reaction catalyzed by an epoxidase to form styrene oxide or a derivative thereof;
(d) generating phenylacetaldehyde or a derivative thereof from styrene oxide or a derivative thereof by conducting an isomerization reaction catalysed by an isomerase to form phenylacetaldehyde or a derivative thereof; and
(e) generating phenylacetic acid or a derivative thereof from phenylacetaldehyde or a derivative thereof by conducting an oxidation reaction catalyzed by an oxidase or an aldehyde dehydrogenase to form phenylacetic acid or a derivative thereof.
30. The method according to Clause 29, wherein: (a) the ammonia lyase is phenylalanine ammonia lyase from Arabidopsis thaliana, or its mutants or similar enzymes with more than 50% identity; and/or
(b) the decarboxylase is phenylacrylic acid decarboxylase from Aspergillus niger, or its mutants or similar enzymes with more than 50% identity; and/or
(a) the epoxidase is a styrene monooxygenase, optionally wherein the styrene monooxygenase is from Pseudomonas sp. VLB120, or its mutants or similar enzymes with more than 50% identity; and/or
(b) the isomerase is a styrene oxide isomerase, optionally wherein the styrene oxide isomerase is from Pseudomonas sp. VLB 120, or its mutants or similar enzymes with more than 50% identity; and/or
(c) the oxidase or aldehyde dehydrogenase is aldehyde dehydrogenase (EcALDH).
31. The method according to any one of Clauses 28 to 30, wherein the method is conducted in one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes), where the recombinant microbial cell is E. coli pACYCDuet- PAL-PAD, pRSFDuet-StyABC-EcALDH.
32. The method according to any one of Clauses 28 to 31 , wherein the method further comprises cells that overexpress the natural L-phenylalanine biosynthetic pathway, which cells convert glucose or glycerol to L-phenylalanine.
33. The method according to Clause 32 wherein the microbial cells producing L- phenylalanine from glucose or glycerol overexpress at least one enzyme is selected from one or more of the group consisting of DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), or their mutants or similar enzymes with more than 50% identity, optionally wherein the microbial cells producing L-phenylalanine from glucose or glycerol overexpress at least one enzyme is:
(a) a feedback inhibition resistant mutant of AroG;
(b) a feedback inhibition resistant mutant of PheA.
34. The method according to Clause 33, wherein the microbial cells producing L- phenylalanine from glucose or glycerol that overexpress at least one enzyme is one in which the microbial cells are mutated for deletion or inactivation of err and/or tyrA genes. 35. The method according to any one of Clauses 28 to 34, wherein the one-pot reaction system comprises an aqueous medium.
36. The method according to any one of Clauses 28 to 34, wherein the one-pot reaction system comprises use of a bi-phasic medium, optionally wherein the bi-phasic medium consists of an aqueous and solid resin medium, or an aqueous and organic solvent medium (e.g. the organic solvent medium is selected from one or more alkanes solvents and/or one or more ester solvents).
37. The method according to any one of Clauses 28 to 36, wherein the one-pot reaction system comprises use of absorbent resins or nanomaterials for in-situ removal of product.
38. An isolated nucleic acid molecule encoding at least one heterologous catalytic enzyme selected from the group comprising:
(a) an epoxidase for conducting an epoxidation reaction to form styrene oxide or a derivative thereof from styrene or a derivative thereof;
(b) an isomerase for generating phenylacetaldehyde or a derivative thereof from styrene oxide by an isomerization reaction; and
(c) a reductase for generating 2-phenethylethanol or a derivative thereof by a reduction reaction from phenylacetaldehyde or a derivative thereof;
(d) an ammonia lyase for generating trans-cinnamic acid or a derivative thereof from L-phenylalanine or a derivative thereof by a deamination reaction;
(e) a decarboxylase for generating styrene or a derivative thereof from trans- cinnamic acid or a derivative thereof in a decarboxylation reaction; and
(f) an oxidase or an aldehyde dehydrogenase for generating phenylacetic acid or a derivative thereof from phenylacetaldehyde by an oxidation reaction.
39. The isolated nucleic acid of Clause 38, encoding a plurality of said catalytic enzymes.
40. The isolated nucleic acid molecule of Clause 39, wherein said plurality of catalytic enzymes is arranged as at least one module selected from the group comprising: i) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms styrene or a derivative thereof to phenylacetaldehyde or a derivative thereof; ii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms phenylacetaldehyde or a derivative thereof to 2-phenylacetic acid or a derivative thereof; iii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms an L-phenylalanine or a derivative thereof to styrene or a derivative thereof, or any combination thereof.
41. An expression construct comprising at least one nucleic acid molecule as described in any one of Clauses 38 to 40.
42. One or more recombinant prokaryotic or eukaryotic cells selected from the group comprising bacterial cells, yeast cells, mammalian cells and insect cells, wherein said cells comprise at least one expression construct as described in Clause 41.
43. The one or more recombinant prokaryotic or eukaryotic cells according to Clause 42, wherein said cells are recombinant bacterial cells.
44. A kit comprising at least one isolated nucleic acid according to any one of Clauses 38 to 40.
45. The method according to any one of Clauses 28 to 37, wherein the at least five enzymes used in the method are provided:
(a) in whole cells genetically engineered to overexpress the at least five enzymes, optionally wherein the at least five overexpressed enzymes are located on one or more plasmids or integrated in the chromosome of each of the one or more recombinant microbial cells;
(b) in a cell-free extract;
(c) as purified enzymes; or
(d) as immobilized enzymes.
Drawings
FIG. 1 depicts the schematic representation of the sustainable bioproduction of natural PEA, PAA, Et-PA and PE-PA from renewable feedstock L-phenylalanine (L-Phe), glucose, and glycerol.
FIG. 2 depicts the synthesis of natural PEA and PAA from bio-based L-Phe and renewable feedstock glucose and glycerol, a) i) The conversion of L-Phe to PEA via one-pot cascade deamination-decarboxylation-epoxidation-isomerization-reduction-transferase reaction. Enzymes involved are phenylalanine ammonia lyase (PAL), phenylacrylic acid decarboxylase (PAD; consisting of PAD and FDC), styrene monooxygenase (SMO, consisting of StyA and StyB), styrene oxide isomerase (SOI), phenylacetaldehyde reductase (PAR), and alcohol acetyl transferase (ATF). ii) The conversion of L-Phe to PAA via one-pot cascade deamination- decarboxylation-epoxidation-isomerization-oxidation reaction. The enzymes involved are PAL, PAD, SMO, SOI, and aldehyde dehydrogenase (EcALDH). b) i) The conversion of glucose or glycerol to PEA via the combination of natural shikimate pathway for the synthesis of L-Phe and artificial enzyme cascade for the conversion of L-Phe to PEA. ii) The conversion of glucose or glycerol to PAA via the combination of natural shikimate pathway for the synthesis of L-Phe and artificial enzyme cascade for the conversion of L-Phe to PAA.
FIG. 3 depicts a) schematic representation of the plasmid expressing ATF and codon- optimized ATF (Opt-ATF). b) Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the soluble and particulate fractions of E. co//-pCDFDuet (Lane 1), E. co//-pCDFDuet-ATF (Lane 2) and E. co//-pCDFDuet-Opt-ATF (Lane 3). The expression of ATF is marked with an arrow, c) Time course of biotransformation of 45 mM 2-phenylethanol (2- PE) to PEA with 10 g cdw/L cells of E. coli Opt-ATF. Reaction conditions: two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose) and n-hexadecane (1 :1 v/v); 30 °C; 220 rpm; and 4 h. Concentrations are normalized to aqueous phase volume. Experiments were performed in duplicates with error bars showing ± s.d.
FIG. 4 depicts the high-performance liquid chromatography (HPLC) chromatograms showing the consumption of 2-PE during the biotransformation of 2-PE to PEA by 10 g cdw/L of a) E. coli T7 express (negative control) and b) E. co//-Opt-ATF. The samples at 0 and 2 h were analysed. Reaction conditions: two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose, 45 mM 2-PE) and n-hexadecane (1 :1 v/v); 30 °C; 220 rpm; and 2 h.
FIG. 5 depicts gas chromatography-mass spectrometry (GC-MS) analysis, a) GC chromatogram showing the production of PEA in biotransformation of 2-PE to PEA with 10 g cdw/L of E. coli Opt-ATF. b) MS profile of the 2-PEA peak eluting at 6.886 min. The sample at 2 h was measured. Reaction conditions: two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose, 45 mM 2-PE) and n-hexadecane (1 :1 v/v); 30 °C; 220 rpm; and 2 h.
FIG. 6 depicts a) E. co//-Sty-PEA harboring three plasmids for the expression of PAL, PAD, SMO, SOI, PAR and ATF. b) SDS-PAGE analysis of soluble fractions of E. co//-Sty-PEA. Lane M, Protein ladder; Lane 1 , E. co//-pCDFDuet (negative control); Lane 2, E. co//-Sty-2-PE (negative control); and Lane 3, E. co//-Sty-PEA. c) Concentration of the accumulated 2-PE and produced PEA at 24 h in the biotransformation of 50, 80 and 100 mM L-Phe, respectively, with 10 g cdw/L cells of E. co//-Sty-PEA. Reaction conditions: two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose) and n-hexadecane (1 :1 v/v); 30 °C; and 24 h. d) Optimization of reaction conditions by modifying the organic phase in the biotransformation of 100 mM L-Phe to PEA with 15 g cdw/L cells of E. co//-Sty-PEA. Reaction conditions: two- phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose; Aq) and n-hexadecane (Hx) or ethyl oleate (EO) in 1 :1 or 1 :0.5 v/v; 30 °C; and 24 h. e, f) Time courses of the cascade conversion of 100 mM L-Phe to PEA with optimized reaction conditions using 15 g cdw/L E. co//-Sty-PEA with (e) ethyl oleate and (f) biodiesel as organic phases. Reaction conditions: two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose) and ethyl oleate/biodiesel (1 :0.5, v/v); 30 °C; 24 h; and additional 2% glucose was added at 6 h. Concentrations are normalized to aqueous phase volume. Experiments were performed in triplicates with error bars showing ± s.d.
FIG. 7 depicts the time courses of the accumulated 2-PE and produced PEA at 24 h in the biotransformation of a) 50 mM, b) 80 mM and c) 100 mM L-Phe to PEA, respectively, with 10 g cdw/L cells of E. co//-Sty-ATF. Reaction conditions: two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose) and n-hexadecane (1 :1 v/v); 30 °C; and 24 h.
FIG. 8 depicts the optimization of reaction conditions for the cascade conversion of L-Phe to PEA. Concentration of the synthesized PEA in the biotransformation with varying a) amount of whole cells and b) glucose addition. Reaction conditions: two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose) and n-hexadecane (1 :1 v/v); 30 °C; and 24 h.
FIG. 9 depicts a) E. co//-Sty-PAA harboring two plasmids for the expression of PAL, PAD, SMO, SOI and EcALDH. b) SDS-PAGE analysis of soluble fractions of E. co//-Sty-PAA. Lane M, Protein ladder; Lane 1 , E. co//-pCDFDuet (negative control); and Lane 2, E. co//-Sty-PAA. c) Time course of the cascade conversion of 100 mM of L-Phe to PAA with 15 g cdw/L cells of E. co//-Sty-PAA. Reaction conditions: two-phase system of phosphate buffer (200 mM, pH 8.0, 2% glucose) and ethyl oleate (1 :0.5 v/v); 30 °C; and 10 h. Concentrations are normalized to aqueous phase volume. Experiments were performed in triplicates with error bars showing ± s.d.
FIG. 10 depicts a) coupled fermentation and biotransformation approach for the production of PEA and PAA from renewable feedstock glucose and glycerol, respectively. Fermentation of glucose and glycerol to L-Phe was performed with E. coli NST74-Phe, followed by biotransformation of 67-78 mM biosynthesized L-Phe to PEA and PAA with 15 g cdw/L E. coli- Sty-PEA and E. co//-Sty-PAA, respectively, in a two-phase system with fermented media containing phosphate buffer and ethyl oleate (1 :0.5, v/v). b) Concentration of PEA produced from glucose and glycerol using coupled fermentation and biotransformation approach and conversion of biosynthesized L-Phe to PEA. c) Concentration of PEA produced from glucose and glycerol using coupled fermentation and biotransformation approach and conversion of biosynthesized L-Phe to PAA. Concentrations are normalized to aqueous phase volume. Experiments were performed in triplicates with error bars showing ± s.d.
FIG. 11 depicts the screening of lipases for the conversion of PAA to Et-PA and PE-PA, respectively.
FIG. 12 depicts the a) production of natural Et-PA and PE-PA from bio-based L-Phe and renewable feedstock glucose, i) Bioproduction of PAA from L-Phe or glucose, followed by lipase-catalyzed esterification of PAA to Et-PA. ii) Bioproduction of PAA and 2-PE from L-Phe or glucose, followed by lipase-catalyzed esterification of PAA and 2-PE to PE-PA. b) Concentration and conversion of Et-PA from 36 h biotransformation of 20 mM of PAA produced from glucose and 40 mM ethanol using lipases TL-Lipozyme and Novozyme 435, respectively, c) Concentration and conversion of PE-PA from 36 h biotransformation of 20 mM PAA produced from glucose and 40 mM 2-PE produced from glucose using lipases TL- Lipozyme and Novozyme 435, respectively. Reaction conditions for the esterification: 0.25 g immobilized enzymes; 5 mL hexane; 30 °C; and 800 rpm. Experiments were performed in triplicates with error bars showing ± s.d.
Description
In a first aspect of the invention, there is provided a method for producing phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof using one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes, which method comprises subjecting styrene or a derivative thereof to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.
In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.
The phrase, “consists essentially of” and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.
In embodiments of the invention, the styrene itself may be produced from L-phenylalanine by a further enzymatic cascade, with the L-phenylalanine being obtained either from the cell’s natural metabolic processes or an enhanced enzymatic pathway that provides an up-regulated production of L-phenylalanine. These multiple reaction steps may be performed simultaneously or sequentially in one reaction vessel, to allow for the green, efficient, and economical production of the desired products directly from styrene or a derivative thereof, phenylalanine or a derivative thereof, glucose or glycerol. Such, one-pot cascade reactions may avoid the expensive and energy-consuming isolation and purification of intermediates, minimize waste generation, and overcome the possible thermodynamic hurdles normally encountered in traditional multi-step synthesis. For example, multiple enzymes may be coexpressed inside one recombinant microbe strain, and the whole cells of the strain may be directly applied as catalysts for a series of cascade reactions in one pot. Alternatively, the enzymes could be separately expressed in cells of different strains, purified individually, or immobilized (the purified enzymes or cells containing all or some of said enzymes). In any event, the biocatalysts (enzymes, cells, immobilized enzymes, and immobilized cells) can be mixed together in any suitable combination to effect the one pot transformation discussed herein.
As discussed hereinbefore, there is provided a method for producing an phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof, which method comprises subjecting styrene or a derivative thereof (or other suitable starting materials mentioned herein) to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.
Styrene and derivatives thereof can be manufactured on very large scale in the petrochemical industry (e.g. by hydrocarbon cracking), and so form easily available and cheap starting materials for organic synthesis of the type discussed herein. For example, many aromatic and aliphatic alkenes are produced in very large amounts and at very low price. As discussed hereinbelow, styrenes and substituted styrenes are very useful substrates to produce various desirable products.
While the use of suitable styrene and its derivatives generated from the petrochemical industry is envisaged, the current invention also allows for the conversion of L-phenylalanine and derivatives thereof into styrene and derivatives thereof by way of further enzyme-catalyzed transformations which will be discussed hereinbelow. This may enable access to styrene derivatives that may otherwise be difficult to obtain access to and provide a greater pool of possible styrene substrates for use in the enzyme-catalyzed transformations described herein.
When used herein, the term “derivative thereof” as applied to styrene and L-phenylalanine relates to a compound where the benzene ring contains one or more substituents (e.g. 1 , 2, 3, 4or 5, such as 1 to 3, such as 1 or 2 substituents) that are not H. Said substituents may be halo, alkyl, cycloalkyl, aryl, heterocyclic, OH, NH2, SH and combinations thereof (e.g. alkyl aryl, Oalkyl, N(alkyl)H, N(alkyl)2, N(alkyl)(aryl) etc).
Unless otherwise stated, the term "alkyl" refers to an unbranched or branched, saturated or unsaturated hydrocarbyl radical (so forming, for example, an alkenyl or alkynyl), which may be substituted or unsubstituted (with, for example, one or more halogen atoms). The alkyl group may be Ci-w alkyl and, more preferably, Ci-e alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl). The terms “alkenyl” and “alkynyl” are to be interpreted accordingly.
Unless otherwise stated, the term "cycloalkyl" refers to an unbranched or branched, saturated or unsaturated hydrocarbyl radical (so forming, for example, a cycloalkenyl group) that may be substituted or unsubstituted. The cycloalkyl group may be C3-12 cycloalkyl and, more preferably, C5-10 (e.g. C5-7) cycloalkyl. The term “cycloalkenyl” is to be interpreted accordingly.
The term "halogen", when used herein, includes fluorine, chlorine, bromine and iodine.
The term "aryl" when used herein includes Ce-i4 (such as Ce-i3 (e.g. Ce- )) aryl groups that may be substituted or unsubstituted. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 14 ring carbon atoms, in which at least one ring is aromatic. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an aromatic ring. Ce-14 aryl groups include phenyl, naphthyl and the like, such as 1 ,2,3,4-tetrahydronaphthyl, indanyl, indenyl and fluorenyl. Most preferred aryl groups include phenyl.
When used herein, the term “aryl alkyl” is to be interpreted in line with the definitions provided hereinbefore for “alkyl” and “aryl”, where the point of attachment of the group to the rest of the compound of formula (I) or (II) is through the alkyl portion of the aryl alkyl group.
When used herein, the term “heterocyclic” refers to a fully saturated, partly unsaturated, wholly aromatic or partly aromatic ring system in which one or more (e.g. one to four) of the atoms in the ring system is other than carbon (i.e. a heteroatom, which heteroatom is preferably selected from N, O and S), and in which the total number of atoms in the ring system is between three and twelve (e.g. between five and ten). The heterocyclic groups may be substituted or unsubstituted. Heterocyclic groups that may be mentioned include 7- azabicyclo[2.2.1]heptanyl, 6-azabicyclo[3.1.1]heptanyl, 6-azabicyclo[3.2.1]octanyl, 8- azabicyclo[3.2.1]octanyl, aziridinyl, azetidinyl, dihydropyranyl, dihydropyridyl, dihydropyrrolyl (including 2,5-dihydropyrrolyl), dioxolanyl (including 1 ,3-dioxolanyl), dioxanyl (including 1 ,3- dioxanyl and 1 ,4-dioxanyl), dithianyl (including 1 ,4-dithianyl), dithiolanyl (including 1 ,3- dithiolanyl), imidazolidinyl, imidazolinyl, morpholinyl, 7-oxabicyclo[2.2.1]heptanyl, 6- oxabicyclo[3.2.1]octanyl, oxetanyl, oxiranyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrrolidinonyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, sulfolanyl, 3-sulfolenyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydropyridyl (such as 1 ,2,3,4-tetrahydropyridyl and 1 , 2,3,6- tetrahydropyridyl), thietanyl, thirranyl, thiolanyl, thiomorpholinyl, trithianyl (including 1 ,3,5- trithianyi), tropanyl, benzothiadiazolyl (including 2,1 ,3-benzothiadiazolyl), isothiochromanyl and, more preferably, acridinyl, benzimidazolyl, benzodioxanyl, benzodioxepinyl, benzodioxolyl (including 1 ,3-benzodioxolyl), benzofuranyl, benzofurazanyl, benzothiazolyl, benzoxadiazolyl (including 2,1 ,3-benzoxadiazolyl), benzoxazinyl (including 3,4-dihydro- 2H- 1 ,4-benzoxazinyl), benzoxazolyl, benzomorpholinyl, benzoselenadiazolyl (including 2,1 ,3- benzoselenadiazolyl), benzothienyl, carbazolyl, chromanyl, cinnolinyl, furanyl, imidazolyl, imidazo[1 ,2-a]pyridyl, indazolyl, indolinyl, indolyl, isobenzofuranyl, isochromanyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiaziolyl, isoxazolyl, naphthyridinyl (including 1 ,6-naphthyridinyl or, preferably, 1 ,5-naphthyridinyl and 1 ,8-naphthyridinyl), oxadiazolyl (including 1 ,2,3-oxadiazolyl,
1.2.4-oxadiazolyl and 1 ,3,4-oxadiazolyl), oxazolyl, phenazinyl, phenothiazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinolizinyl, quinoxalinyl, tetrahydroisoquinolinyl (including 1 , 2,3,4- tetrahydroisoquinolinyl and 5,6,7,8-tetrahydroisoquinolinyl), tetrahydroquinolinyl (including
1.2.3.4-tetrahydroquinolinyl and 5,6,7,8-tetrahydroquinolinyl), tetrazolyl, thiadiazolyl (including 1 ,2,3-thiadiazolyl, 1 ,2,4-thiadiazolyl and 1 ,3,4-thiadiazolyl), thiazolyl, thiochromanyl, thiophenetyl, thienyl, triazolyl (including 1 ,2,3-triazolyl, 1 ,2,4-triazolyl and 1 ,3,4-triazolyl) and the like. Substituents on heterocyclic groups may, where appropriate, be located on any atom in the ring system including a heteroatom. The point of attachment of heterocyclic groups may be via any atom in the ring system including (where appropriate) a heteroatom (such as a nitrogen atom), or an atom on any fused carbocyclic ring that may be present as part of the ring system. Heterocyclic groups may also be in the N- or S- oxidised form. Heterocyclic groups that may be mentioned herein include cyclic amino groups such as pyrrolidinyl, piperidyl, piperazinyl, morpholinyl or a cyclic ether such as tetrahydrofuranyl.
When used herein, the term “heterocyclic alkyl” is to be interpreted in line with the definitions provided hereinbefore for “alkyl” and “heterocyclic”, where the point of attachment of the group to the rest of the compound of formula (I) or (II) is through the alkyl portion of the heterocyclic alkyl group.
The substituents mentioned herein may be substituted or unsubstituted. When the substituents are substituted, they may be substituted with one or more of the groups selected from the group of halogen (e.g., a single halogen atom or multiple halogen atoms forming, in the latter case, groups such as CF3 or an alkyl group bearing Cl3), cyano, nitro, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, aryl, ORa, SRa, S(=O)Re, S(=O)2Re, P(=O)2Re, S(=O)2ORe, P(=O)2ORe, NRbRc, NRbS(=O)2Re, NRbP(=O)2Re, S(=O)2NRbRc, P(=O)2NRbRc, C(=O)ORe, C(=O)Ra, C(=O)NRbRc, OC(=O)Ra, OC(=O)NRbRc, NRbC(=O)ORe, NRdC(=O) NRbRc, NRdS(=O)2NRbRc, NRdP(=O)2NRbRc, NRbC(=O)Ra, or NRbP(=O)2Re, wherein Ra is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl; Rb, Rcand Rd are independently hydrogen, alkyl, cycloalkyl, heterocycle, aryl, or said Rb and Rc together with the N to which they are bonded optionally form a heterocycle; and Re is alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl. It will be appreciated that these substituted groups may be unsubstituted or are themselves substituted with one or more halogen atoms.
For the avoidance of doubt, in cases in which the identity of two or more may be the same, the actual identities of the respective substituents are not in any way interdependent unless otherwise specified.
Specific styrene and L-phenylalanine derivatives that may be mentioned herein include those in which the phenyl ring of styrene and/or phenylalanine is mono- or disubstituted by (a) substituent(s) selected from F, Cl, Br, CH3 or OCH3. For example, the phenyl ring of styrene and/or phenylalanine may be monosubstituted by o-F, m-F, p-F, m-CI, p-CI, m-Br, p-Br, m- CH3, p-CH3, or p-OCHs. In particular embodiments of the invention that may also be mentioned herein, the styrene derivative may be indene which is either unsubstituted or substituted as described for styrene above.
The terms "amino acid" or "amino acid sequence," as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
The term "isolated" is herein defined as a biological component (such as a nucleic acid, peptide or protein) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins which have been isolated thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
The phrases "nucleic acid" or "nucleic acid sequence," as used herein, refer to an oligonucleotide, nucleotide, polynucleotide, or any fragment thereof, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material. In the context of the invention, "fragments" refers to those nucleic acid sequences which are greater than about 60 nucleotides in length, and most preferably are at least about 100 nucleotides, at least about 1000 nucleotides, or at least about 10,000 nucleotides in length which are not full-length native sequence but retain catalytic enzyme activity.
The term "oligonucleotide," as used herein, refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray. As used herein, the term "oligonucleotide" is substantially equivalent to the terms "amplimers," "primers," "oligomers," and "probes," as these terms are commonly defined in the art. The terms ‘variant’ and ‘mutant’ are used interchangeably herein. The at least one nucleic acids encoding at least one catalytic enzyme may encode a variant or mutant of the exemplified catalytic enzyme which retains activity. A "variant" of a catalytic enzyme, as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing catalytic activity may be found using computer programs well known in the art, for example, DNASTAR software. In some embodiments, variant enzymes are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, homologous or identical at the amino acid level to an exemplary amino acid sequence described herein (e.g., catalase, alcohol dehydrogenase, a-transaminase) or a functional fragment thereof — e.g., over a length of about: 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the length of the mature reference sequence.
The terms ‘phenylacetaldehyde reductase’ (PAR) and ‘alcohol dehydrogenase’ (ADH), as referred to herein, are used interchangeably.
A vector can include one or more catalytic enzyme nucleic acid(s) in a form suitable for expression of the nucleic acid(s) in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence(s) to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences such as the T7 IPTG-inducible promoters disclosed in the Examples herein. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., catalytic enzyme proteins, fusion proteins, and the like).
The recombinant expression vectors of the invention can be designed for expression of catalytic enzyme proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in bacteria (e.g., E. coli), insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector(s) can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. Gene 1988, 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
To maximize recombinant protein expression in E. coli is to express the protein in a host bacterium with an impaired capacity to proteolytical ly cleave the recombinant protein (Gottesman, S. Gene Expression Technology: Methods in Enzymology 1990 185, Academic Press, San Diego, Calif. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., Nucleic Acids Res. 199220: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques and is described in the Examples.
The catalytic enzyme expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector, a vector for expression in bacterial cells, e.g. a plasmid vector, or a vector suitable for expression in mammalian cells. When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
In a preferred embodiment, the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g., by means of a signal transduction pathway), by a chemical (e.g., Isopropyl p-D-1 -thiogalactopyranoside (IPTG)) or by a heterologous polypeptide.
The methods described herein make use of enzymes to catalyse a sequence of reactions. While these reactions may be performed individually or, more particularly, two or more of them in combination, all of the reactions may be combined into a cascade reaction sequence that provides the product from the initial starting material in one pot, thereby eliminating the need for isolation of the intermediates and, potentially, increasing the overall yield of the reaction sequence. These cascade reactions may involve the use of one or more reactive components selected from the group consisting of cells, immobilized cells, cell extracts, isolated enzymes and immobilized enzymes in said reaction vessel.
Another aspect the invention provides at least one expression construct comprising at least one nucleic acid sequence that is heterologous according to any aspect of the invention. Preferably the at least one construct comprises a plasmid suitable for expression of at least one catalytic enzyme in a bacterium.
Another aspect the invention provides a host cell which includes at least one nucleic acid molecule described herein, e.g., at least one catalytic enzyme nucleic acid molecule within a recombinant expression vector or a catalytic enzyme nucleic acid molecule containing sequences which allow homologous recombination into a specific site of the host cell's genome. The terms "host cell" and "recombinant host cell" are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, at least one catalytic enzyme protein can be expressed in bacterial cells (such as E. coli), insect cells (such as Spodoptera frugiperda Sf9 cells), yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman Cell 1981 , I23: 175-182)). Other suitable host cells are known to those skilled in the art.
One or more vector DNAs can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. For example, according to the invention a host cell may comprise one, two, three, four or more plasmids, each of which may express at least one catalytic enzyme directed to a chemical transformation in the pathway from an alkene starting material to the desirable products mentioned herein. The host cell may also include a vector to express catalytic enzymes for providing the alkene by, for example, generating a vinyl carboxylic acid from an a-amino acid by a deamination reaction catalyzed by an ammonia lyase and generating the alkene from the vinyl carboxylic acid in a decarboxylation reaction catalyzed by a decarboxylase.
In a preferred embodiment, the catalytic enzymes required to transform an alkene starting material to phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof are arranged on expression vectors as modules, wherein each module comprises a combination of catalytic enzymes to perform specific reactions within the overall system. For example, a first module may comprise enzymes for the epoxidation and isomerization of a terminal alkene to an aldehyde co-expressed on a plasmid; a second module may comprise enzymes for the reduction-esterification of an aldehyde to phenethyl acetate co-expressed on a plasmid; a third module may comprise enzymes for the deamination-decarboxylation of an a-amino acid to an alkene. Arrangements of such enzymes as modules allow flexibility in constructing a serial cascade of reactions in one pot. One or more modules may be engineered onto the same plasmid. For example, a host cell comprising the said first and third modules, on the same or separate plasmids, is capable of catalyzing the conversion of a terminal alkene to phenethyl acetate. Likewise a host cell comprising said first, second and third module is capable of catalyzing the conversion of a terminal alkene to phenethyl acetate, or a derivative thereof. If module 4 is co-expressed in the host cell, an a-amino acid feed stock can be provided for the cell to generate its own terminal alkene for the cascade reactions.
In one aspect of the invention there is provided one or more recombinant prokaryotic or eukaryotic cells selected from the group comprising bacterial cells, yeast cells, mammalian cells and insect cells, wherein said cells comprise at least one expression construct and/or heterologous nucleic acid molecule that encodes at least one catalytic enzyme required in the pathway from alkene to phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof.
The cells may contain a single expression vector or construct, such as a plasmid, which expresses a single catalytic enzyme or co-expresses a plurality of catalytic enzymes as described herein under the control of at least one regulatory element. The catalytic enzymes may be arranged in the plasmid as an individual artificial operon under the control of a promoter with one ribosome-binding site before every gene, or arranged with individual promoters. Accordingly, a one-pot synthesis cascade may be achieved with cells expressing all required catalytic enzymes on one or several plasmids, or with different recombinant cells which each express a specific repertoire of catalytic enzymes, providing the necessary cells are included for a particular chemical transformation.
In another preferred embodiment, said catalytic enzymes for use in the invention have at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, homology or amino acid identity with at least one enzyme mentioned herein.
A host cell of the invention can be used to produce (i.e. , express) one or more catalytic enzyme proteins. Accordingly, the invention further provides methods for producing one or more catalytic enzyme proteins, e.g., one or more catalytic enzyme proteins described herein, using the host cells of the invention. In one embodiment, the method includes culturing the host cell of the invention (into which one or more recombinant expression vector(s) encoding one or more catalytic enzyme proteins has/have been introduced) in a suitable medium such that one or more catalytic enzyme proteins is/are produced. In another embodiment, the method further includes isolating one or more catalytic enzyme proteins from the medium or the host cell.
According to another aspect of the invention there is provided a kit comprising at least one recombinant cell, expression construct or isolated nucleic acid according to any aspect of the invention. Preferably, the kit can be used to provide one or more components for a one-pot system to enzyme-catalyze the production of phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof from a suitable starting material.
Further aspects of the invention may relate to a method for producing phenylacetic acid (PAA) or a derivative thereof using five or more enzymes, which method comprises subjecting L- phenylalanine or a derivative thereof to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system. This may be achieved using broadly the same technology as discussed hereinbefore and/or throughout the specification.
In aspects and embodiments of the invention where a lipase is required to access the final desired products. The lipase may form part of the enzymatic cascade within one or more recombinant cells, or it may be provided as an immobilised enzyme. For example, the immobilised lipase may be provided on a support - such as described in Facin, etal., Ind. Eng. Chem. Res. 2019, 58, 14, 5358-5378.
Advantages associated with aspects an embodiments of the invention include the following.
Production of phenethyl acetate using one of styrene, L-phenylalanine, glucose or glycerol as the feedstock via an enzyme cascade, providing “natural” phenethyl acetate can be produced with high yield and titer, no (or substantially reduced) by-products by accumulation.
Production of ethyl phenylacetate using one of styrene, L-phenylalanine, glucose or glycerol as the feedstock via an enzyme cascade, providing “natural” phenethyl acetate can be produced with high yield and titer, no (or substantially reduced) by-products by accumulation.
Production of phenylethyl phenylacetate using one of styrene, L-phenylalanine, glucose or glycerol as the feedstock via an enzyme cascade, providing “natural” phenethyl acetate can be produced with high yield and titer, no (or substantially reduced) by-products by accumulation.
It is noted that chemical-based methods are low-yielding, and suffer from by-product accumulation. These by-products can affect the aroma and organoleptic profile of the product. The currently disclosed enzyme cascade avoids the accumulation of by-products, thereby overcoming this issue. In addition, aroma chemicals produced from chemical-based methods are not preferred in the food industry. The disclosed enzyme cascade produces ‘natural’ products with a higher market value and will be more readily accepted for use in the food industry. Chemical-based methods are also multi-step in nature and involve purification of intermediates at each step, which is an energy-intensive process. The current invention is performed in one-pot, making it much more energy- and material-efficient and easy to scale up.
The currently disclosed method to make the above-mentioned materials use chemicals and acidic or basic catalysis at high temperatures. This is avoided by the current invention, which uses mild reaction conditions only. In addition, current synthetic methods use petroleum- dervied chemicals which are unsustainable. The current invention uses renewable feedstocks such as styrene (which can be derived from L-phenylalanine), L-phenylalanine, glucose or glycerol and “greener” solvents, thereby minimising environmental impact.
Finally, current bioproduction methods are low-yielding. The current invention provides a method that is efficient and can provide a high product titer (e.g. 13.6 g/L) and yield (e.g. >80% conversion).
Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.
Examples
Materials
L-Phe (99%), 2-PE (99%), PEA (>98%), PAA (99%), Et-PA (>98%), PE-PA (>98%), D-glucose (99%), Glycerol (>98%), Kanamycin sulfate (95%), ampicillin sodium salt (96%), streptomycin sulfate (95%), ammonium persulfate (>98%), sodium citrate (>98%), thiamine HCI (98%) ferrous sulfate (99%), ammonium hydroxide (28% NH3 in H2O), NaCI (99.5%), betaine (99%), n-hexadecane (99%), ethyl oleate (natural, >85%) KH2PO4 (99%), trifluoro acetic acid (99%), sulphuric acid (>99%), acetophenone (99%), Na2HPO4.2H2O (99%), NH4CI (99%), NaOH (99%), Na2SO4 (99%), benzyl alcohol (99%), MgSO4 (99%), MnSO4 (99%), CaCI2 (99%), FeCI3 (99%), COCI2.6H2O (99%), CuCI2 (99%), ZnCI2.4H2O (98%), NaMoO4 (99%), H3BO4 (99%), trifluoroacetic acid (TFA), hexane, chloramphenicol and HCI (99%) were purchased from Sigma-Aldrich. Isopropyl p-D-1 -thiogalactopyranoside (IPTG, 99%) was purchased from 1st base Singapore. Biodiesel (B100) was purchased from Alpha Biofuels Singapore. Ethyl acetate (EA, HPLC grade) and acetonitrile (HPLC grade) were obtained from Tedia. Luria broth (LB, Miller) powder and Bacto™ yeast extract were purchased from Becton Dickinson Germany. 5x SDS sample loading dye was purchased from GenScript (USA). Plasmid isolation kit and gel extraction kit were purchased from Qiagen USA. All DNA-modifying enzymes including DNA polymerases and restriction digestion enzymes required in gene cloning were purchased from Thermo Fisher Scientific USA.
Preparation of media used for cultivation in shaking flask or bioreactor
Seed medium for the production of L-Phe in shaking flask’, glucose/glycerol 20 g/L, (NH4)2SO4 10 g/L, KH2PO4 1.5 g/L, MgSO4 5 g/L, yeast extract 4 g/L, FeSO4 15 mg/L, sodium citrate 0.5 g/L and thiamine HCI 0.1 g/L. The pH was adjusted to 7.0 with NH4OH. Fermentation medium for the production of L-Phe in bioreactor, glucose/glycerol 10 g/L, (NH4)2SO4 10 g/L, KH2PO4 5 g/L, MgSC 5 g/L, yeast extract 5 g/L, FeSC 15 mg/L, MnSC 15 mg/L, and betaine 1 g/L. The pH was maintained at ~6.8 with NH4OH.
Modified M9 medium: glucose 20 g/L, yeast extract 6 g/L, Na2HPO4 6 g/L, KH2PO4 3 g/L, NH4CI 1 g/L, NaCI 0.5 g/L, MgSC 1 mM, CaCh 0.1 mM, and trace metal solution 1 mL/L. The composition of trace metal solution (per litre): 0.1 M HCI solution, 8.3 g FeCL, 0.84 g ZnCh, 0.13 g CUCI2’2H2O, 0.1 g CoCI2’2H2O, 0.1 g H3BO3, 0.1 g Na2MoO4 and 0.016 g MnCI2.
Analytical techniques
Cell growth measurement
Cell growth was monitored by measuring the optical density (OD6oo) at 600 nm using NanoDrop™ (Thermo Fisher Scientific Inc., Massachusetts, USA).
HPLC analysis of the concentration of L-Phe, 2-PE, and PAA in the aqueous phase
L-Phe, 2-PE and PAA in the aqueous phase were measured by HPLC (LC-20AD, Prominence, Shimadzu Corporation). Detector, Photodiode array (DAD); Column, Agilent Poroshell 120 SB-C18 (150 x 4.6 mm, 2.7 pm); Eluent, 30% acetonitrile and 70% ultrapure water containing 0.1 % TFA; Flow rate, 0.5 mL/min; Column temperature, 30 °C. Acetophenone was used as an internal standard. Retention time: L-Phe, 3.5 min; 2-PE, 9.6 min; PAA, 9.2 min; and acetophenone, 15.9 min.
Gas chromatography (GC) analysis of the concentration of PEA, Et-PA and PE-PA in the organic phase
PEA, Et-PA and PE-PA in organic phases were analyzed by GC (Agilent 7890A). Detector, FID; Column, Agilent HP-5 column (30 m x 0.32 mm x 0.25 mm); Temperature programme: 70 °C, hold for 1 min; increase 25 °C/min to 200 °C, hold for 1 min; increase 50 °C/min to 280 °C, hold for 1 min. Benzyl alcohol was used as an internal standard. Retention time: PEA, 8.15 min; 8.06 Et-PA, min; 11.09 PE-PA, min; and benzyl alcohol, 5.9 min.
GC-MS
The organic phase containing PEA, Et-PA and PE-PA were subjected to GC/MS analysis using a 7890B GC system with an Agilent 5977A MSD and an HP-5MS column (30 m x 0.32 mm x 0.25 mm); Temperature programme: 70 °C, hold for 1 min; increase 25 °C/min to 200 °C, hold for 1 min; and increase 50 °C/min to 280 °C, hold for 1 min. HPLC analysis of the concentration of glucose, glycerol, and acetate
Glucose, glycerol, and acetate levels during fed-batch fermentation was monitored by HPLC (LC-20AD, Prominence, Shimadzu Corporation). Detector, refractive index detector; Column, Aminex-HPX87H column (Biorad, USA); Eluent, 5 mM sulfuric acid in ultrapure water; Flow rate, 0.5 mL/min; Column temperature, 30 °C. Retention time: glucose, 10.1 min; glycerol, 15.2 min; and acetate, 17.4 min.
General procedure for SDS-PAGE analysis of soluble fractions of recombinant E. coli
The cell pellets were normalized to 10 ODeoo by resuspending in 20 mM potassium phosphate buffer (pH 8.0). 1mL of resuspended cells were taken in 2 mL lysis tube followed by the addition of 1 g of 0.1 mm glass beads. The cells were broken using FastPrep-24 homogenizer for 20 s and 6 cycles. The broken cells were centrifuged at 4,000 x g for 5 min to remove unbroken cells and glass beads. 500 pL of supernatant was further centrifuged at 13,000 x g for 30 min to separate soluble and particulate fractions. The soluble fractions (supernatant) were mixed with 5x SDS sample loading dye, heated at 95 °C for 15 min, and resolved in 12% SDS-PAGE gel.
Example 1. List of strains and plasmids used
Table 1. List of strains and plasmids used in this study.
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Example 2. Design of enzyme cascade for the production of natural PEA from bio-based L-Phe and selection of appropriate enzymes
Here, we report the novel and sustainable synthesis of natural PEA, PAA, Et-PA and PE-PA from bio-based L-Phe and renewable feedstock glucose and glycerol, respectively, via new artificial enzyme cascades (FIG. 1) with high product titer.
A six-enzyme artificial cascade was designed for the conversion of L-Phe to PEA (FIG. 2ai). The cascade consists enzymes for the following reactions: deamination of L-Phe to transcinnamic acid using PAL; decarboxylation of trans-cinnamic acid to styrene using PAD; epoxidation of styrene to styrene oxide using SMO; isomerization of styrene oxide to phenylacetaldehyde using SOI; reduction of phenylacetaldehyde to 2-PE using PAR; and transfer reaction of 2-PE to PEA using ATF.
PAL of Arabidopsis thaliana, PAD of Aspergillus niger, SMO and SOI of Pseudomonas sp. VLB120 and PAR of Solanum lypersicum were selected for establishing the newly proposed cascade to convert L-Phe to PEA. As ATF of Saccharomyces cerevisiae (S. cerevisiae) was reported to synthesize various esters using alcohols and acyl-CoAs (A. B. Mason & J. P. Dufour, Yeast 2000, 16, 1287-1298; and W. Li et a!., Eur. Food Res. Technol. 2018, 244, 555- 564), it was thus examined for the production of PEA from 2-PE.
Engineering of E. coli expressing ATF, cell growth, and whole-cell biotransformation of 2-PE to PEA
The gene coding for ATF was amplified from the genome of S. cerevisiae using PCR and cloned into pCDFDuet plasmid (pCDF-ATF) for expression in E. coli. As the codon preference is different for S. cerevisiae and E. coli, ATF gene was also codon-optimized (Opt-ATF), synthesized, and cloned into pCDFDuet plasmid (pCDF-Opt-ATF). The recombinant plasmids pCDF-ATF and pCDF-Opt-ATF were transformed into individual E. coli T7 express strains to construct E. coli ATF and E. coli Opt-ATF, respectively. The glycerol stock of recombinant E. coli strain was inoculated in 2 mL LB medium with appropriate antibiotics and grown at 37 °C and 220 rpm for 6-8 h. 1 mL of the culture was added to 50 mL of modified M9 medium with appropriate antibiotics (50 pg/mL streptomycin) in a 250 mL flask and grown at 37 °C and 220 rpm. At 2 h, 0.5 mM IPTG was added and the growth temperature was set at 22 °C. After 16 h of growth at 22 °C, the cells were harvested by centrifugation at 2,000 x g for 10 min. The harvested cells were used for SDS-PAGE analysis and biotransformation of 2-PE to PEA.
10 g cdw/L of cells were resuspended in 10 mL 200 mM phosphate buffer (pH 8.0) containing 45 mM of 2-PE and 2% glucose. 10 mL n-hexadecane was added, and the reaction was performed at 30 °C and 220 rpm for 4 h. The aqeuous and organic phase were collected at different time points by centrifuging at 10,000 x g for 10 min and analyzed using reverse phase HPLC and GC, respectively, to quantify PEA and 2-PE concentration.
Genetic engineering of recombinant E. coli for the cascade conversion of L-Phe to PEA
The genes coding for PAL and PAD were amplified using PCR and cloned in pRSFDuet plasmid. The genes coding for SMO, SOI and PAR were amplified using PCR and cloned in pETDuet plasmid. The genes coding for ATF was amplified from S. cerevisiae. The codon- optimized ATF was synthesized by Genscript and cloned in pCDFDuet plamid. The presence of recombinant genes in the engineered strains were confirmed using colony PCR and the expression of recombinant genes were confirmed using SDS-PAGE analysis. The confirmed strains were grown in 2 mL LB medium with respective antibiotics at 37 °C for 12 h. Glycerol stocks were made for all recombinant strains and stored at -80 °C until further usage.
Preparation of natural PEA by biotransformation
50 mL phosphate buffer (200 mM, pH 8) containing 15 g cdw/L of E. coli-Sty-PEA, 100 mM L- Phe, and 2% glucose were mixed with 50 mL ethyl oleate. The reaction was performed at 30 °C and 220 rpm. 2% of glucose was added to the reaction at 6 h. At 24 h, the reaction mixtures were centrifuged at 10,000 x g for 10 min to separate the aqueous and ethyl oleate phases. The ethyl oleate phase containing PEA was dried over Na2SO4, filtered, and loaded onto a silica column. The column was washed with hexane to remove ethyl oleate, and the crude product was eluted with hexane:EA of 15:1 as eluent (Rf ~ 0.3). The collected organic phase was subjected to evaporation, giving PEA as a colorless oil (543 mg, purity >99% by GC, 79% isolated yield). 1H NMR (400 MHz, CDCI3) 6 7.31 - 7.10 (m, 3H), 4.21 (t, J = 7.1 Hz, 1 H), 2.86 (t, J = 7.1 Hz, 1 H), 1.96 (s, 1 H). 13C NMR (101 MHz, CDCh) 6 171 .06, 137.84, 128.90, 128.52, 126.58, 64.95, 35.10, 20.99.
Results and discussion
S. cerevisiae is known to produce various phenylesters during the fermentation process to produce wines. Therefore, alcohol acetyl transferases from S. cerevisiae were analyzed for the production of PEA. Based on literature (K. J. Verstrepen et al., Appl. Environ. Microbiol. 2003, 69, 5228-5237; and P. T. Adeboye, M. Bettiga & L. Olsson, Sci. Rep. 2017, 7, 42635), ATF was reported to be highly active in S. cerevisiae. ATF was amplified from S. cerevisiae genome and cloned under the control of T7 promoter into pCDFDuet plasmid.
The recombinant E. coli was grown and the expression of ATF was studied (FIG. 3b). The expression of ATF was checked in E. coli in soluble and particulate fractions. It was identified that most of the expression was present in the particulate fractions, which denotes the production of enzymes as inactive inclusion bodies. The expression of ATF in soluble fraction was weak, therefore, the ATF gene was codon-optimized for efficient expression in E. coli, synthesized, cloned into pCDFDuet plasmid, and expressed in the recombinant strain E. coli Opt-ATF. The soluble expression of ATF significantly improved in E. coli Opt-ATF, as shown in SDS-PAGE (FIG. 3b).
The biotransformation of 2-PE to PEA was studied with resting cells of E. coli Opt-ATF in a two-phase system consisting of phosphate buffer containing 45 mM 2-PE and n-hexadecane at 30 °C for 4 h. The analysis of both aqueous and organic phases confirmed the production of 41 .7 mM PEA from 45 mM 2-PE with 92.6% conversion (FIGS. 3c and 4-5). Thus, Opt-ATF is an excellent enzyme for the conversion of 2-PE to PEA in the proposed enzyme cascade.
Example 3. Engineering of E. coli expressing six-enzyme cascade and one-pot biotransformation of L-Phe to PEA
The plasmid expressing optimized ATF was transformed into E. coli which contained recombinant plasmids expressing PAL, PAD, StyA, StyB, StyC and PAR and named as E. co//-Sty-PEA (FIG. 6a).
Engineering of E. coli expressing six-enzyme cascade, cell growth, and one-pot whole-cell biotransformation of L-Phe to PEA The plasmids, pRSF-PE-l, pCDF-Opt-ATF and pET-PE-ll, were used for the expression of PAL, PAD, styrene monooxygenase (StyA, StyB), styrene oxide isomerase (StyC), and PAR. pRSF-PE-l (pRSFDuet-padl-fcfcl-pa/) and pRSF-PE-ll (pETDuet-styA-styB-styC-par) plasmids were transformed into competent cells of E. coli Opt-ATF to construct E. coli-Sty- PEA, a recombinant strain harboring three recombinant plasmids expressing a six-enzyme cascade.
E. co//-Sty-PEA was inoculated in 2 mL LB medium with appropriate antibiotics (25 pg/mL streptomycin, 25 pg/mL kanamycin or 50 pg/mL ampicillin) and grown at 37 °C and 220 rpm for 6-8 h. 1 mL of the culture was added to 50 mL modified M9 medium with appropriate antibiotics (25 pg/mL streptomycin, 25 pg/mL kanamycin or 50 pg/mL ampicillin) in a 250 mL flask and grown at 37 °C and 220 rpm. At 2 h, 0.5 mM IPTG was added and the growth temperature was set at 22 °C. After 16 h of growth, the cells were harvested by centrifugation at 2000 x g for 10 min and used for SDS-PAGE analysis and biotransformation of L-Phe to PEA.
10 mL suspension of E. co//-Sty-PEA (15 g cdw/L) in phosphate buffer (200 mM, pH 8) containing 2% glucose and 100 mM L-Phe were mixed with 5 mL ethyl oleate or biodiesel. The reaction was incubated at 30 °C and 220 rpm for 24 h. Additional glucose of 2% was added to the reaction at 6 h. 200 pL of the reaction mixture were collected at various time points of the reaction and centrifuged at 10,000 x g for 10 min to separate the aqueous and organic phase. The aqueous phase was analyzed using reverse phase HPLC to measure the concentration of L-Phe and 2-PE, whereas the organic phase was analyzed using GC to measure the concentration of 2-PE and PEA.
Biotransformation of 2-PE to PEA by E. coli-ATF
The resting cells of E. coli-ATF was resuspended in phosphate buffer containing 50 mM 2-PE and biotransformation was performed in a two-phase system (phosphate buffer and n- hexadecane in 1 :1 v/v) for 6 h at 30 °C.
Biotransformation of L-Phe to PEA by E. co//-Sty-PEA
The biotransformation of L-Phe to PEA was performed with the resting cells of E. co//-Sty-PEA in phosphate buffer containing 50, 80 or 100 mM L-Phe and n-hexadecane (1:1 v/v) for 24 h at 30 °C. Samples were analyzed at various time points.
Results and discussion After successful expression of ATF in E. coli, the biotransformation of 2-PE to PEA was studied. The resting cells of E. coli- TF was resuspended in phosphate buffer containing 50 mM 2-PE and biotransformation was performed in a two-phase system for 6 h at 30 °C. The analysis of aqueous phase confirmed the consumption of 2-PE whereas the analysis of organic phase with GC-MS confirmed that PEA was produced. GC-FID analysis of the organic phase confirmed that 49.1 mM PEA was produced from 50 mM 2-PE.
The expression of all the seven recombinant proteins were confirmed by SDS-PAGE analysis (FIG. 6b). The recombinant strain was used for the biotransformation of L-Phe to PEA. Samples were analyzed at various time points and time-course of the biotransformation is shown in FIGS. 6c and 7. 36, 49.3, and 52.8 mM PEA were produced and 10, 15.6, and 16.1 mM 2-PE were accumulated from 50, 80, and 100 mM L-Phe, respectively. As shown in FIGS. 6c and 7, in all three experiments, significant amount of 2-PE was accumulated thus reducing the conversion efficiency of L-Phe to PEA.
The reason for the accumulation of 2-PE in the biotransformation of L-Phe to PEA could be due to the low availability of acetyl-CoA which is the cofactor for the final step of the cascade. The reactions were investigated by modifying the amount of cells used and amount of glucose added to the reaction (FIG. 8). Increasing the amount of whole cells from 10 to 15 g cdw/L increased the PEA production from 52.8 to 68.4 mM. However, further increase to 20 g cdw/L reduced the production to 64.7 mM. Since glucose could be used for acetyl-CoA biosynthesis and the initial 2% glucose added could not be sufficient, additional glucose (2%) was added to the reaction at 6 h. Upon this addition, L-Phe to PEA biotransformation with 15 g cdw/L E. co//-Sty-PEA increased PEA production from 68.4 to 79.3 mM giving rise to 79.3% conversion.
Example 4. Biotransformation of L-Phe to PEA in two phase system with green solvent
Although using n-hexadecane as an organic phase can give higher titer in the biotransformation of L-Phe to PEA, the process is not sustainable as n-hexadecane is a petrochemical. In order to enhance sustainability, replacing n-hexadecane with greener ethyl oleate with a reduced amount of organic phase (from 1 :1 to 1 :0.5) was attempted (FIG. 6d).
Biotransformation of 100 mM L-Phe with the resting cells of E. co//-Sty-PEA (15 g cdw/L) was performed in 10 mL of phosphate buffer and 10 mL ethyl oleate (compared with 10 mL n- hexadecane) at 30 °C for 24 h, producing PEA in 79.4 mM (79.3 mM for n-hexadecane). This result demonstrates that this green solvent is a suitable organic solvent for such two phase biotransformation. The reduction of the amount of organic solvents was explored by using aqueous and organic phase ratio of 1 :0.5 (v/v). The use of reduced amounts of ethyl oleate gave nearly the same higher product concentration (80.1 mM vs. 79.4 mM). Unexpectedly, the use of greener solvent ethyl oleate showed better performance than the use of n- hexadecane (80.1 mM vs. 67.5 mM). Thus, the use of ethyl oleate not only increased the sustainability but also the efficiency of the biotransformation. Furthermore, the green solvent, biodiesel, which was produced from waste cooking oil, was used for the two-phase biotransformation at aqueous and organic phase ratio of 1 :0.5 (v/v), giving nearly identical results as ethyl oleate. As shown in the time course profiles (FIGS. 6e and f), the highest production of 83.1 mM (13.6 g/L) PEA was achieved at 24 h from 100 mM L-Phe, corresponding to 83.1% conversion. Moreover, there is no accumulation of 2-PE at the end of the reaction. To the best of our knowledge, this is the highest production of PEA achieved from L-Phe in E. coli. In comparison, the use of the Ehrlich pathway in E. coli for the production of PEA resulted in only 687 mg/L (D. Guo et al., J. Agric. Food Chem. 2018, 66, 5886-5891).
Thus, a novel, green, and efficient artificial six-enzyme cascade was designed and developed for the production of PEA from L-Phe. ATF was proven to be a good catalyst for the conversion of 2-PE to PEA, a key reaction in the six-enzyme cascade. The cascade reactions were optimized by the addition of glucose to generate acetyl-CoA and the use of aqueous-organic two phase system (1 :0.5 v/v) to minimize the toxicity of intermediates and product. The green solvents (ethyl oleate and biodiesel) were proven to be better than n-hexadecane for the same biotransformation, resulting in high-yielding production of 83.1 mM (13.6 g/L) of PEA from L- Phe with 83.1% conversion. Therefore, the present disclosure is a simple and high-yielding process (13.6 g/L titer; >80% yield) and do not use any toxic materials or petroleum-derived substrates, which is attractive for industries.
Example 5. Design of five-enzyme cascade, engineering of E. coli expressing the cascade, and one-pot biotransformation of L-Phe to PAA
A five-enzyme artificial cascade was designed for the conversion of L-Phe to PAA by overexpressing enzymes for the following reactions: deamination of L-Phe to trans-cinnamic acid using PAL; decarboxylation of trans-cinnamic acid to styrene using PAD; epoxidation of styrene to styrene oxide using SMO; isomerization of styrene oxide to phenylacetaldehyde using SOI; and oxidation of phenylacetaldehyde to PAA using EcALDH (FIG. 2aii). SMO and SOI of Pseudomonas sp. VLB120 and ALDH of Escherichia coli enzymes were used for establishing the proposed cascades. For the conversion of L-Phe to styrene, PAL of Arabidopsis thaliana, and PAD of Aspergillus niger were chosen. The recombinant strain expressing PAL, PAD, SMO, SOI, and EcALDH from two plasmids was constructed as E. coli- Sty-PAA for the conversion of L-Phe to PAA (FIG. 9a).
Genetic engineering of recombinant E. coli for the cascade conversion of L-Phe to PAA
The genes coding for PAL and PAD were amplified using PCR and cloned in pACYCDuet plasmid. The genes coding for SMO, SOI and EcALDH were amplified using PCR and cloned in pRSFDuet plasmid. The presence of recombinant genes in the engineered strains were confirmed using colony PCR and the expression of recombinant genes were confirmed using SDS-PAGE analysis. The confirmed strains were grown in 2 mL LB medium with respective antibiotics at 37 °C for 12 h. Glycerol stocks were made for all recombinant strains and stored at -80 °C until further usage.
Engineering of E. coli expressing five-enzyme cascade, cell growth, and one-pot whole-cell biotransformation of L-Phe to PAA
The plasmids, pACYC-PAA-l and pRSF-PAA-ll, were used for the expression of PAL, PAD (FDC), styrene monooxygenase (StyA, StyB), styrene oxide isomerase (StyC), and aldehyde dehydrogenase (ALDH). pACYCDuet expressing pad1, fdc1, and pal and pRSFDuet expressing styA, styB, styC and EcALDH were transformed into E. coli T7 express and the recombinant strain was named as E. co//-Sty-PAA.
E. co//-Sty-PAA was inoculated in 2 mL LB medium with appropriate antibiotics (50 pg/mL kanamycin or 25 pg/mL chloramphenicol) and grown at 37 °C and 220 rpm for 8 h. 1 mL of the culture was added to 50 mL modified M9 medium with appropriate antibiotics (50 pg/mL kanamycin or 25 pg/mL chloramphenicol) in a 250 mL flask and grown at 37 °C and 220 rpm. At 2 h, 0.5 mM IPTG was added and the growth temperature was set at 22 °C. After 16 h of growth, the cells were harvested by centrifugation at 2000 x g for 10 min and used for SDS- PAGE analysis and biotransformation of L-Phe to PAA.
L-Phe to PAA biotransformation was performed in a two-phase reaction with 5 mL ethyl oleate and 10 mL phosphate buffer (200 mM, pH 8.0) containing 15 g cdw/L of E. co//-Sty-PAA cells, 100 mM L-Phe, and 2% glucose. The reaction was performed at 30 °C and 220 rpm for 10 h. 200 pL of the reaction mixture was collected at various time points of the reaction and centrifgued at 10,000 x g for 10 min and the aqueous phase was analyzed using reverse phase HPLC to measure the concentration of L-Phe and PAA.
Preparation of natural PAA by biotransformation 50 mL phosphate buffer (200 mM, pH 8) containing 15 g cdw/L of E. coli-Sty-PAA, 100 mM L- Phe, and 2% glucose were mixed with 25 mL ethyl oleate. The reaction was performed at 30 °C and 220 rpm for 12 h, followed by centrifugation at 10,000 x g for 10 min to separate the aqueous and organic phases. The aqueous phase was used for the isolation of PAA. pH of the aqueous phase was adjusted to <2.0 with concentrated HCI, and PAA from the mixture was extracted with EA (50 mL x 3 times). The organic phase was collected and dried over Na2SC>4, filtered, and evaporated. The crude PAA was purified by flash chromatography on silica gel column with n-Hexane:EA:TFA of 100:5:0.1 as eluent (Rf ~ 0.3) to give PAA as a white solid (430.2 mg, purity >99% by HPLC, 74% isolated yield).
1H NMR (400 MHz, CDCh): 5 11.41 (s, 1 H), 7.25 - 7.11 (m, 5H), 3.53 (s, 2H). 13C NMR (101 MHz, CDCh): 5 178.45, 133.34, 129.49, 128.75, 127.46, 41.19.
Results and discussion
The SDS-PAGE analysis of E. co//-Sty-PAA confirmed the expression of all heterologous enzymes in soluble fraction (FIG. 9b). The biotransformation of L-Phe to PAA was performed on the same conditions that was optimized for the conversion of L-Phe to PEA. The conversion of 100 mM L-Phe with 15 g cdw/L of E. co//-Sty-PAA at 30 °C and 220 rpm in a two-phase system containing ethyl oleate as organic phase (Aqueous:Organic, 1 :0.5 v/v) afforded 87.1 mM PAA in 87.1% conversion (FIG. 9c). This high-yielding synthesis provided green, sustainable, and efficient production of this natural compound.
Thus, a novel, green, and efficient artificial five-enzyme cascade was designed and developed for the production of PAA from L-Phe. Biotransformation of 100 mM L-Phe with the engineered E. coli cells expressing the five enzymes in two-phase system (aqueous-organic ratio, 1 :0.5 v/v) containing ethyl oleate as organic phase produced 87.1 mM (11.6 g/L) PAA with 87.1% conversion. This is the first report for the bioproduction of PAA from L-Phe using artificial enzyme cascade.
Example 6. Production of natural PEA and PAA from renewable feedstock glucose and glycerol by coupling L-Phe biosynthesis pathway and artificial enzyme cascades
After successful conversion of L-Phe to PEA in Examples 2 to 4, we attempted to produce PEA using coupled-cells approach. L-Phe is produced naturally by E. coli via growing cell fermentation of sugars such as glucose and glycerol through shikimate pathway. As our artificial enzyme cascades could convert L-Phe to PEA and PAA, it is desirable to combine it with the natural L-Phe biosynthesis pathway (shikimate pathway) to produce PEA and PAA directly from glucose or glycerol (FIG. 2b). The fermentative production of L-Phe from glucose or glycerol (renewable feedstocks) was thus coupled with the artificial cascades biotransformation of L-Phe to PEA and PAA, respectively (FIG. 10a). Since the production of L-Phe from glucose or glycerol is strictly regulated in E. coli by feedback regulation, an engineered E. coli (E. coli NST74-Phe) (Y. Zhou et al., Biotechnol. Bioeng. 2020, 117, 2340- 2350; and Y. Zhou et al., ChemSusChem 2018, 11, 2221-2228) with relaxed feedback regulation was used as the production host for this experiment. The biosynthesized L-Phe will be transformed into PEA by performing whole-cells catalyzed biotransformation using fermented media and recombinant E. coli expressing artificial enzyme cascades.
One-pot conversion of glucose and glycerol to PEA by coupled fermentation and biotransformation
Firstly, fermentative production of L-Phe from glucose and glycerol was performed using E. coli NST74-Phe (B. S. Sekar, B. R. Lukito & Z. Li, ACS Sustain. Chem. Eng. 2019, 7, 12231- 12239) to achieve 67 and 78 mM L-Phe, respectively, and then the fermentation culture containing biosynthesized L-Phe and E. coli NST74-Phe cells was stored at 4 °C or used directly in biotransformation. E. co//-Sty-PEA was grown as described above in Example 5 to prepare the cells for biotransformation. 0.6 g of wet cells of E. co//-Sty-PEA, 0.378 g of phosphate powder (0.36 g of K2HPO4 and 18.2 mg KH2PO4), and 0.2 g glucose were added to 10 mL of fermentation broth (containing 67 or 78 mM L-Phe) to form 11 mL of aqueous system containing 15 g cdw/L of cells, 61 or 71 mM L-Phe, 2% glucose and 200 mM phosphate buffer. 5 mL ethyl oleate was added, and biotransformation was performed at 30 °C and 220 rpm. Additional glucose (2%) was added at 6 h. At 24 h, the organic phase was collected to quantify the concentration of PEA by GC.
One-pot conversion of glucose and glycerol to PAA by coupled fermentation and biotransformation
Fermentative production of L-Phe from glucose and glycerol was performed using E. coli NST74-Phe to achieve 67-80 mM L-Phe and the fermentation culture containing biosynthesized L-Phe and E. coli NST74-Phe cells was stored at 4 °C or used directly in biotransformation. E. co//-Sty-PAA was grown as described above in Example 5 to prepare the cells for biotransformation. 0.6 g of wet cells of E. co//-Sty-PAA, 0.378 g of phosphate powder (0.36 g of K2HPO4 and 18.2 mg KH2PO4), and 0.2 g glucose were added to 10 mL of fermentation broth (containing 67 or 78 mM L-Phe) to form 11 mL of aqueous system containing 15 g cdw/L of cells, 61 or 71 mM L-Phe, 2% glucose and 200 mM phosphate buffer. 5 mL ethyl oleate was added, and biotransformation was performed at 30 °C and 220 rpm. At 12 h, the reaction mixture was centrifuged at 10,000 x g for 10 min and the aqueous phase was collected to quantify the concentration of PAA by reverse phase HPLC.
Results and discussion
At first, fermentation of glucose or glycerol with E. coli NST74-Phe overexpressing five key enzymes of shikimate pathway (AroG*, AroK, YdiB, PheA*, and TyrB) (B. S. Sekar, B. R. Lukito & Z. Li, ACS Sustain. Chem. Eng. 2019, 7, 12231-12239) produced 67-78 mM L-Phe. Resting cells of E. coli overexpressing the artificial cascade (E. co//-Sty-PEA or E. coli-Sty- PAA) was then added to fermentation broth containing L-Phe to a cell density of 15 g cdw/L, followed by the addition of ethyl oleate to form two-phase system (aqueous and organic phase ratio of 1 :0.5, v/v). Biotransformation for 24 h gave 53.7-63.1 mM (8.8-10.4 g/L) PEA (FIG. 10b) and 57.6-67.8 mM (7.8-9.2 g/L) PAA (FIG. 10c), respectively. Thus, the one-pot production of natural PEA and PAA from low-cost renewable feedstocks such as glucose and glycerol were successfully demonstrated. The titer of PEA and PAA achieved in our processes from glucose or glycerol is 13-fold (D. Guo et al., J. Agric. Food Chem. 2018, 66, 5886-5891) and 158-fold (L. Zhang et al., Amb Express 2017, 7, 1-7) higher than those from the reported productions from glucose (no report from glycerol), respectively. Green, sustainable, and efficient bioproduction of natural PEA and PAA from renewable feedstock glucose and glycerol were thus established.
Therefore, one-pot production of natural PEA and PAA from low-cost renewable feedstocks such as glucose and glycerol were successfully demonstrated by coupling of artificial enzyme cascade and L-Phe biosynthesis pathway. Using this coupled fermentation and biotransformation approach, 63.1 mM (10.4 g/L) of PEA and 67.8 mM (9.2 g/L) PAA were produced from glucose or glycerol, respectively. The high-yielding one-pot syntheses of PEA and PAA from L-Phe, glucose, or glycerol using novel enzyme cascades, greener solvents, and renewable resources provide green, sustainable and efficient production of PEA and PAA as high-value natural aroma compounds.
Example 7. Bioproduction of natural Et-PA from L-Phe and glucose
A six-enzyme artificial cascade was designed for the conversion of L-Phe to Et-PA: deamination of L-Phe to trans-cinnamic acid using PAL; decarboxylation of trans-cinnamic acid to styrene using PAD; epoxidation of styrene to styrene oxide using SMO; isomerization of styrene oxide to phenylacetaldehyde using SOI; oxidation of phenylacetaldehyde to PAA using EcALDH; and esterification of PAA to Et-PA using lipase. Two commercially available immobilized lipases such as Novozyme 435 (Candida Antarctica lipase B) (B. M. Lue et al., J. Chem. Technol. Biotechnol. 2005, 80, 462-468; and B. Sandig & M. R. Buchmeiser, ChemSusChem 2016, 9, 2917-2921) and TL lipozyme (Thermomyces lanuginosus lipase) (A.-F. Hsu et al., Biotechnol. Lett. 2004, 26, 917-921) were examined for the conversion of PAA to Et-PA.
Biotransformation of PAA to Et-PA with immobilized lipases
0.25 g of Novozyme 435 or TL lipozyme were resuspended in 5 mL hexane containing 20 mM bio-produced PAA and 40 mM ethanol. While the Novozyme 435 reaction was performed in anhydrous condition, 1% of water was added for the reaction with TL lipozyme. The reaction was performed at 40 °C with stirring at 800 rpm. After 36 h of reaction, the hexane phase was analyzed in GC for the concentration of Et-PA.
Cascade conversion of L-Phe to Et-PA
Recombinant E. coli expressing PAL, PAD, SMO, SOI, EcALDH was used for the biotransformation of L-Phe to PAA. The biotransformation was performed at 30 °C, 220 rpm in a two-phase system (phosphate buffer and ethyl oleate in 1 :0.5 v/v). The biosynthesized PAA was isolated from the biotransformation using silica column chromatography by following the preparation of natural PAA by biotransformation protocol in Example 5, and used for the production of Et-PA. The biotransformation protocol was optimized by increasing the stirrer speed.
Preparation of natural Et-PA by biotransformation
20 mL hexane containing 0.5 g Novozyme 435, 20 mM biosynthesized PAA produced from L- Phe by using E. co//-Sty-PAA, and 40 mM ethanol were stirred at 40 °C and 800 rpm for 36 h. After filtration, the hexane phase was concentrated, and the crude product was purified by flash chromatography on a silica gel column with n-hexane:EA:TFA of 100:10:0.1 as eluent (Rf ~ 0.3) to give Et-PA as a colourless oil (53.6 mg, purity >99% by GC, 96% isolated yield).
1H NMR (400 MHz, CDCh) 57.26 - 7.18 (m, 5H), 4.08 (q, J = 7.2 Hz, 2H), 3.54 (s, 2H), 1.18 (t, J = 7.2 Hz, 2H). 13C NMR (101 MHz, CDCh) 5 171.76, 134.32, 129.38, 128.69, 127.18, 60.99, 41.60, 14.32.
Results and discussion
We screened the two commercially available immobilized lipases Novozyme 435 and TL lipozyme. Both Novozyme 435 and TL lipozyme were able to convert PAA to Et-PA in the presence of ethanol. Novozyme 435 produced Et-PA with a conversion of 56.7% and TL lipozyme produced Et-PA with a conversion of 51.7% (FIG. 11).
Recombinant E. coli expressing PAL, PAD, SMO, SOI, EcALDH was used for the biotransformation of L-Phe to PAA. 59 mM of PAA was produced from 70 mM L-Phe in 84% conversion. Novozyme 435 efficiently converted 20 mM of biosynthesized PAA to 15.8 mM of Et-PA in 79% conversion.
Natural PAA was prepared from glucose using the coupled fermentation-biotransformation approach, isolated and used in this esterification reaction to produce natural Et-PA (FIGS. 12ai and b). Both Novozyme 435 and TL lipozyme were able to convert PAA to Et-PA. For the esterification of 20 mM natural PAA and 40 mM ethanol, Novozyme 435 produced 16.6 mM (2.7 g/L) Et-PA with a conversion of 83.1 % while TL lipozyme gave 11.1 mM (1.8 g/L) Et-PA with the conversion of 55.5%. Accordingly, Novozyme 435 is a better catalyst for the production of Et-PA from PAA which was derived from glucose. Therefore, Novozyme 435 was chosen for the production of Et-PA. Thus, the novel bioproduction of Et-PA directly from glucose was achieved, demonstrating the first example of sugar-derived Et-PA production.
Example 8. Bioproduction of natural PE-PA from L-Phe and glucose
The lipases Novozyme 435 and TL lipozyme were also examined for the esterification of natural PAA to natural PE-PA.
A six-enzyme artificial cascade was designed for the conversion of L-Phe to PE-PA: deamination of L-Phe to trans-cinnamic acid using PAL; decarboxylation of trans-cinnamic acid to styrene using PAD; epoxidation of styrene to styrene oxide using SMO; isomerization of styrene oxide to phenylacetaldehyde using SOI; oxidation of phenylacetaldehyde to PAA using EcALDH; and esterification of PAA to PE-PA using lipase.
Biotransformation of PAA to PE-PA with immobilized lipases
0.25 g of Novozyme 435 or TL lipozyme were resuspended in 5 mL hexane containing 20 mM bio-produced PAA and 40 mM bio-produced 2-PE (B. S. Sekar, B. R. Lukito & Z. Li, ACS Sustain. Chem. Eng. 2019, 7, 12231-12239). While the Novozyme 435 reaction was performed in anhydrous condition, 1% of water was added for the reaction with TL lipozyme. The reaction was performed at 40 °C with stirring at 800 rpm. After 36 h of reaction, the hexane phase was analyzed in GC for the concentration of PE-PA. Cascade conversion of L-Phe to PE-PA
Recombinant E. coli expressing PAL, PAD, SMO, SOI, EcALDH was used for the biotransformation of L-Phe to PAA. The biotransformation was performed at 30 °C and 220 rpm in a two-phase system (phosphate buffer and ethyl oleate in 1 :0.5 v/v). The biosynthesized phenylacetic acid was isolated from the biotransformation using silica column chromatography by following the preparation of natural PAA by biotransformation protocol in Example 5, and used for the production of PE-PA. The biotransformation protocol was optimized by increasing the stirrer speed and reducing the water content from 2% to 1%.
Preparation of natural PE-PA by biotransformation
20 mL hexane (1% water; v/v) containing 0.5 g TL-Lipozyme, 20 mM biosynthesized PAA produced from L-Phe using E. co//-Sty-PAA, and 40 mM natural 2-PE produced from L-Phe (B. S. Sekar, B. R. Lukito & Z. Li, ACS Sustain. Chem. Eng. 2019, 7, 12231-12239) using E. co//-Sty-2-PE were stirred at 40 °C and 800 rpm. After 36 h of reaction, the mixture was filtered, concentrated, and subjected to flash chromotagraphy with n-hexane:EA:TFA of 100:10:0.1 as eluent (Rf = 0.25) to give PE-PA as a colourless oil (83.2 mg, purity >99% by GC, 89.5% isolated yield).
1H NMR (400 MHz, CDCI3): 6 7.64 - 7.13 (m, 8H), 7.11 - 6.93 (m, 2H), 4.23 (t, J = 7.0 Hz, 2H), 3.53 (s, 2H), 2.84 (t, J = 7.0 Hz, 2H). 13C NMR (101 MHz, CDCI3) 6 171.63, 137.88, 134.13, 129.43, 129.06, 128.69, 128.61 , 127.19, 126.66, 65.47, 41.58, 35.18.
Results and discussion
We screened two commercially available immobilized lipases such as Novozyme 435 and TL lipozyme. Both Novozyme 435 and TL lipozyme were able to convert PAA to PE-PA in the presence of 2-PE. Novozyme 435 produced PE-PA with the conversion of 51.7% and TL lipozyme produced PE-PA with the conversion of 77.3%.
Recombinant E. coli expressing PAL, PAD, SMO, SOI, EcALDH was used for the biotransformation of L-Phe to PAA. 59 mM of PAA was produced from 70 mM L-Phe in 84% conversion. The biosynthesized PAA was isolated from the biotransformation using silica column chromatography and used for the production of PE-PA. TL lipozyme efficiently converted 20 mM of biosynthesized PAA to 19.1 mM of PE-PA in 95% conversion.
PAA and 2-PE (B. S. Sekar, B. R. Lukito & Z. Li, ACS Sustain. Chem. Eng. 2019, 7, 12231- 12239) were first prepared from glucose using the coupled fermentation-biotransformation approach, isolated, and then used for the esterification reaction to produce natural PE-PA (FIGS. 12aii and c). Both Novozyme 435 and TL lipozyme were able to convert PAA to PEPA. Esterification of 20 mM biosynthesized PAA with 40 mM biosynthesized 2-PE by using Novozyme 435 produced 11.3 mM (2.7 g/L) PE-PA with a conversion of 56.5%. On the other hand, the same biotransformation performed with TL lipozyme gave 19.3 mM (4.6 g/L) PE-PA with a conversion of 96.5%. Obviously, TL lipozyme is much more suitable lipase for the production of PE-PA from PAA and 2-PE which were derived from glucose. Therefore, TL lipozyme was chosen for the production of PE-PA. Thus, the novel bioproduction of PE-PA directly from glucose was demonstrated, as the first example of sugar-derived PE-PA production.
The first example of L-Phe or sugar-derived Et-PA and PE-PA production have been demonstrated in Examples 7 and 8. The novel syntheses were achieved by coupling bioproduction of PAA from L-Phe or glucose with enzymatic esterification of PAA with ethanol and biosynthesized 2-PE, respectively. While Novozyme 435 is a better catalyst for the production of Et-PA (16.6 mM; 2.7 g/L; 83.1% conversion), TL lipozyme is a much more suitable lipase for the production of PE-PA from PAA (19.3 mM; 4.6 g/L; 96.3% conversion). Our new methods for producing Et-PA and PE-PA from PAA and ethanol or 2-PE derived from sugar provide green, sustainable, and efficient productions of Et-PA and PE-PA as high-value natural aroma compounds.
Therefore, this work discloses the design and development of an artificial enzyme cascade for the efficient production of natural PEA, Et-PA and PE-PA from biobased L-Phe via artificial enzyme cascade and from renewable feedstocks such as glucose and glycerol via the combination of natural biosynthesis pathway and artificial enzyme cascade. This work is also simple, green, sustainable and uses low-cost renewable feedstocks for the efficient production of natural aroma chemicals (PEA, Et-PA and PE-PA), thus providing an economical and sustainable approach for the production of these high value natural aroma chemicals, which is in agreement with the current market and societal needs. Therefore, our work will attract great interest from industries to develop commercial production of natural PEA, Et-PA and PEPA.
Example 9. Production of Et-PA and PE-PA from glucose or glycerol
The strain for the conversion of L-Phe to PAA was constructed by transforming the plasmid overexpressing PAL and PAD E. co//-StyABC-EcALDH. Recombinant E. coli expressing PAL, PAD, StyA, StyB, StyC and EcALDH was inoculated in 2 mL LB medium containing appropriate antibiotics and grown at 37 °C, 220 rpm for 6-8 h. 1 mL of the culture was added to 50 mL of modified M9 medium with appropriate antibiotics in a 250 mL flask. After 2 h of growth at 37 °C, 0.1 mM IPTG and the growth temperature was set at 22 °C. After 16 h of growth at 22 °C, the cells were harvested by centrifugation at 4000 rpm for 10 min. To 5 mL of fermentation broth containing 70-80 mM biosynthesized L-Phe, phosphate buffer (200 mM, pH 8), 2% glucose, and 1 mL of ethyl oleate were added. Harvested cells of E. coli (15 g cdw/L) were added and biotransformation was performed at 30 °C, 220 rpm in a two-phase system (phosphate buffer and ethyl oleate in 1 :0.5 v/v). The produced PAA was extracted from aqueous phase using silica gel column chromatography by following the preparation of natural PAA by biotransformation protocol in Example 5 and used in the biotransformation of PAA to Et-PA and PE-PA using Novozyme 435 and TL lipozyme, respectively.

Claims

Claims
1. A method for producing phenethyl acetate (PEA) or a derivative thereof, ethyl phenylacetate (Et-PA) or a derivative thereof, or phenylethyl phenylacetate (PE-PA) or a derivative thereof using four or more enzymes, which method comprises subjecting styrene or a derivative thereof to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.
2. The method of Claim 1 , wherein the method produces producing phenethyl acetate or a derivative thereof, comprising the steps of:
(a) generating styrene oxide or a derivative thereof from styrene or a derivative thereof by conducting an epoxidation reaction catalyzed by an epoxidase to form styrene oxide or a derivative thereof;
(b) generating phenylacetaldehyde or a derivative thereof from styrene oxide or a derivative thereof by conducting an isomerization reaction catalysed by an isomerase to form phenylacetaldehyde or a derivative thereof;
(c) generating 2-phenethylethanol or a derivative thereof from phenylacetaldehyde or a derivative thereof by conducting a reduction reaction catalyzed by a reductase to form 2- phenethylethanol or a derivative thereof; and
(d) generating phenethyl acetate or a derivative thereof from 2-phenethylethanol or a derivative thereof by conducting an esterification reaction catalysed by a transferase to form phenethyl acetate or a derivative thereof.
3. The method according to Claim 2, wherein:
(a) the epoxidase is a styrene monooxygenase, optionally wherein the styrene monooxygenase is from Pseudomonas sp. VLB120, or its mutants or similar enzymes with more than 50% identity; and/or
(b) the isomerase is a styrene oxide isomerase, optionally wherein the styrene oxide isomerase is from Pseudomonas sp. VLB 120, or its mutants or similar enzymes with more than 50% identity; and/or
(c) the reductase is a phenylacetaldehyde reductase, optionally wherein the phenylacetaldehyde reductase is from Solanum lypersicum, or its mutants or similar enzymes with more than 50% identity; and/or
(d) the transferase is an alcohol acetyl transferase, optionally wherein the alcohol acetyl transferase is from Saccharomyces cerevisiae, or its mutants or similar enzymes with more than 50% identity.
4. The method according to Claim 3, wherein the method is conducted in one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes), where the recombinant microbial cell is E. coli pETDuet-StyAB-StyC-PAR, pCDFDuet-Opt-ATF.
5. The method of Claim 1 , wherein the method produces producing ethyl phenylacetate or a derivative thereof or phenylethyl phenylacetate or a derivative thereof, comprising the steps of:
(a) generating styrene oxide or a derivative thereof from styrene or a derivative thereof by conducting an epoxidation reaction catalyzed by an epoxidase to form styrene oxide or a derivative thereof;
(b) generating phenylacetaldehyde or a derivative thereof from styrene oxide or a derivative thereof by conducting an isomerization reaction catalysed by an isomerase to form phenylacetaldehyde or a derivative thereof;
(c) generating phenylacetic acid or a derivative thereof from phenylacetaldehyde or a derivative thereof by conducting an oxidation reaction catalyzed by an oxidase or an aldehyde dehydrogenase to form phenylacetic acid or a derivative thereof; and
(d) generating ethyl phenylacetate or a derivative thereof from phenylacetic acid or a derivative thereof by conducting an esterification reaction using a lipase in a reaction medium comprising ethanol to form ethyl phenylacetate or a derivative thereof; or
(e) generating phenylethyl phenylacetate or a derivative thereof from phenylacetic acid or a derivative thereof by conducting an esterification reaction using a lipase in a reaction medium comprising 2-phenylethanol to form ethyl phenylacetate or a derivative thereof.
6. The method according to Claim 5, wherein:
(a) the epoxidase is a styrene monooxygenase, optionally wherein the styrene monooxygenase is from Pseudomonas sp. VLB120, or its mutants or similar enzymes with more than 50% identity; and/or
(b) the isomerase is a styrene oxide isomerase, optionally wherein the styrene oxide isomerase is from Pseudomonas sp. VLB 120, or its mutants or similar enzymes with more than 50% identity; and/or
(c) the oxidase or aldehyde dehydrogenase is aldehyde dehydrogenase (EcALDH) from Escherichia coli K12, or its mutants or similar enzymes with more than 50% identity; and/or
(d) the lipase is Novozyme 435 from Candida Antarctica or TL lipozyme from Thermomyces lanuginosus, or their mutants or similar enzymes with more than 50% identity.
7. The method according to Claim 6, wherein the method is conducted in one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes), where the recombinant microbial cell is E. coli pRSFDuet-StyABC-EcALDH in the presence of an immobilised lipase.
8. The method according to any one of the preceding claims, wherein the method further comprises providing styrene or a derivative thereof by generating trans-cinnamic acid or a derivative thereof from L-phenylalanine or a derivative thereof by a deamination reaction catalyzed by an ammonia lyase and generating styrene or a derivative thereof from the trans- cinnamic acid or a derivative thereof in a decarboxylation reaction catalyzed by a decarboxylase.
9. The method according to Claim 8, wherein:
(a) the ammonia lyase is phenylalanine ammonia lyase from Arabidopsis thaliana, or its mutants or similar enzymes with more than 50% identity; and/or
(b) the decarboxylase is phenylacrylic acid decarboxylase from Aspergillus niger, or its mutants or similar enzymes with more than 50% identity.
10. The method according to any one of Claims 2 to 4, wherein the method is conducted in one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes), where the recombinant microbial cell is E. coli pRSFDuet- PAL-PAD, pETDuet-StyAB-StyC-PAR, pCDFDuet-Opt-ATF.
11. The method according to any one of Claims 8 to 10, wherein the method is conducted in one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes), where the recombinant microbial cell is E. coli pACYCDuet- PAL-PAD, pRSFDuet-StyABC-EcALDH in the presence of an immobilised lipase.
12. The method according to any one of Claims 8 to 11 , wherein the method further comprises cells that overexpress the natural L-phenylalanine biosynthetic pathway, which cells convert glucose or glycerol to L-phenylalanine.
13. The method according to Claim 12 wherein the microbial cells producing L- phenylalanine from glucose or glycerol overexpress at least one enzyme is selected from one or more of the group consisting of DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), or their mutants or similar enzymes with more than 50% identity, optionally wherein the microbial cells producing L-phenylalanine from glucose or glycerol overexpress at least one enzyme is:
(a) a feedback inhibition resistant mutant of AroG;
(b) a feedback inhibition resistant mutant of PheA.
14. The method according to Claim 13, wherein the microbial cells producing L- phenylalanine from glucose or glycerol that overexpress at least one enzyme is one in which the microbial cells are mutated for deletion or inactivation of err and/or tyrA genes.
15. The method according to any one of the preceding claims, wherein the one-pot reaction system comprises an aqueous medium.
16. The method according to any one of Claims 1 to 15, wherein the one-pot reaction system comprises use of a bi-phasic medium, optionally wherein the bi-phasic medium consists of an aqueous and solid resin medium, or an aqueous and organic solvent medium (e.g. the organic solvent medium is selected from one or more alkanes solvents and/or one or more ester solvents).
17. The method according to any one of the preceding claims, wherein the one-pot reaction system comprises use of absorbent resins or nanomaterials for in-situ removal of product.
18. An isolated nucleic acid molecule encoding at least one heterologous catalytic enzyme selected from the group comprising:
(a) an epoxidase for conducting an epoxidation reaction to form styrene oxide or a derivative thereof from styrene or a derivative thereof;
(b) an isomerase for generating phenylacetaldehyde or a derivative thereof from styrene oxide by an isomerization reaction; and
(c) a reductase for generating 2-phenethylethanol or a derivative thereof by a reduction reaction from phenylacetaldehyde or a derivative thereof;
(d) a transferase for generating phenethyl acetate or a derivative thereof by an esterification reaction from 2-phenethylethanol or a derivative thereof;
(e) an ammonia lyase for generating trans-cinnamic acid or a derivative thereof from L-phenylalanine or a derivative thereof by a deamination reaction; (f) a decarboxylase for generating styrene or a derivative thereof from trans- cinnamic acid or a derivative thereof in a decarboxylation reaction;
(g) an oxidase or an aldehyde dehydrogenase for generating phenylacetic acid or a derivative thereof from phenylacetaldehyde by an oxidation reaction; and
(h) a lipase for generating ethyl phenylacetate or phenylethyl phenylacetate, or derivatives thereof, from phenylacetic acid or a derivative thereof in an esterification reaction.
19. The isolated nucleic acid of Claim 18, encoding a plurality of said catalytic enzymes.
20. The isolated nucleic acid molecule of Claim 19, wherein said plurality of catalytic enzymes is arranged as at least one module selected from the group comprising: i) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms styrene or a derivative thereof to phenylacetaldehyde or a derivative thereof; ii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms phenylacetaldehyde or a derivative thereof to 2-phenylethanol or a derivative thereof; iii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms 2-phenylethanol or a derivative thereof to phenethyl acetate or a derivative thereof; and iv) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms an L-phenylalanine or a derivative thereof to styrene or a derivative thereof, or any combination thereof.
21. The isolated nucleic acid molecule of Claim 19, wherein said plurality of catalytic enzymes is arranged as at least one module selected from the group comprising: i) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms styrene or a derivative thereof to phenylacetaldehyde or a derivative thereof; ii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms phenylacetaldehyde or a derivative thereof to 2-phenylacetic acid or a derivative thereof; iii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms 2-phenylacetic acid or a derivative thereof to phenylethyl phenylacetate or a derivative thereof or phenethyl acetate; and iv) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms an L-phenylalanine or a derivative thereof to styrene or a derivative thereof, or any combination thereof.
22. An expression construct comprising at least one nucleic acid molecule as described in any one of Claims 19 to 21.
23. One or more recombinant prokaryotic or eukaryotic cells selected from the group comprising bacterial cells, yeast cells, mammalian cells and insect cells, wherein said cells comprise at least one expression construct as described in Claim 22.
24. The one or more recombinant prokaryotic or eukaryotic cells according to Claim 23, wherein said cells are recombinant bacterial cells.
25. A kit comprising at least one isolated nucleic acid according to any one of Claims 19 to 21.
26. The method according to Claim 4, wherein the method further comprises acetyl CoA.
27. The method according to any one of Claims 1 to 17 and 27, wherein the at least four enzymes used in the method are provided:
(a) in whole cells genetically engineered to overexpress the at least four enzymes, optionally wherein the at least four overexpressed enzymes are located on one or more plasmids or integrated in the chromosome of each of the one or more recombinant microbial cells;
(b) in a cell-free extract;
(c) as purified enzymes; or
(d) as immobilized enzymes.
28. A method for producing phenylacetic acid (PAA) or a derivative thereof using five or more enzymes, which method comprises subjecting L-phenylalanine or a derivative thereof to multiple enzyme-catalyzed chemical transformations in a one-pot reaction system.
29. The method of Claim 28, wherein the method produces producing phenylacetic acid or a derivative thereof, comprising the steps of: (a) generating trans-cinnamic acid or a derivative thereof from L-phenylalanine or a derivative thereof by a deamination reaction catalyzed by an ammonia lyase;
(b) generating styrene or a derivative thereof from the trans-cinnamic acid or a derivative thereof in a decarboxylation reaction catalyzed by a decarboxylase;
(c) generating styrene oxide or a derivative thereof from styrene or a derivative thereof by conducting an epoxidation reaction catalyzed by an epoxidase to form styrene oxide or a derivative thereof;
(d) generating phenylacetaldehyde or a derivative thereof from styrene oxide or a derivative thereof by conducting an isomerization reaction catalysed by an isomerase to form phenylacetaldehyde or a derivative thereof; and
(e) generating phenylacetic acid or a derivative thereof from phenylacetaldehyde or a derivative thereof by conducting an oxidation reaction catalyzed by an oxidase or an aldehyde dehydrogenase to form phenylacetic acid or a derivative thereof.
30. The method according to Claim 29, wherein:
(a) the ammonia lyase is phenylalanine ammonia lyase from Arabidopsis thaliana, or its mutants or similar enzymes with more than 50% identity; and/or
(b) the decarboxylase is phenylacrylic acid decarboxylase from Aspergillus niger, or its mutants or similar enzymes with more than 50% identity; and/or
(a) the epoxidase is a styrene monooxygenase, optionally wherein the styrene monooxygenase is from Pseudomonas sp. VLB120, or its mutants or similar enzymes with more than 50% identity; and/or
(b) the isomerase is a styrene oxide isomerase, optionally wherein the styrene oxide isomerase is from Pseudomonas sp. VLB 120, or its mutants or similar enzymes with more than 50% identity; and/or
(c) the oxidase or aldehyde dehydrogenase is aldehyde dehydrogenase (EcALDH).
31 . The method according to any one of Claims 28 to 30, wherein the method is conducted in one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes (i.e. the one or more recombinant microbial cells genetically engineered to overexpress multiple enzymes), where the recombinant microbial cell is E. coli pACYCDuet- PAL-PAD, pRSFDuet-StyABC-EcALDH.
32. The method according to any one of Claims 28 to 31 , wherein the method further comprises cells that overexpress the natural L-phenylalanine biosynthetic pathway, which cells convert glucose or glycerol to L-phenylalanine.
33. The method according to Claim 32 wherein the microbial cells producing L- phenylalanine from glucose or glycerol overexpress at least one enzyme is selected from one or more of the group consisting of DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), or their mutants or similar enzymes with more than 50% identity, optionally wherein the microbial cells producing L-phenylalanine from glucose or glycerol overexpress at least one enzyme is:
(a) a feedback inhibition resistant mutant of AroG;
(b) a feedback inhibition resistant mutant of PheA.
34. The method according to Claim 33, wherein the microbial cells producing L- phenylalanine from glucose or glycerol that overexpress at least one enzyme is one in which the microbial cells are mutated for deletion or inactivation of err and/or tyrA genes.
35. The method according to any one of Claims 28 to 34, wherein the one-pot reaction system comprises an aqueous medium.
36. The method according to any one of Claims 28 to 34, wherein the one-pot reaction system comprises use of a bi-phasic medium, optionally wherein the bi-phasic medium consists of an aqueous and solid resin medium, or an aqueous and organic solvent medium (e.g. the organic solvent medium is selected from one or more alkanes solvents and/or one or more ester solvents).
37. The method according to any one of Claims 28 to 36, wherein the one-pot reaction system comprises use of absorbent resins or nanomaterials for in-situ removal of product.
38. An isolated nucleic acid molecule encoding at least one heterologous catalytic enzyme selected from the group comprising:
(a) an epoxidase for conducting an epoxidation reaction to form styrene oxide or a derivative thereof from styrene or a derivative thereof;
(b) an isomerase for generating phenylacetaldehyde or a derivative thereof from styrene oxide by an isomerization reaction; and
(c) a reductase for generating 2-phenethylethanol or a derivative thereof by a reduction reaction from phenylacetaldehyde or a derivative thereof;
(d) an ammonia lyase for generating trans-cinnamic acid or a derivative thereof from L-phenylalanine or a derivative thereof by a deamination reaction; (e) a decarboxylase for generating styrene or a derivative thereof from trans- cinnamic acid or a derivative thereof in a decarboxylation reaction; and
(f) an oxidase or an aldehyde dehydrogenase for generating phenylacetic acid or a derivative thereof from phenylacetaldehyde by an oxidation reaction.
39. The isolated nucleic acid of Claim 38, encoding a plurality of said catalytic enzymes.
40. The isolated nucleic acid molecule of Claim 39, wherein said plurality of catalytic enzymes is arranged as at least one module selected from the group comprising: i) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms styrene or a derivative thereof to phenylacetaldehyde or a derivative thereof; ii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms phenylacetaldehyde or a derivative thereof to 2-phenylacetic acid or a derivative thereof; iii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms an L-phenylalanine or a derivative thereof to styrene or a derivative thereof, or any combination thereof.
41 . An expression construct comprising at least one nucleic acid molecule as described in any one of Claims 38 to 40.
42. One or more recombinant prokaryotic or eukaryotic cells selected from the group comprising bacterial cells, yeast cells, mammalian cells and insect cells, wherein said cells comprise at least one expression construct as described in Claim 41 .
43. The one or more recombinant prokaryotic or eukaryotic cells according to Claim 42, wherein said cells are recombinant bacterial cells.
44. A kit comprising at least one isolated nucleic acid according to any one of Claims 38 to 40.
45. The method according to any one of Claims 28 to 37, wherein the at least five enzymes used in the method are provided:
(a) in whole cells genetically engineered to overexpress the at least five enzymes, optionally wherein the at least five overexpressed enzymes are located on one or more plasmids or integrated in the chromosome of each of the one or more recombinant microbial cells;
(b) in a cell-free extract;
(c) as purified enzymes; or
(d) as immobilized enzymes.
PCT/SG2023/050036 2022-01-19 2023-01-19 Bioproduction of natural phenethyl acetate, phenylacetic acid, ethyl phenylacetate, and phenylethyl phenylacetate from renewable feedstock WO2023140793A2 (en)

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