Engineered limonene synthase and use thereof for limonene production

Abstract

The invention relates to improved limonene synthases for biosynthetic production of limonene. engineered limonene synthases with improved characteristics compared to reference limonene synthases are provided. Some engineered limonene synthases have improved limonene production yield. Other engineered limonene synthases have switched enantioselectivity. Still other engineered limonene synthases have improved yield with specific substrates. Some engineered limonene synthases have improvements in a combination of one or more of limonene production yield, enantioselectivity and substrate specificity. Also provided herein is a method of screening limonene synthase, e.g., for characterization of limonene synthases. Further provided is a method of producing limonene using the engineered limonene synthases provided herein.

Description

ENGINEERED LIMONENE SYNTHASE AND USE THEREOF FOR LIMONENE PRODUCTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore application No. 10202402813 S, filed 10 September 2024, the contents of it being hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The invention is in the field of biotechnology. The invention relates to improved enzymes for producing limonene, including improved limonene synthase enzymes and methods of screening for such improved enzymes.BACKGROUND OF THE INVENTION

[0003] Limonene is a chiral, high-demand and high-value monoterpene that has wide applications in the flavors, fragrances, therapeutics, cosmetics, biofuels, agri-food, biomaterials, and solvent industries. Currently, extraction from citrus rinds which are byproducts of juice industries, is the primary source of limonene production. Citrus fruits are produced in abundance, however, maj or citrus fruit producers, especially in Asia, are interested in the whole fruits market compared to the juice market; for example, 95% of total orange production in China is consumed as whole fruits. Challenges in citrus rind recycling limit limonene production and developing countries' citrus juice industries lack infrastructure for onsite recycling. Additionally, seasonal fluctuations, and diseases like Huanglongbing strongly affect the supply and quality of fruits for limonene extraction. Further, part of the limonene produced from citrus fruits is not food-grade as it contains traces of pesticides used in agricultural fields, limiting its application in household and food products. The total chemical synthesis of limonene has been explored. However, this requires harsh conditions like high temperatures and pressures, making it energy intensive and produces environmentally damaging toxic waste products, restricting large-scale production. In terms of bioproduction, bacteria, yeast and fungi have been successfully used as microbial limonene production hosts. However, limonene biosynthesis in microbes is often limited by the poor activity of limonene synthases (LS). The best yields achieved in microbial biosynthesis of limonene to-date range between about 1.35 g / L to about 3.6 g / L of (-)-limonene using Mentha spicata limonene synthase expressed in recombinant E. coli. There is therefore a need for improved limonenesynthases for boosting limonene production which can improve the commercial viability of the flavor and fragrance industries.SUMMARY

[0004] In one aspect, provided herein is an engineered limonene synthase with increased limonene production relative to a reference limonene synthase, wherein the engineered limonene synthase comprises one or more mutations relative to the reference limonene synthase.

[0005] In another aspect, provided herein is an engineered limonene synthase with switched enantioselectivity relative to a reference limonene synthase, wherein the engineered limonene synthase comprises one or more mutations relative to the reference limonene synthase.

[0006] In another aspect, provided herein is a polypeptide sequence encoding the engineered limonene synthase as described herein.

[0007] In another aspect, provided herein is a polynucleotide sequence encoding the polypeptide sequence as described herein.

[0008] In another aspect, provided herein is a vector comprising the polynucleotide sequence as described herein.

[0009] In another aspect, provided herein is a host cell comprising the vector as described herein.

[0010] In another aspect, provided herein is a method of determining a characteristic of a limonene synthase comprising the steps of:(a) expressing in a host cell:(i). a first expression vector comprising the genes encoding the enzymes of the mevalonate pathway and a geranyl pyrophosphate synthase (GPPS) gene or a neryl pyrophosphate synthase (NPPS) gene; and(ii). a second expression vector encoding a limonene synthase gene, wherein the genes encoding the enzymes of the mevalonate pathway are acetyl- CoA acetyltransferase (atoB), 3 -hydroxy-3 -methylglutaryl-CoA synthase HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase PMK), mevalonate diphosphate decarboxylase (PMD) and isopentenyl-diphosphate isomerase (ztZz);(b) culturing the host cell to produce limonene; and(c) analyzing the limonene product to determine the characteristic of the limonene synthase.

[0011] In another aspect, provided herein is a method of producing limonene comprising the steps of:(a) expressing in a host cell:(i). genes encoding the enzymes of the mevalonate pathway;(ii). a gene encoding a geranyl pyrophosphate synthase (GPPS) or a gene encoding a neryl pyrophosphate synthase (NPPS); and(iii). a polynucleotide sequence encoding an engineered limonene synthase as described herein;(b) culturing the host cell in a culture medium under conditions suitable for producing limonene; and(c) extracting the limonene from the culture medium.DEFINITIONS

[0012] As used herein, “equivalent” in the context of amino acid positions refers to an amino acid position in one amino acid sequence that corresponds to an equivalent position in another amino acid sequence. For example, if the first 10 amino acid residues of an amino acid sequence consisting of 100 residues are removed, then the last amino acid in the resultant amino acid sequence, i.e., 90th amino acid, will be equivalent to the 100th amino acid in the original sequence. Thus, the position number 90 in the resultant amino acid sequence is said to be “equivalent” to position number 100 in the original amino acid sequence. Those skilled in the art will understand how to determine equivalent positions between any two or more amino acid sequences. For example, two or more amino acid sequences may be aligned in order to determine the equivalent positions among the two or more sequences. Methods of sequence alignment are well understood by those skilled in the art, and tools are readily accessible on the Internet, for example, the Clustal Omega sequence alignment tool.

[0013] As used herein, “enantioselectivity” in the context of an enzyme such as limonene synthase, refers to the preferential formation of one enantiomer of a compound, such as limonene, over another. For example, an enzyme that is enantioselective for a (+) enantiomer is one that preferentially produces the (+) enantiomer, and vice versa for the (-) enantiomer. In cases where an enzyme exclusively or nearly exclusively produces only one enantiomer, that enzyme is considered as “enantiospecific” for that enantiomer.

[0014] As used herein, “switched enantioselectivity” refers to a change in the preferential formation of one enantiomer of a compound over another. For example, a limonene synthase that originally produces (+) limonene may produce (-) limonene when its enantioselectivity isswitched. A limonene synthase may produce one enantiomer exclusively, for example only (+) limonene or only (-) limonene, or it may produce a mixture of both enantiomers, for example, a mixture of both (+) and (-) limonene in varying proportions. A switch in enantioselectivity can mean that a limonene synthase originally producing predominantly (+) limonene, switches to produce predominantly (-) limonene, or vice versa. A switch in enantioselectivity can also mean that a limonene synthase originally producing a mixture of (+) and (-) limonene in a certain proportion, switches to produce a mixture of (+) and (-) limonene in a different proportion, or switches to produce either (+) or (-) limonene exclusively. A switch in enantioselectivity can also mean that a limonene synthase originally producing either (+) or (- ) limonene exclusively, switches to produce a mixture of (+) and (-) limonene.

[0015] As used herein, the term "about", in the context of concentrations of components of the formulations, optical density, time period, typically means + / - 10% of the stated value.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0017] FIG. 1 is a schematic diagram of the limonene biosynthetic pathway, beginning with the conversion of acetyl-CoA into mevalonate (mevalonate or MVA pathway), which is then converted to isopentenyl pyrophosphate (1PP) and dimethylallyl pyrophosphate (DMAPP), which are terpene precursors. IPP and DMAPP are condensed to geranyl pyrophosphate (GPP) via geranyl pyrophosphate synthase (GPPS) or to neryl pyrophosphate (NPP) via neryl pyrophosphate synthase (NPPS). GPP and NPP in turn are the direct substrates of limonene synthase. AtoB, acetyl-CoA acetyltransferase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, HMG-CoA reductase; MK, mevalonate kinase; PMK, phosphomevalonate kinase; PMD, mevalonate diphosphate decarboxylase; idi, isopentenyl -diphosphate isomerase; LS, limonene synthase. The exemplary LS depicted in FIG. 1 is truncated Citrus sinensis limonene synthase (tCsLs, alternatively abbreviated as “tCITSI” throughout this disclosure).

[0018] FIG. 2 depicts the developed plasmid system for the expression of limonene from acetyl-CoA. This plasmid system can be used for screening limonene synthases. EcoCTs- CPMS6a and EcoCTs-CPMS6b are used in parallel to synthesize GPP or NPP from acetyl- CoA respectively. EcoCTs-CPMS5 is used to synthesize limonene from GPP or NPP. Piacuvs, mutated promoter from Escherichia coh lac operon; Puc, trc promoter, pl5A ori, pl5A origin of replication; pl5A, pl5A plasmid backbone; colEl, ColEl plasmid backbone. In the plasmidsystem depicted, the atoB and idi coding sequences are obtained from Escherichia coli, the HMGS and HMGR coding sequences are obtained from Staphylococcus aureus, the MK, PMK and PMD coding sequences are obtained from Saccharomyces cerevisiae, trGPPS is truncated GPPS from Abies grandis, trNPPS is truncated NPPS from Solatium lycopersicum, and LS represents limonene synthase from various organisms (i.e., limonene synthases to be screened).

[0019] FIG. 3 depicts the results of an analysis of 10 limonene synthase homologues from various organisms for limonene production (FIG. 3A and 3B) and for enantioselectivity (FIG. 3C and 3D).

[0020] FIG. 4 is a graphical representation of a sequence alignment between the N-terminal truncated A. rugosa limonene synthase (tAGARU) and N-terminal truncated N. tenuifolia limonene synthase (tNEPTE). The truncation is a deletion of deletion of residues 2 - 69 at the N-terminal of AGARU (SEQ ID NO: 12) and a deletion of residues 2 - 72 at the N-terminal of NEPTE (SEQ ID NO: 13). The alpha-helical regions with respect to tAGARU are indicated above the aligned sequence. With respect to tAGARU, the 4 residues (S8, 117, 1265, P530) and 1 loop (E276-I286, also referred to in this disclosure as “loopl”) identified to be involved in substrate specificity are indicated with black arrows and with a bracket, respectively.

[0021] FIG. 5 are charts showing the effects of various mutations on limonene production of various limonene synthases. “tArLS” refers to truncated A. rugosa limonene synthase and “tNtLS” refers to truncated N. tenuifolia limonene synthase, “loopl” refers to E276-I286. FIGs. 5A and 5B depict the specific production of various mutant tArLS compared to wildtype tArLS (leftmost bars in each of FIG. 5 A and 5B, “tArLS-WT”), using GPP or NPP as substrate, respectively. FIG. 5C depicts the effect of the “loop!” mutant tNtLS (right bars, “tNtLS-loopl”) compared to wild-type (left bars, “tNtLS”), as well as the effect of switching substrate from GPP to NPP. FIG. 5D depicts the ratio of ratio of limonene produced between the NPP pathway and the GPP pathway for the various tArLS mutants compared to wild-type (leftmost bar, “tArLS”), and essentially represents the ratio of production values in FIG. 5B to production values in FIG. 5A. FIG. 5E depicts the effect of the ispA S80F mutation on limonene production, using E. coli MG1655 as an example host cell. The amino acid residue positions in FIGs. 5A, 5B and 5D are indicated relative to truncated AGARU limonene synthase (tArLS) (SEQ ID NO: 12). The “loopl” in FIG. 5C refers to residues in truncated NEPTE limonene synthase (tNtLS) that correspond to E276-I286 in tArLS. As can be seen in the sequence alignment in FIG. 4, the corresponding tNtLS loopl mutations are P280E-R281P- K285A-T286N-Q289I-L290I.

[0022] FIG. 6 shows the kinetics analysis of purified N-tenninal truncated wild-type AGARU limonene synthase and N-terminal truncated wild-type NEPTE limonene synthase using the GPP and NPP pathways. V (pM / min) was obtained from the measurement of pyrophosphate production over time and plotted against the substrate concentration [S], The Michaelis-Menten kinetic equation was used to calculate KM and kcat of each enzyme.

[0023] FIG. 7 shows the improvements in (+)-limonene production of mutants generated via site-saturation mutagenesis (SSM) of N-terminal truncated C. sinensis limonene synthase compared to truncated wild-type CITSI (tCITSI). FIG. 7A is a three-dimensional representation of the tCITSI polypeptide structure, in complex with GPP (top) or NPP (bottom). The enzyme is represented as ribbons, whereas GPP / NPP are represented as sticks. The amino acid positions selected for SSM are shaded in a different colour than the ribbons and indicated with lines. FIG. 7B is a heatmap showing the yield improvement of tCITSI mutants compared to wild-type tCITSI after site-saturation mutagenesis on 12 selected sites. Culture was performed in deep well plates and analyzed by LC-MS. The vertical axis indicates the amino acid position of the mutation with respect to wild-type tCITSI selected for SSM, using GPP or NPP as the substrate. The horizontal axis indicates the amino acid substituted at each selected position. Lighter shading represents higher limonene yields. Amino acid substitutions marked with “X” indicate where the substitution is the same as the original residue in the wild-type tCITSI sequence, thus, not tested. FIG. 7C is the specific limonene yield of the best-performing mutants and their combinations, assessed via GC-MS.

[0024] FIG. 8 shows the enantioselectivity of tCITSI mutants. The limonene enantiomers produced were determined by a GC chiral column.

[0025] FIG. 9 shows the outcome of the optimization of shake flask conditions for enhanced (+)-limonene production. FIG. 9A is a schematic showing the two-plasmid system used for flask optimization of limonene production. In the plasmid system depicted, the atoii and idi coding sequences are obtained from Escherichia coli, the HMGS and HMGR coding sequences are obtained from Staphylococcus aureus, the MK, PMK and PMD coding sequences are obtained from Saccharomyces cerevisiae, trGPPS is truncated GPPS from Abies grandis, and tCsLS represents limonene synthase from Citrus sinensis. Glycerol is the carbon source for limonene production and (+)-limonene is produced. FIG. 9B is a chart showing the effect of shaking speed and amino acid (a.a.) supplementation on (+)-limonene production. FIG. 9C is a chart showing the effect of IPTG concentrations for inducing the pathway gene expression The floating squares in both charts represent the OD600 of the culture.

[0026] FIG. 10 shows the effect of various optimizations on limonene production. FIG. 10A is a chart showing the effect of the MG1655 ispA S80F, cultured in shake flasks and using auto-induction to induce limonene production. FIG. 10B is a chart showing the effect of different carbon sources on (+)-limonene yield. FIG. 10C is a chart showing the improvement of specific limonene production using the MG1655 ispA S80F host strain and the tCsLS-Q8K mutant in shake flasks with 200 pM PTG induction, comprising the pEcoCTS-CPMSNl and pEcoCTs-CPMS2(-Q8K) plasmids.

[0027] FIG. 11 is a chart showing the limonene production yield results of fed-batch production of (+)-limonene in a 5 L bioreactor using tCsLS-Q8K under optimized culture conditions.DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides improved enzymes for bioproduction of limonene. The improvement may be an improvement in the limonene production yield. The improvement may also be a switched enantioselectivity. The improvement may also be both an improvement in limonene production yield and a switched enantioselectivity

[0029] In one aspect, provided herein is a engineered limonene synthase with increased limonene production relative to a reference limonene synthase, wherein the engineered limonene synthase comprises one or more mutations relative to the reference limonene synthase.

[0030] The engineered limonene synthase can be obtained by mutation of a reference limonene synthase. Mutation of a reference limonene synthase refers to mutation of a reference limonene synthase sequence. A reference limonene synthase sequence may be a reference limonene synthase amino acid sequence or a reference limonene synthase nucleic acid sequence. A reference limonene synthase nucleic acid sequence may encode a reference limonene synthase polypeptide (i.e., amino acid sequence). Those skilled in the art will understand that mutation of a reference limonene synthase nucleic acid sequence may result in mutation of the polypeptide (amino acid sequence) encoded by the mutated reference nucleic acid sequence. An engineered limonene synthase obtained by mutation of a reference limonene synthase may also be referred to as a “mutant limonene synthase”.

[0031] Mutation of a reference limonene synthase can be performed by any one or more of several methods known in the art, such as via random mutagenesis or site-directed mutagenesis. A mutation of a reference limonene synthase may be an insertion, deletion, and / or substitution of one or more residues of the reference limonene synthase sequence. For example, mutationof a reference limonene synthase amino acid sequence may be an insertion, deletion, and / or substitution of one or more amino acid residues in the reference amino acid sequence. Mutation of a reference limonene synthase nucleic acid sequence may be an insertion, deletion, and / or substitution of one or more nucleic acid residues in the reference amino acid sequence. Those skilled in the art will understand that due to codon degeneracy, mutation of a reference nucleic sequence may or may not result in mutation of the polypeptide (amino acid sequence) encoded by the mutated nucleic acid sequence.

[0032] A reference limonene synthase sequence can be an amino acid sequence or a nucleic acid sequence obtained from a reference limonene synthase. An engineered limonene synthase enzyme can be obtained by mutating a reference limonene synthase amino acid sequence and / or by mutating a reference limonene synthase nucleic acid sequence. The reference limonene synthase can be a limonene synthase from any organism that possesses a limonene synthase. For example, the reference limonene synthase can be a Citrus sinensis limonene synthase, an Agastache rugosa limonene synthase, a Nepeta tenuifolia limonene synthase, a. Mentha spicata limonene synthase, an Abies grandis limonene synthase, a Picea sitchensis limonene synthase, a Cannabis sativa limonene synthase, a Lavandula angustifolia limonene synthase, a Coffee arabica limonene synthase, or a Perilla frutescens limonene synthase.

[0033] The reference limonene synthase can be any naturally occurring limonene synthases e.g., wild-type limonene synthases or it can be another engineered limonene synthase, e.g., a mutant limonene synthase. It will be understood by those skilled in the art that any limonene synthase amino acid sequence or nucleic acid sequence can be a reference limonene synthase sequence.

[0034] In one example, the engineered limonene synthase is a Citrus sinensis limonene synthase, an Agaslache rugosa limonene synthase, a Nepeta tenuifolia limonene synthase, a Mentha spicata limonene synthase, an Abies grandis limonene synthase, a Picea sitchensis limonene synthase, a Cannabis sativa limonene synthase, a Lavandula angustifolia limonene synthase, a Coffee arabica limonene synthase, or a Perilla frutescens limonene synthase.

[0035] Reference limonene synthase amino acid sequences and reference limonene synthase nucleic acid sequences may be obtained from a public repository of amino acid and / or nucleic acid sequences. Some examples of such repositories include NCBI GenBank and UniProtKB. Any limonene synthase amino acid or limonene synthase nucleic acid sequence may serve as a reference sequence for an engineered limonene synthase.

[0036] In one example, the reference limonene synthase is a wild-type Citrus sinensis limonene synthase (SEQ ID NO: 1), a wild-type Agastache rugosa limonene synthase (SEQID NO: 2), a wild-type Nepeta tenuifolia limonene synthase (SEQ ID NO: 3), a wild-type Mentha spicata limonene synthase (SEQ ID NO: 4), a wild-type Abies grandis limonene synthase (SEQ ID NO: 5), a wild-type Picea sitchensis limonene synthase (SEQ ID NO: 6), a wild-type Cannabis sativa limonene synthase (SEQ ID NO: 7), a wild-type Lavandula angustifolia limonene synthase (SEQ ID NO: 8), a wild-type Coffee arabica limonene synthase (SEQ ID NO: 9), or a wild-type Perilla frutescens limonene synthase (SEQ ID NO: 10).

[0037] In some examples, the reference limonene synthase is any limonene synthase enzyme with a known production of limonene. Production of limonene can be assessed using various methods, including but not limited to specific yield, i.e., weight of limonene produced per volume of the culture per optical density of the culture (e.g., mg / L / OD), weight of limonene produced per dry cell weight (e g , g / gDCW), rate of limonene production, namely weight of limonene produced per culture volume per elapsed time (e.g., mg / L / h), rate of utilization of substrate, rate of utilization of carbon source, etc. Methods of measuring production of limonene will be well-understood by those skilled in the art.

[0038] It will be understood by those skilled in the art that the term “wild-type” when used to refer to an enzyme, generally refers to an enzyme that occurs in nature. Thus, a “wild-type amino acid sequence” refers to the amino acid sequence of an enzyme that occurs in nature, and a “wild-type nucleic acid sequence” refers to the nucleic acid sequence that encodes the polypeptide (amino acid sequence) of a wild-type enzyme. Thus, for example, “wild-type Citrus sinensis limonene synthase” refers to any Citrus sinensis limonene synthase enzyme that occurs in nature, and a “wild-type Citrus sinensis limonene synthase sequence” refers to an amino acid sequence of a wild-type Citrus sinensis limonene synthase and / or a nucleic acid sequence encoding a wild-type Citrus sinensis limonene synthase polypeptide (amino acid sequence).

[0039] It will also be understood by those skilled in the art that the term “mutant” when used to refer to an enzyme, generally refers to an enzyme that comprises one or more mutations compared to a reference enzyme, for example, a reference wild type enzyme. Thus, for example, “mutant Agastache rugosa limonene synthase” refers to any Agastache rugosa limonene synthase that comprises one or more mutations compared to a reference Agastache rugosa limonene synthase, such as a wild type Agastache rugosa limonene synthase.

[0040] In one example, the engineered limonene synthase is a Citrus sinensis limonene synthase comprising a substitution at one or more amino acid positions equivalent to Q59, Q576, 1451, N307, E322, L449, 1450, S454, T578, 1336, or combinations thereof of a wildtype Citrus sinensis limonene synthase (SEQ ID NO: 1). In some examples, the substitution atone or more amino acid positions equivalent to Q59, 1451, Q576, N307, E322, L449, 1450, S454, T578, 1336, or combinations thereof is Q59A, Q59E, Q59K, Q59L, Q59N, Q59S, Q59P, Q59T, I451A, I451C, I451F, I451L, I451M, I451S, I451T, I451V, I451W, I451Y, Q576A, Q576D, Q576G, Q576I, Q576L, Q576M, Q576N, Q576P, Q576S, Q576T, Q576Y, N307D, E322R, L449A, L449C, I450A, S454C, T578V, I336V, or combinations thereof.

[0041] In one example, the substitution is at amino acid positions equivalent to Q59 and 1451. In some examples, the substitution at amino acid positions equivalent to Q59 and 1451 is selected from the group consisting of: (i) Q59K and I451A; (ii) Q59K and I451T; (iii) Q59A and 1451 A; and (iv) Q59A and 145 IT.

[0042] In other examples, the substitution at one or more amino acid positions equivalent to Q59, 1451, Q576, N307, E322, L449, 1450, S454, T578, 1353, 1336, or combinations thereof, is any amino acid substitution at one or more of said amino acid positions or combinations thereof that increases the limonene production yield of the mutant Citrus sinensis limonene synthase compared to a wild-type Citrus sinensis limonene synthase (SEQ ID NO: 1).

[0043] In another example, the engineered limonene synthase is a Agastache rugosa limonene synthase comprising a substitution at one or more amino acid positions equivalent to S76, 1333, E344, P345, A349, N350, 1353, 1354, 185, Q380, E383, P598, or combinations thereof of a wild-type Agastache rugosa limonene synthase (SEQ ID NO: 2). In some examples, the substitution at one or more amino acid positions equivalent to S76, 1333, E344, P345, A349, N350, 1353, 1354, 185, Q380, E383, P598, or combinations thereof is S76K, I333V, E344P, P345R, A349K, N350T, I353Q, I354L, I85M, Q380L, E383D, P598A, or combinations thereof.

[0044] In one example, the substitution is at amino acid positions equivalent to: (i) E344, P345, A349, N350, 1353, and 1354, (ii) S76, E344, P345, A349, N350, 1353 and 1354; (iii) S76, 1333, E344, P345, A349, N350, 1353 and 1354; (iv) 185 and 1333; or (v) Q380 and E383. In some examples, the substitution at amino acid positions equivalent to (i) - (v) is selected from the group consisting of: E344P, P345R, A349K, N350T, I353Q, and I354L; S76K, E344P, P345R, A349K, N350T, I353Q, and I354L; S76K, I333V, E344P, P345R, A349K, N350T, I353Q, and I354L; I85M and I333V; and Q380L and E383D.

[0045] In other examples, the substitution at one or more amino acid positions equivalent to S76, 1333, E344, P345, A349, N350, 1353, 1354, 185, Q380, E383, P598, or combinations thereof, is any amino acid substitution at one or more of said amino acid positions or combinations thereof that increases the limonene production yield of the mutant Agastacherugosa limonene synthase compared to a wild-type Agastache rugosa limonene synthase (SEQ ID NO: 2).

[0046] In another example, the engineered limonene synthase is a Nepeta tenuifolia limonene synthase comprising a substitution at one or more amino acid positions equivalent to A605, K79, M88, V340, L387, D390, or combinations thereof of a wild-type Nepeta tenuifolia limonene synthase (SEQ ID NO: 3). In some examples, the substitution at one or more amino acid positions equivalent to A605, K79, M88, V340, L387, D390, or combinations thereof of a wild-type Nepeta tenuifolia is A605P, K79S, M88I, V340I, L387Q, D390E, or combinations thereof.

[0047] In one example, the substitution is at amino acid positions equivalent to: (i) L387 and D390; or (ii) M88 and V340 In some examples, the substitution at amino acid positions equivalent to (i) or (ii) is selected from the group consisting of: (i) L387Q and D390E, and (ii) M88I and V340I.

[0048] In other examples, the substitution at one or more amino acid positions equivalent to K79, M88, V340, A605, L387, D390, or combinations thereof, is any amino acid substitution at one or more of said amino acid positions or combinations thereof that increases the limonene production yield of the mutant Nepeta tenuifolia limonene synthase compared to a wild-type Nepeta tenuifolia limonene synthase (SEQ ID NO: 3)

[0049] In another aspect, provided herein is a engineered limonene synthase with switched enantioselectivity relative to a reference limonene synthase, wherein the engineered limonene synthase comprises one or more mutations relative to the reference limonene synthase.

[0050] Limonene synthase enzymes from different organisms may have different enantioselectivity in producing each of the (+) or (-) enantiomers of limonene. Any one limonene synthase may have an enantioselectivity that is specific to that limonene synthase. A limonene synthase may produce one or the other enantiomer exclusively (i.e., it is enantiospecific for a particular enantiomer), or it may produce a mixture of (+) and (-) enantiomers in a particular proportion. The enantioselectivity of any given limonene synthase may be different compared to a different limonene synthase. Thus, a suitable limonene synthase for limonene production would be selected based on the desired production of the (+) and / or (- ) enantiomer. However, due to differences in limonene production yield between different limonene synthases, it may be desirable to switch the enantioselectivity of a higher-producing limonene synthase to obtain a engineered limonene synthase with higher production yield of a limonene enantiomer compared to another limonene synthase that produces the same enantiomer. While in some cases an increase in limonene production yield of a limonenesynthase is desirable for the bioproduction of limonene, in other cases the enantioselectivity of the limonene synthase may be more important. Thus, the present invention also relates to an engineered limonene synthase with switchable enantioselectivity for producing limonene enantiomers as desired.

[0051] In some examples, the switch in enantioselectivity is a complete switch. In other examples, the switch in enantioselectivity is a partial switch.

[0052] A complete switch in enantioselectivity may refer to when a limonene synthase switches from producing only one enantiomer to producing only another enantiomer, for example, a switch from 100% (+)-limoneneto 100% (-)-limonene. A complete switch may also refer to when a limonene synthase switches from producing a mixture of enantiomers to producing only one enantiomer, for example, a switch from 50% (+)-limonene and 50% (-)- limonene to 100% (+)-limonene or 100% (-)-limonene. Generally, the result of a “complete switch” is that the engineered limonene synthase produces only one enantiomer compared to before the switch.

[0053] A partial switch may refer to when a limonene synthase switches from producing a certain proportion of enantiomers to another proportion of enantiomers, for example from 80% (+)-limonene and 20% (-)-limonene to 50% (+)-limonene and 50% (-)-limonene. A partial switch may refer to when a limonene synthase switches from producing only one enantiomer to producing a mixture of enantiomers, for example a switch from 100% (+)-limonene to 50% (+)-limonene and 50% (-)-limonene or a switch from 100% (-)-limonene to 40% (-)-limonene and 60% (+)-limonene, etc. It would be apparent to those skilled in the art that a production of “100%” of one enantiomer can also be represented as a proportion of “100% / 0%” of two enantiomers. For example, a limonene synthase producing 100% (+)-limonene may also be said to produce a proportion of 100% (+)-limonene / 0% (-)-limonene. Generally, the result of a “partial switch” is that the engineered limonene synthase produces a mixture of enantiomers in a different proportion compared to before the switch. It would generally be understood that the above examples are for illustration and other percentages and ratios may be acceptable.

[0054] In one example, the engineered limonene synthase with switched enantioselectivity is a Citrus sinensis limonene synthase, a Agastache rugosa limonene synthase, a Nepeta tenuifolia limonene synthase, a Mentha spicata limonene synthase, a Abies grandis limonene synthase, a Picea sitchensis, limonene synthase a Cannabis sativa limonene synthase, a Lavandula angustifolia limonene synthase, a Coffee arabica limonene synthase, or a Perilla jrutescens limonene synthase. In some examples, the reference limonene synthase is a wildtype limonene synthase from the same organism.

[0055] In one example, the engineered limonene synthase is a Citrus sinensis limonene synthase comprising a substitution at one or more amino acid positions equivalent to S312, 1336, T446, 1450, F484 and combinations thereof of a wild-type Citrus sinensis limonene synthase (SEQ ID NO: 1). In some examples, the substitution at one or more amino acid positions equivalent to S312, 1336, T446, 1450, F484 and combinations thereof is S312C, I336N, T446S, I450M and F484L.

[0056] In one example, the substitution is at amino acid positions equivalent to: (i) S312 and 1336; (ii) S312 and 1450; (iii) 1336 and 1450; (iv) 1450 and T446; (v) S312, 1336 and 1450; (vi) S312, 1450 and T446; (vii) 1336, 1450 and T446; (viii) 1336, 1450 and F484; (ix) 1450, T446 and F484; (x) S312, 1450, T446 and F484; (xi) 1336, 1450, T446 and F484; or (xii) 1336, 1450, T446, F484 and S312. In some examples, the substitution at amino acid positions equivalent to (i) - (xii) is selected from the group consisting of: (i) S312C and I336N; (ii) S312C and I450M; (iii) I336N and I450M; (iv) T446S and I450M; (v) S312C, I336N and I450M; (vi) S312C, T446S, and I450M; (vii) I336N, T446S, and I450M; (viii) I336N, I450M and F484L; (ix) T446S, I450M and F484L; (x) S312C, T446S, I450M and F484L; (xi) I336N, T446S, I450M and F484L; or (xii) S312C, I336N, T446S, I450M and F484L.

[0057] There are various suitable substrates for limonene production by limonene synthase. Generally, either geranyl pyrophosphate (GPP) and / or neryl pyrophosphate (NPP) are suitable substrates for limonene synthases. The choice of substrate can have an effect on the limonene production yield. Some limonene synthases have higher production yield when using GPP as the substrate while other limonene synthases have higher production yield when using NPP as the substrate. Still other limonene synthases have comparable production yield regardless of whether GPP or NPP is used as the substrate.

[0058] In one example, limonene yield is increased when geranyl pyrophosphate (GPP) is the substrate for the engineered limonene synthase. In other examples, limonene yield is increased when neryl pyrophosphate (NPP) is the substrate for the engineered limonene synthase. In yet other examples, limonene yield is increased when GPP or NPP is the substrate for the limonene synthase.

[0059] It would be understood by those skilled in the art that the increased limonene yield of a engineered limonene synthase provided herein compared to its reference limonene synthase may depend on the substrate. For example, an engineered limonene synthase provided herein may have increased limonene yield compared to its reference limonene synthase when GPP is the substrate for the engineered limonene synthase; the limonene yield of said engineered limonene synthase may or may not also be increased compared to said referencelimonene synthase when NPP is the substrate. An engineered limonene synthase provided herein may have increased limonene yield compared to its reference limonene synthase when NPP is the substrate for the engineered limonene synthase; the limonene yield of said engineered limonene synthase may or may not also be increased compared to said reference limonene synthase when GPP is the substrate. An engineered limonene synthase provided herein may also have increased limonene yield compared to its reference limonene synthase when either GPP or NPP is the substrate.

[0060] In one example, the mutant enzyme is truncated at the N-terminal or the C-terminal relative to a wild-type limonene synthase.

[0061] Truncation of the engineered limonene synthase may confer additional desirable properties to the engineered limonene synthase. For example, truncation of a peptide signaling sequence may improve the limonene yield of the engineered limonene synthase when the engineered limonene synthase is expressed in a host cell for producing limonene. Examples of signal sequences include but are not limited to plastid targeting sequences and endoplasmic reticulum trafficking sequences.

[0062] Those skilled in the art will understand how to identify signaling sequences in a limonene synthase enzyme, for example, by performing a sequence alignment of a limonene synthase sequence (amino acid or nucleic acid sequence) against known signaling sequences (amino acid or nucleic acid sequences) and looking for sequences in the limonene synthase sequence that align with high identity against the known signaling sequence. Tools such as signalP can also be used to detect signal peptides in a given amino acid sequence. It will be understood by those skilled in the art that signaling sequences may be located at the N-terminal of a full-length polypeptide (enzyme).

[0063] In one example, the engineered limonene synthase enzyme is: (a) a truncated Citrus sinensis limonene synthase comprising a deletion of residues 2 - 52 at the N-terminal; (b) a truncated Agastache rugosa limonene synthase comprising a deletion of residues 2 - 69 at the N-terminal; (c) a truncated Nepeta tenuifolia limonene synthase comprising a deletion of residues 2 - 72 at the N-terminal; (d) a truncated Citrus sinensis, Agastache rugosa, or Nepeta tenuifolia limonene synthase comprising a deletion of the amino acids upstream of the position equivalent to R58 in a wild-type Mentha spicata limonene synthase except for the first methionine residue at the start of the N-terminal polypeptide sequence; or (e) a truncated Citrus sinensis, Agastache rugosa, or Nepeta tenuifolia limonene synthase comprising a deletion of the N-terminal plastid targeting sequence except for the first methionine residue at the start of the N-terminal polypeptide sequence.

[0064] The amino acid positions in the engineered limonene synthases described herein may take into account whether the mutant is a full-length mutant or a truncated mutant. Unless otherwise specified, the amino acid positions in the limonene synthases described herein refer to the positions with respect to the full-length, untruncated limonene synthase. Corresponding amino acid positions in a truncated limonene synthase can be determined by simply subtracting the number deleted (truncated) amino acids from the position with respect to the full-length limonene synthase. For example, the amino acid position Q58 in a full-length Citrus sinensis limonene synthase corresponds to amino acid position Q8 in a Citrus sinensis limonene synthase with a deletion (truncation) of residues 2-52 (50 amino acids) at the N-terminal (i.e., a subtraction of 50 amino acids upstream from position 58 in the full-length sequence).

[0065] Tn another aspect, provided herein is a polypeptide sequence encoding the engineered limonene synthase as described herein.

[0066] It will be understood by those skilled in the art that an enzyme (polypeptide), e g., the engineered limonene synthase described herein, may be represented as a polypeptide sequence, i.e., an amino acid sequence.

[0067] In another aspect, provided herein is a polynucleotide sequence encoding the polypeptide sequence as described herein.

[0068] In another aspect, provided herein is a vector comprising the polynucleotide sequence described herein.

[0069] In another aspect, provided herein is a host cell comprising the vector as described herein.

[0070] The host cell may be any cell capable of expressing the engineered limonene synthase polypeptide described herein. Some examples of host cells capable of expressing the polypeptides described herein include but are not limited to eukaryotic host cells such as animal cells, yeast cells and fungal cells, and prokaryotic host cells such as bacterial cells.

[0071] In one example, the host cell is a bacterial cell.

[0072] Suitable bacterial cells for expressing the engineered limonene synthase polypeptide described herein include but are not limited to Escherichia coli (E. coh). Examples of suitable E. coli cells include but are not limited to E. coli MG1655, E. coli BL21, E. coli K12 and any strains suitable for industrial bioproduction such as E. coli BL21(DE3), RV308(DE3), and HMS174(DE3).

[0073] In one example, the bacterial cell is an Escherichia coli cell

[0074] It will be understood by those skilled in the art that a polypeptide may be obtained by expressing the polypeptide in a host cell capable of expressing the polypeptide. Apolynucleotide sequence encoding said polypeptide may be introduced into a host cell to express the polypeptide in the host cell. Some examples of methods for introducing the polynucleotide encoding the polypeptide include but are not limited to transformation, transfection, transduction, and electroporation Suitable host cells for expressing polypeptides and methods for introducing polynucleotides encoding polypeptides into such host cells are discussed in detail elsewhere, for example vnMolecular cloning: a laboratory manual, Michael R. Green and Joseph Sambrook; Cold Spring Harbor Laboratory Press, 2012.

[0075] In another aspect, provided herein is a method of determining a characteristic of a limonene synthase comprising the steps of:(a) expressing in a host cell:(i). a first expression vector comprising the genes encoding the enzymes of the mevalonate pathway and a geranyl pyrophosphate synthase (GPPS) gene or a neryl pyrophosphate synthase (NPPS) gene; and(ii). a second expression vector encoding a limonene synthase gene, wherein the genes encoding the enzymes of the mevalonate pathway are acetyl- CoA acetyltransferase (atoB), 3 -hydroxy-3 -methylglutaryl-CoA synthase (HMGS), HMG-CoA reductase (HMGR}, mevalonate kinase (MK), phosphomevalonate kinase PMK), mevalonate diphosphate decarboxylase (PMD) and isopentenyl-diphosphate isomerase (A / / )(b) culturing the host cell to produce limonene; and(c) analyzing the limonene product to determine the characteristic of the limonene synthase.

[0076] The genes on the first and / or second expression vector may be under operable control of a promoter. In some examples, the genes of the mevalonate pathway and the GPPS or NPPS gene are under operable control of a promoter and are located in a single operon. In other examples, the genes of the mevalonate pathway and the GPPS or NPPS gene are under operable control of a promoter and are located in separate operons. When the genes of the mevalonate pathway and the GPPS or NPPS gene are located in separate operons, the promoter in each operon may be the same promoter (i.e., the promoters have the same nucleic acid sequence) or a different promoter (i.e., the promoters have different nucleic acid sequences). Each operon can also be located on the same or different vector.

[0077] The genes of the mevalonate pathway enzymes may be obtained from any organism that possesses mevalonate pathway enzymes. Examples of such organisms include but are notlimited to Escherichia coli, Staphylococcus aureus, Streptomyces sp. and Saccharomyces cerevisiae.

[0078] Limonene synthases may utilize either geranyl pyrophosphate (GPP) or neryl pyrophosphate as substrate for limonene production. Limonene production in E. coli through the expression of a heterologous mevalonate (MVA) pathway is a viable strategy for biosynthetic production of limonene. The MVA pathway ends with the production of isoprenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In the GPP route of limonene production, GPPS condenses IPP and DMAPP to GPP and in the NPP route, NPPS condenses IPP and DMAPP to NPP. GPP is the canonical substrate of limonene synthase; however, the IPP, DMAPP and subsequently GPP synthesized can be taken up by the E. coli host cell’s native enzymes causing flux loss and toxicity to the host cell. Thus, it may be desirable to obtain limonene synthase with substrate specificity for NPP instead of GPP for improved limonene production.

[0079] In one example, the genes encoding the enzymes of the mevalonate pathway and the GPPS or NPPS gene are in separate operons in the first expression vector. In a further example, the operon containing the GPPS or NPPS gene is located between the origin of replication and the antibiotic resistance marker in the first expression vector.

[0080] The arrangement of operons containing the biosynthetic pathway genes for limonene production can be advantageous when screening for engineered limonene synthases. For example, when the operons are on separate vectors but under control of the same promoter, plasmid recombination events during host cell replication may result in plasmid instability leading to impaired limonene synthase expression and / or limonene production yield. However, the resultant host cell with impaired limonene synthase expression and / or limonene production yield would still be viable; it would not be possible to exclude these host cells from further screening based on a simple viability check, leading to inefficiencies in the screening process. In contrast, when the operons are located on separate vectors, and arranged in such a way that if a plasmid recombination event happens, the antibiotic resistance is lost, host cells with impaired limonene synthase expression and / or limonene production yield can be excluded from further screening as they would no longer be viable.

[0081] In one example, the characteristic of the limonene synthase is limonene production, enantioselectivity, substrate specificity or combinations thereof.

[0082] As screening of limonene synthases may require considerable time and effort, the present invention also relates to an improved method for screening limonene synthases to identify limonene synthases with improved and / or desirable characteristics.

[0083] The limonene synthase screening method described herein can be used to screen for limonene synthases with any desired characteristics for limonene production. For example, the screening can be for improved production, (+) or (-) limonene production, or specificity for GPP or NPP, compared to a reference limonene synthase The screening can also be for limonene production yield and enantioselectivity, e.g. for improved production of (-) limonene compared to a reference limonene synthase. The screening can also be for limonene production yield and specificity for GPP as a substrate or NPP as a substrate, e.g. improved production with substrate specificity for NPP compared to a reference limonene synthase. The screening can also be for limonene production yield, enantioselectivity and substrate specificity compared to a reference limonene synthase e.g., for improved production of (+) limonene with substrate specificity for GPP compared to a reference limonene synthase

[0084] In one example, the limonene synthase is a wild-type limonene synthase or an engineered limonene synthase.

[0085] Any limonene synthase, whether wild-type or engineered, can be screened using the screening method described herein. For example, an engineered limonene synthase can be screened for limonene production compared to a wild-type limonene synthase In another example, a wild-type limonene synthase from one organism can be screened for limonene production compared to a wild-type limonene synthase from a different organism. In another example, more than one engineered limonene synthases can be screened for limonene production or for enantioselectivity compared to a wild-type limonene synthase or compared to an engineered limonene synthase. In another example, more than one engineered limonene synthases can be screened for limonene production and enantioselectivity compared to a wildtype limonene synthase or compared to an engineered limonene synthase. In another example the substrate specificity of more than one limonene synthase, including wild-type and engineered limonene synthases, can be screened.

[0086] In one example, the host cell is an Escherichia coli cell.

[0087] It will be understood by those skilled in the art that in addition to an Escherichia coli host cell, any host cell that is capable of expressing the limonene synthase polypeptide can be used in the limonene synthase screening method described herein. Some examples of suitable E. coli cells include but are not limited to E. coli MG1655, E. coli BL21, E. coli K12 and any strains suitable for industrial bioproduction such as E. coli BL21(DE3), RV308(DE3), and HMS174(DE3).

[0088] In another aspect, provider herein is a method of producing limonene comprising the steps of:(a) expressing in a host cell:(i) genes encoding the enzymes of the mevalonate pathway;(ii). a gene encoding a geranyl pyrophosphate synthase (GPPS) or a gene encoding a neryl pyrophosphate synthase (NPPS); and(iii). a polynucleotide sequence encoding an engineered limonene synthase as described herein;(b) culturing the host cell in a culture medium under conditions suitable for producing limonene; and(c) extracting the limonene from the culture medium.

[0089] In one example, the genes encoding the enzymes of the mevalonate pathway are acetyl-CoA acetyltransferase (atoB 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), mevalonate diphosphate decarboxylase (PMD) and isopentenyl-diphosphate isomerase (idi).

[0090] The genes encoding the enzymes of the mevalonate pathway may be obtained from any organism that possesses a mevalonate pathway, including but not limited to Escherichia coli (E. coli), Saccharomyces cerevisiae ( . cerevisiae), and Staphylococcus aureus (S. aureus).

[0091] The gene coding a GPPS or the gene coding a NPPS may be a wild type GPPS or NPPS gene or a truncated GPPS (trGPPS) or truncated NPPS (trNPPS) gene.

[0092] In one example, the gene coding a GPPS or the gene coding a NPPS are a truncated GPPS (trGPPS) or truncated NPPS (trNPPS), respectively. In some examples, the trGPPS or trNPPS comprises the sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 22, respectively.

[0093] The genes encoding the mevalonate pathway enzymes, genes encoding the GPPS or NPPS, and polynucleotide sequence encoding an engineered limonene synthase as described herein may be codon optimized for expression in the host cell.

[0094] It will be understood by those skilled in the art that codon optimization may be performed when expressing a heterologous gene in a host cell so as to improve expression of said heterologous gene in the host cell. For example, if the host cell is an E. coli host cell and the gene to be expressed in the host cell is a S. cerevisiae gene, the S. cerevisiae gene would be heterologous to the E. coli host cell. Codon optimization of the S. cerevisiae gene for expression in the E. coli host cell may be performed so as to improve expression of said . cerevisiae gene in the E. coli host cell. The principles and methods of codon optimization are well understood by those skilled in the art, and tools for performing codon optimization are readily available on the Internet. Codon optimization can be performed for expression of any heterologous gene in a host cell. For example, genes encoding mevalonate pathway enzymes,genes encoding a GPPS or a NPPS, and polynucleotide sequences encoding an engineered limonene synthase as described herein, may all be codon optimized for expression in any host cell. As an example, genes encoding mevalonate pathway enzymes and genes encoding GPPS and NPPS that have been codon-optimized for expression in E. coll are provided in Table 1. Table 1: Exemplary genes encoding the enzymes of the mevalonate pathway, GPPS andNPPS.Table 2: Polypeptides encoded by the exemplary genes (coding sequences) in Table 1.* denotes a STOP codonTable 3: Other exemplary polypeptides

[0095] In one example, the host cell is an Escherichia coli (E. coli) host cell. In some examples, the E. coli host cell is an E. coli MGI655, an E. coli K12 or an E. coli BL21(DE3) host cell. In other examples, the E. coli host cell is an E. coli RV308(DE3) or E. coli HMS174(DE3) host cell.

[0096] In one example, the E. coli host cell comprises a mutated famesyl diphosphate synthase (ispA) gene. In some examples, the mutation is a S80F mutation. In one example, the mutated ispA gene comprises the sequence set forth in SEQ ID NO: 24.

[0097] The host cell may be cultured in a culture medium. The culture medium may be any medium that is suitable for culturing the host cell for producing limonene. Examples of suitable culture media include but are not limited to Luria-Bertani (LB) media, R-media, ZYM-media, auto-induction media, Terrific Broth (TB), M9 media and 2xPY media. Formulations of these culture media are well -understood by those skilled in the art. Those skilled in the art will also understand that any culture media can be modified to suit the culture conditions of the host cell to be cultured. The culture medium may also be a feed solution. It will be understood by those skilled in the art that a feed solution refers to a medium that is used to feed host cells for biosynthetic production of natural products, such as limonene, in a production setting. For example, a feed solution may be a concentrated mixture of nutrients (such as carbon sources, nitrogen sources, minerals, and sometimes inducers or precursors) that is added to the bioreactor during cultivation to support cell growth and product formation.

[0098] In one example, the culture medium is R-media or a feed solution.

[0099] In one example, the culture medium is a feed solution. In some examples, the feed solution comprises glycerol and MgSC . For example, the concentration of glycerol in the feed solution may be from about 500-800 g / L glycerol, and the concentration of MgSCL in the feed solution may be from about 5-8 g / L MgSO4. Generally, the more concentrated the feed solution, the lesser the volume of feed solution will be used. In one example, the concentration of glycerol in the feed solution is 800 g / L and the concentration of MgSOi in the feed solution is 8 g / L.

[0100] The dissolved oxygen (DO) in the culture medium may be maintained at a level such as to promote growth and / or limonene production. For example, the dissolved oxygen may be maintained at about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0101] In one example, the dissolved oxygen (DO) of the culture medium is maintained at about 30%.

[0102] The pH of the culture medium may be maintained at a level such as to promote growth and / or limonene production. For example, the pH may be maintained at about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0.

[0103] In one example, the pH of the culture medium is maintained at about 7.0.

[0104] The temperature of the culture medium may be maintained at a level such as to promote growth and / or limonene production. For example, the temperature may be maintained at about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, or about 50°C.

[0105] In one example, the temperature of the culture medium is maintained at about 30°C.

[0106] In one example, the genes encoding the enzymes of the mevalonate pathway, the GPPS orNPPS gene, the polynucleotide sequence encoding the engineered limonene synthase, and combinations thereof, are operably linked to an inducible promoter.

[0107] The genes encoding the enzymes of the mevalonate (MVA) pathway, the GPPS or NPPS gene, and the polynucleotide sequence encoding the engineered limonene synthase may be linked to a same inducible promoter. For example, all of the genes may be operably linked to a lacZ promoter. The genes may also be operably linked to different inducible promoters. For example, the genes encoding the enzymes of the MVA pathway may be operably linked to a lacZ promoter and the GPPS orNPPS gene is operably linked to a promoter other than a lacZ promoter. The genes may be located on same or separate operons regardless of the inducible promoter that they are operably linked to.

[0108] Suitable inducible promoters include but are not limited to a lacZ promoter, a Trc promoter, a lac promoter, a lacUV5 promoter, a T7 promoter, and an arabinose-induced promoter.

[0109] In one example, the culture medium is supplemented with an inducer. In some examples, the inducer is supplemented at the beginning of the culture process. In other examples, the inducer is supplemented to the culture medium when the cells are at a predetermined cell density (OD600). For example, the inducer is supplemented to the culture medium when the cells are at an OD600 of between about 0.05 to 10.

[0110] In one example, the inducer is isopropyl |3-D-l-thiogalactopyranoside (IPTG) or lactose.

[0111] The culture medium may be supplemented with the inducer to reach a concentration of inducer in the medium that is useful for inducing expression of the genes encoding the mevalonate pathway enzymes, the gene encoding a GPPS or a NPPS, the polynucleotide sequence encoding an engineered limonene synthase, and combinations thereof.

[0112] In some examples, the inducer is lactose and the culture medium is supplemented with lactose to a concentration of lactose in the medium of about 0.5 mM to about 25 mM, for example about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, 1.5 mM , about 2 mM, 2.5 mM, about 3 mM, about 4 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM or about 30 mM. In one example, the lactose is supplemented at a concentration of about 2.5 mM.

[0113] In some examples, the inducer is IPTG and the culture medium is supplemented with IPTG to a concentration of IPTG in the medium of about 0.5 pM to about 500 pM, for example about 0.5 pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, or about 500 pM. In one example, the concentration of IPTG in the medium is about 200 pM.

[0114] The culturing of the host cell may be culturing in a culture vessel. Suitable culture vessels include but are not limited to a tube, a flask or a bioreactor.

[0115] The culture vessel may be kept stationary during the culturing of the host cell, or it may be shaken during the culturing of the host cell. Shaking of the vessel during the culturing of the host cell may be done to improve growth of the host cell and / or production of limonene by the host cell. Shaking of the vessel may be rotational shaking or lateral (i.e., side-to-side horizontally) shaking.

[0116] In one example, the culture vessel is shaken rotationally during the culturing. In some examples, the rotational shaking is at about 50 to about 500 rpm, for example about 50 rpm, about 60 rpm, about 70 rpm, about 80 rpm, about 90 rpm, about 100 rpm, about 150 rpm, about 200 rpm, about 250 rpm, about 300 rpm, about 350 rpm, about 400 rpm or about 500 rpm. In one example, the rotational shaking is at about 300 rpm.

[0117] When the culturing of the host cell is culturing in a bioreactor culture vessel, the rate that culture medium is provided to the host cell culture or to the bioreactor culture vessel can be adjusted.

[0118] In one example, the culture vessel is a bioreactor, and the culture medium is provided continuously to the culture at a rate of 12.7 mL / h.

[0119] The limonene produced by the method as described herein can be extracted from the culture medium. Suitable methods of extracting limonene from the culture medium include but are not limited to organic solvents such as dodecane, isopropyl myristate and sunflower oil.

[0120] In one example, the limonene is extracted from the culture medium using dodecane.

[0121] In one example, the host cell is cultured in a bioreactor, the culture medium is a feed solution comprising 800 g / L glycerol and 8 g / L MgSO4, the culture medium is supplemented with IPTG to a concentration of IPTG in the medium of about 200 pM when the cells are at an OD600 of 10, the culture vessel is shaken rotationally during the culturing at about 300 rpm, the culture medium is fed at a rate of 12.7 mL / h, the dissolved oxygen (DO) is maintained at about 0%, the pH of the culture is maintained at about pH 7 using ammonium hydroxide, the temperature of the culture is maintained at about 30°C, and the limonene is extracted from the culture medium using dodecane.

[0122] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0123] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0124] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.EXPERIMENTAL SECTION

[0125] Non-limiting examples of the invention and comparative examples will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

[0126] Example 1: 2-plasmid system for parallel screening for GPP and NPP pathways

[0127] E. colt production of limonene is usually performed by using the 1 -plasmid system pJBEI-6409, which produces limonene from acetyl-CoA via GPP, by overexpressing the enzymes of the mevalonate pathway, as well as geranyl pyrophosphate synthase (GPPS) and limonene synthase (LS). This system can be split into a 2-plasmid system, overexpressing the same enzymes separately, with the mevalonate pathway on the first plasmid (pJBEI-3122) and GPPS and LS on the second plasmid (pJBEI-3101) (plasmids pJBEI-6409, pJBEI-3101 and pJBEI-3122 from Alonso-Gutierrez, J. et al., Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production; Metab Eng 19, 33-41 (2013)). However, it was discovered that due to the repetition of the trc promoters, the plasmids pJBEI-6409 and pJBEI- 3101 might be unstable, as plasmid recombination can happen during bacterial replication Thus, a new 2-plasmid system was developed that includes the mevalonate pathway in the first plasmid, as well as either truncated GPPS (EcoCTs-CPMS6a) or truncated neryl pyrophosphate synthase (NPPS) (EcoCTs-CPMS6b), and LS on the second plasmid (EcoCTs-CPMS5). A schematic representation of the plasmids is provided in FIG. 2.

[0128] The new plasmids were made using restriction-free cloning. EcoCTs-CPMS6a was created by deleting the ter promoter, truncated GPPS and the rrnB T1 and T2 terminators from the pJBEI-6409 plasmid, then re-introducing them in between the p15A origin of replication and the chloramphenicol acetyltransferase expression site. In EcoCTs-CPMS6b, the truncated GPPS is replaced with truncated NPPS. This way, if recombination happens during replication,either the origin of replication or the antibiotic resistance is lost, which makes the strain that had undergone plasmid recombination unable to survive. EcoCTs-CPMS5 was created by deleting GPPS from the pJBEI-3101 plasmid. Using EcoCTs-CPMS6a (or 6b) and EcoCTs- CPMS5 together allows production of limonene from acetyl-CoA in an E. coli host cell. EcoCTs-CPMS6a and 6b can be used in parallel to screen for limonene production from either GPP or NPP.

[0129] For subsequent limonene production experiments, purified plasmids were cotransformed by heat shock in E. coli MG1655 chemically competent cells, plated on Luria- Bertani (LB) agar with chloramphenicol and ampicillin and incubated overnight at 37°C. Three freshly grown colonies were picked and inoculated in 1 mL LB media with chloramphenicol and ampicillin, and incubated at 37°C with shaking at 300 rpm overnight. OD600 of the preculture was measured. Each pre-culture was inoculated in 1 mL R-media (2 g / L ammonium sulphate, 4.2 g / L KH2PO4, 11.24 g / L K2HPO4, 1.86 g / L citric acid, and 10 mL / L of lOOx trace element solution). The lOOx trace element solution consists of C0CI2 6H2O (0.25 g / L), MnSCL 4H2O (1.5 g / L), CuSO^I (0.15 g / L), H3BO3 (0.3 g / L), Na2MoO4-2H2O (0.25 g / L), Zn(CH3COO)2 (0.8 g / L), Fe(III) citrate (5 g / L), and EDTA (0.84 g / L) at pH 8). Magnesium sulphate solution containing magnesium sulphate (0.5 g / L) and thiamine (0.0045g / L) was supplemented to the media. The carbon sources (1 g / L glucose, 10 g / L glycerol) and an inducer (2.5 mM lactose) were added just before using media for bacterial culture together with two times the normal working concentration of chloramphenicol and ampicillin and bacterial pre-culture diluted to a starting OD600 of 0.1 in a 14 mL-snap cap tube. A 200 pL dodecane overlay was added to each tube. No dodecane was used for chiral column cultures. Tubes were placed in a test tube rack and incubated at 30°C with shaking at 300 rpm for 72 hrs. After 72 hrs, the OD of the solution was measured, and the dodecane layer was extracted and diluted in hexane, for limonene to be quantified by liquid-injection GC-MS (DB-5 column, limonene quantification) or SPME GC-MS (Agilent 112-6632 Cyclosil-B column).

[0130] Example 2: Exploring natural diversity

[0131] We first searched the Pfam database for terpene synthase domain, selected reviewed LS-annotated enzymes, and clustered them based on their sequence identity percentage to select in total 10 limonene synthases that were described in the literature to be enantioselective, either for (L)- or (D)-limonene production (Table 1).Table 4: Limonene synthase homologues used in the invention

[0132] The newly developed 2-plasmid system was used to assess the effect of truncation on the limonene production. It was found that for each of the 10 limonene synthase homologues, truncating the N-terminal peptide sequence up to the tandem pair of arginines was beneficial for limonene production (FIG. 3A). This was done by aligning the Alphafold2-predicted 3D structures of the 10 limonene synthases and manually truncating the peptide sequence at the tandem arginine. The truncations were performed on the EcoCTs-CPMS5 plasmid, and the enzymes were assessed for limonene production. The assessment found that ABIGR, CANSA, LAV AN, COFAR and PERFR had very low production of limonene. Additionally, it was found that truncated MENSP limonene synthase (tMENSP LS) was produced (-) limonene with a 91% increase compared to MENSP LS, and truncated C1TS1 limonene synthase (tCITSI LS) produced (+) limonene with a 83% increase compared to CITSI LS. The greatest increase was found to be for truncated PICSI limonene synthase (tPICSI), with a 230-fold increase.

[0133] Using the truncated limonene synthases, production of limonene through the GPP and NPP pathways was compared (FIG. 3B). It was found that tMENSP was the highest producer for (-) limonene, and tCITSI for (+) limonene. Both have similar production levels of limonene through either one of the GPP or NPP pathways. On the other hand, the homologue tAGARU was found to have a very different limonene production between the GPP and NPP pathways, which was not the case for the other homologues. Surprisingly, it was found that tAGARU had a 13 -fold greater limonene production through the NPP pathway compared to the GPP pathway. To the inventors’ best knowledge, tAGARU is the first limonene synthase reported to show such stark differences in limonene production based on on whether it uses NPP or GPP as the substrate.

[0134] To confirm the enantioselectivity of the homologues, the limonene produced was run through a chiral column, and the enantioselectivity of the enzymes agreeing with that reported in the literature was observed (FIG. 3C). For the low producers, the background signal prevented accurate measurement of the precise levels of (+) and (-) limonene in the sample. It was found that when a limonene synthase naturally producing a limonene enantiomer is used with NPP as the substrate, about 10-15% of the limonene produced was of the opposite enantiomer (FIG. 3D).

[0135] Example 3: Limonene substrate specificity

[0136] Although N-terminal truncated AGARU (tAGARU) and NEPTE (tNEPTE) limonene synthase homologues have a sequence similarity of 96% and sequence identity of 88%, they display different behavior in terms of both limonene production efficiency (limonene titer) and enantioselectivity. Using NPP (i.e., NPP pathway) instead of GPP (i.e., GPP pathway) as substrate for limonene production did not improve limonene production to a similar extent; the NPP pathway improved limonene production to a greater extent in tAGARU than in tNEPTE (FIG. 3B). To determine the factors behind such a difference, the alignment of thesequences was analyzed. First, it was found that the active site residues are completely conserved between the two homologues (R261, S267, W270, 1294, T295, D298, D302, Y373, E376, S398, V399, S400, V404, L438, R439, D442, T446, D455, K458, F517, D523 and H525 - amino acid positions are based on truncated AGARU (SEQ ID NO: 12). Structural analysis revealed that the active site residues (first shell around the GPP -bound enzyme) were conserved between tAGARU and tNEPTE, thus, residues further from the catalytic domain were investigated. It was found that 4 residues and 1 loop differed between the two enzymes: S8, 117, 1265, E276-I286 (hereinafter referred to as “loop!”) and P530 (amino acid positions based on truncated AGARU (SEQ ID NO: 12) (corresponding to S76, 185, 1333, E344-I354 and P598 respectively on full length wild-type Agastache rugosa limonene synthase (SEQ ID NO: 2)). These key residues are either located sequentially in the C-terminal RR(Xs)W region (S8, 117), or in the N-terminal catalytic domain (1265, loopl, P53O). The amino acids at these positions in tAGARU were substituted with amino acids at the corresponding positions in tNEPTE. Namely, the tAGARU mutations were: S8K, I17M, I265V, loopl (E276-I286, specific mutations within loopl being E276P, P277R, A281K, N282T, I285Q and I286L) and P530A. The corresponding mutations with respect to full length wild-type Agastache rugosa limonene synthase (SEQ ID NO: 2) are S76K, I85M, I333V, loopl (E344-I354, specific mutations within loop 1 being E344P, P345R, A349K, N350T, I353Q and I354L) and P598 respectively.

[0137] The results of these mutations on limonene production are shown in FIG. 5A - 5D. Limonene synthase with mutations in these amino acid positions were cloned in the EcoCTs- CPMS5 plasmid and analyzed for limonene production accordingly. As shown in FIG. 5A and 5B, certain mutations at these positions were found to improve limonene production for the GPP pathway (S8K, I265V, loopl, S8K-loopl) and for the NPP pathway (I265V, loopl, S8K- loopl) in tAGARU. As noted above, the specific mutations in loopl are E276P, P277R, A281K, N282T, T285Q and I286L Additionally, the I265V, loopl , and S8K-loopl mutations were found to have an impact on the ratio between limonene produced through the NPP over the GPP pathway, while the S8K single mutation did not have such an impact (FIG. 5D). Nevertheless, none of the tAGARU single mutants had a limonene production yield to comparable to the level of tNEPTE. Two single mutations (S8K and I265V), as well as the loopl substitutions, increased limonene production from GPP and NPP Mutants S8K-loopl and S8K-I265V-loopl, which combine the previous beneficial mutations, showed improved ability to produce limonene. The mutant S8K-I265 V-loopl led to a limonene titer enhanced by 4.8-fold from GPP and 1.9-fold from NPP. Nevertheless, limonene titers obtained in E. coli whole cell production with mutant S8K-1265V-loopl did not reach that of tNtLS which wasstill 1.4-fold higher from substrate NPP (FIG. 5B and FIG. 5C). Interestingly, the limonene production gap between tNtLS and tArLS limonene production from GPP was greatly reduced by the introduction of the mutations into wild-type tArLS, from 14.8 - to 3.1-fold. (FIG. 5A and FIG. 5C). These mutations in tArLS also reduced the difference between limonene titer obtained from NPP and GPP (FIG. 5D). Indeed, the ratio of limonene produced from NPP and that from GPP (NPP / GPP ratio), reflecting substrate specificity in vivo, dropped from 11.2 (wild-type tArLS) to 4.6 (tArLS- S8K-I265V-loopl), reaching a value close to that of tNtLS, which had a value of 2.0. Thus, these results indicate that selected residues, surrounding the conserved active site, participate in the limonene production using either GPP or NPP as substrate. To confirm the role of the loop! in limonene production, we swapped the amino acid residues in this region from tArLS to tNtLS. Specifically, the corresponding “loopl” mutations in tNtLS (SEQ ID NO: 13) were P280E-R281P-K285A-T286N-Q289I-L290I (P351, R352, K356, T357, Q360 and L361 with respect to the full-length NEPTE limonene synthase (SEQ ID NO: 3)). The corresponding tNtLS mutant showed a significant decrease in limonene production from both GPP and NPP (FIG. 5C). Interestingly, it also increased the NPP / GPP ratio to 3.4 (FIG. 5D), showing that the swap of this region is key to modifying the substrate selectivity of either AGARU or NEPTE limonene synthases. Additional combinations of mutations are currently being investigated for benefit to limonene production.

[0138] In order to confirm the differences in limonene production between the GPP and NPP pathways for tAGARU, as well as the differences in limonene production between tAGARU and tNEPTE despite their high identity, both enzymes were purified and their enzyme kinetics parameters were characterized. This also provided information on which of the enzymes’ properties (binding or activity) need to be improved. Both enzymes were cloned in a psY7 vector which is derived from pET-21d (Novagen®), which includes a maltose binding protein (MBP), a 8xHis tag, an HRV 3C protease cleavage site, and the protein of interest (i.e., the limonene synthase). The plasmids psY7-tAGARU and psY7-tNEPTE were transformed into E. coll BL21 (DE3) cells. Proteins were overexpressed in ZYM media. Briefly, a single colony for the LB petri dish was picked and incubated overnight in LB supplemented with ampicillin to generate a bacterial pre-culture. QD600 of the overnight grown pre-culture was measured. 50 mL of ZYM media supplemented with 30 mM lactose and ampicillin was added to a 250ml sterile glass conical flask with a steel breathable cap. Preculture was added to the media to a starting OD600 of 0.1. The flask was then incubated at 28°C with shaking at 300 rpm for 24hrs. QD600 of the culture was measured. Cells were harvested by centrifuging the culture at 3900 rpm, 4°C for 45 mins. The cell pellet wassuspended in 2.5 mL of His-lysis buffer (50 mM Tris / HCl pH 8.0, 500 mM NaCl, 20 mM imidazole, 1 mg / tnL lysozyme (BioBasic®), 20 U / mL DNasel (Merck®) and 5% glycerol), and lyzed by freeze-thaw cycles. The mixture was frozen at -80°C overnight. Subsequently, it was thawed at room temperature for 1 hour and re-frozen at -80°C for 1-2 hours, thawed at room temperature for 1 hour, and re-frozen again -80°C for 1-2 hours. The cells were subsequently thawed at room temperature to obtain the final cell lysate. The lysate was centrifugated at 15,000 rpm at 4°C for 40 mins. The supernatant was added to 1 mL of Ni-NTA beads which were equilibrated in His-binding buffer (50 mM Tris / HCl pH 8.0, 500 mM NaCl, 20 mM imidazole, 5% glycerol), and the solution was incubated at 4°C overnight with end-to- end mixing. The supernatant solution was then passed through a 10 mL spin column, and the beads were washed with 25 mL His-Binding buffer. An equal amount of 2X protease buffer (50 mM Tris / HCl pH 8.0, 20mM imidazole, 2 mM DTT, 5% glycerol), was added to the column, as well as 30 pL (18 IU) of 3C protease, His, Human (Genscript®). The column was incubated at 4°C overnight with end-to-end mixing. The cleaved proteins were eluted with 3 x 0.5 mL His-binding buffer, assessed for purity with SDS-PAGE, and concentration was checked using a spectrophotometer. The activity of purified limonene synthases was characterized using the EnzChek® Pyrophosphate Assay Kit from Thermo Fisher Scientific® (FIG. 7). For tAGARU, the binding efficiency was found to be higher (lower Km) for GPP than NPP, whereas the activity (kcat) was lower with GPP (Table 4). Overall, the catalytic efficiency (kcat / Ku) of the purified tAGARU was found to be 2-fold higher with NPP than GPP, which confirms the limonene production data. Moreover, purified tNEPTE was found to have a higher limonene production efficiency compared to purified tAGARU, corroborating with the limonene production data.Table 4: Kinetics of wild-type tAGARU (tArLs), wild-type tNEPTE (tNtLS), and tAGARU mutants using NPP or GPP as substrates. tArLS tArLS-S8K tArLS-loopl ^r?J'.S8KJI265V-looplGPP NPP GPP NPP GPP NPP GPP NPP33.3 44.3 18.1 20.1 10.8 14.8 8.0 8.5KM( iiM )± 11.4 ± 5.4 ± 2.4 ± 5.2 ± 2.6 ± 2.0 ± 0.9 ± 0.80.053 0.169 0.022 0.043 0.004 0.007 0.011 0.015 kcat(sec1)± 0.008 ± 0.010 ± 0.001 ± 0.004 ± 0.000 ± 0.000 ± 0.000 ± 0.000Catalytic 1.60 3.81 1.23 2.38 0.39 0.47 1.32 1.78 efficiency (mM-1sec-1) ± 0.6 ± 0.51 ± 0.18 ± 0.39 ± 0.10 ± 0.07 ± 0.15 ± 0.18Catalytic efficiency 2.38 1.93 1.19 1.35NPP / GPP ratioTable 4 (continued) tNtLSGPP NPP12.7 16.0KM (pM)± 1.2 ± 1.50.037 0.079 kCat (sec-1)± 0.001 ± 0.003Catalytic 2.95 4.98 efficiency (niM-1sec-1) ± 0.30 ± 0.1Catalytic efficiency 1.69NPP / GPP ratio

[0139] Example 4: Medium-throughput screening

[0140] To screen truncated CTTST (tCITST) mutants generated to improve limonene titer, a2 mL 96-deep-well plate format was used. The use of dodecane as a second phase to extract limonene was found to be inadequate because of its reaction with the polypropylene material of the plates. Thus, limonene was expressed in 800 pL R-media with a 160 pL solvent phase of isopropyl myristate (IM). After transformation, colony-picking and preculture, limonene productions cultures were started at OD600 0.1 with autoinduction. Autoinduction was initiated via addition of 1 g / L glucose, 2.5 mM lactose, and 9 g / L glycerol to the R-media. Thedeep well plates were incubated at 30°C with shaking at 400 rpm and 90% humidity for 72 hrs. Subsequently, 240 pL of IM was added to each well, the plate was shaken, and IM was extracted, diluted with isopropanol and assessed for limonene presence by LC-QqQ.

[0141] Using structure-based in silica analyses, in combination with stability prediction, and the sequence alignment of tAGARU and tNEPTE, 12 amino acid positions in tCITSI were investigated for improvement of limonene production, namely Q8, S261, 1285, 1394, T395, L398, 1399, 1400, F433, G449, Q525 and T527 (amino acid positions based on the sequence of truncated CITSI (SEQ ID NO: 11)) (FIG. 7A). The corresponding amino acid positions in full length wild-type Citrus sinensis limonene synthase (SEQ ID NO: 1) are Q59, S312, 1336, 1445, T446, L449, 1450, 1451, F484, G500, Q576 and T578, respectively. Site-saturation mutagenesis was performed and a total of 228 mutants were analyzed by producing in deep well plates and analysis with LC-MS. The summary of this analysis is shown in FIG. 7B. The best producing mutants were selected and further analyzed by GC-MS. Namely, the best producing mutants are Q8A, Q8K, Q8N, I285V, S261C, L398A, L398V, I399A, I399M, I400T, I400A, I400V, Q525H, Q525K, T527M, and T527N (amino acid positions based on the sequence of truncated CITSI (SEQ ID NO: 11)). The corresponding mutations in full length wild-type Citrus sinensis limonene synthase (SEQ ID NO: 1) are Q59A, Q59K, Q59N, I336V, S312C, L449A, L449V, I450A, I450M, 145 IT, 1451 A, 145 IV, Q576H, Q576K, T578M, and T578N respectively. Q8K was found to improve specific limonene production in CITSI limonene synthase by 2-fold compared to wild-type CITSI limonene synthase (FIG. 7C).

[0142] Based on further in silica analyses and experimental results, additional tCITSI mutations were investigated: N256D, K243R, E271R, P272R, G407C, S403C, I268M, R279Q, L515H / H516Y, E271R / T498E, H520V, H520I, LI 7M, I268M / I399V (amino acid positions based on the sequence of truncated CITSI (SEQ ID NO: 11). The corresponding mutations in full length wild-type Citrus sinensis limonene synthase (SEQ ID NO: 1) are N307D, K294R, E322R, P323R, G458C, S454C, I319M, R330Q, L566H / H567Y, E322R / T549E, H571V, H571I, L68M, I319M / 1450V respectively. The results found N256D, E271R and S403C to improve tCITSI limonene production.

[0143] Example 5: Controlling enantioselectivity

[0144] Limonene synthase is a divalent metal-dependent class I terpene cyclase. Limonene synthase catalyzes the first committed step in limonene synthesis. The active site of the limonene synthase has an aspartate rich DDxxD motif which is highly conserved among monoterpene synthases. The motifs aspartate residues bind to a trinuclear cluster of divalent metal ions (Mg2+or Mn2-). This cluster helps form a complex with the diphosphate moiety thathas been released, resulting in the creation of characteristic carbocation intermediates. The substrate or the early-stage intermediate is likely responsible for determining the enantioselectivity of limonene product, where one intermediate would produce (+) limonene and the other would produce (-) limonene. It was proposed that the M458 / I450 and N345 / I336 in the (-) LS from MENSP and (+) LS from CITSI were the principal determinants of product enantiospecificity. Although others have attempted to switch the enantioselectivity of (-)M spicata limonene synthase to produce (+) limonene by exchanging sites observed in (+) LS from C. sinensis have been made, no significant shift was observed when utilizing N345I and M458I mutations in M. spicata limonene synthase. In the present invention, the following tCITSI mutants were tested for switched enantioselectivity: S261C, I285N, I399M, S261C- I285N, S261C-I399M, I285N-I399M, I399M-T395S, I399M-T395S-F433L, S261C-I285N- 1399, S261C-I399M-T395S, I285N-I399M-T395S, I285N-I399M-F433L, S261C-I399M- T395S-F433L, I285N-I399M-T395S-F433L, I285N-I399M-T395S-F433L-S261C(corresponds to S312C, I336N, I450M, S312C-I336N, S312C-I450M, I336N-I450M, I450M- T446S, I450M-T446-F484L, S312C-I336N-I450, S312C-I450M-T446S, I336N-I450M- T446S, I336N-I450M-F484L, S312C-I450M-T446S-F484L, I336N-I450M-T446-F484L, I336N-I450M-T446S-F484L-S312C respectively on full length wild-type Citrus sinensis limonene synthase (SEQ ID NO: 1). They were assessed for enantioselectivity (FIG. 7). Surprisingly, some tCITSI mutants display different enantioselectivity depending on whether GPP or NPP was used as the substrate for limonene synthase (1285N-I399M-F433L, I285N- I399M-T395S-F433L). A switch in enantioselectivity of tCITSI to (-) limonene of up to 50% (-) limonene produced when using GPP as the substrate and 65% (-) limonene produced when using NPP as the substrate was attained. However, there was some reduction in limonene production titer in the tCITSI mutants with switched enantioselectivity.

[0145] Example 6: Flask optimization to improve limonene production

[0146] A two-plasmid system was utilized to optimize the biosynthesis of (+)-limonene. The first plasmid, pEcoCTs-CPMSNl, contains the genes encoding the enzymes of the mevalonate pathway, ensuring an efficient supply of precursors. The second plasmid, pEcoCTs-CPMS2, harbours the genes for geranyl pyrophosphate synthase (GPPS) and tCsLS- Q8K, which are converting these precursors into (+)-limonene. E. coli was used as the host cell for limonene production. Introduction of an S80F mutation in the E. coll farnesyl diphosphate synthase gene (ispA) improved limonene production in shake flask cultures by 79% compared to E. coll with a wild-type ispA gene. Based on this result, the host cell strain MG1655ispA- N12Q8K harboring the ispA S80F mutation was used for subsequent optimization.

[0147] Using this strain, the shaking speed was optimized to enhance (+)limonene production. For this, lactose induction was utilized to induce the production of limonene, which was also used during the site-saturation mutagenesis initial screening. Limonene production at two shaking speeds was compared: 100 rpm and 300 rpm. The results showed that 300 rpm yielded a significant 2.6-fold increase in limonene production compared to 100 rpm (FIG. 9B), suggesting that an improved oxygen supply due to higher shaking speed could be beneficial for limonene production. Supplementation of the cultures with an amino acid cocktail was also investigated, but this did not result in additional improvement (Figure 4a). Consequently, it was concluded that the higher shaking speed, providing better oxygenation, was optimal for limonene production.

[0148] Next, IPTG induction was tested, as autoinduction mediais not easily scalable. IPTG concentration was optimized using shake flasks with the MG1655ispA-N12Q8K strain, 300 rpm shaking speed, and glycerol as the carbon source. Under 200 pM IPTG induction, indeed, a higher specific titer for limonene was obtained using glycerol substrate, although there was no statistical difference compared to that with glucose (FIG. 10B). IPTG concentrations from 1 to 400 pM were then tested. It was found that IPTG at 200 pM not only produced the highest limonene titer but also the most reproducible results (FIG. 9C). Notably, the limonene titers obtained with 200 pM IPTG were comparable to those obtained with autoinduction media, indicating that IPTG induction at this concentration is effective for scaling up production.

[0149] Based on the above optimizations, wild-type (WT) tCsLS was compared against the Q8K tCsLS mutant under the best-performing shake flask conditions (ispA S80F strain, 300 rpm, 10 g / L glycerol feed, and 200 pM IPTG). The Q8K mutant produced significantly more limonene than the WT, confirming that the increase in titer is primarily driven by the mutation rather than the conditions alone (FIG. 10C); a 2-fold improvement was achieved with the Q8K mutant. These optimal conditions were employed in the next stage of process optimization for bioreactor production.

[0150] Example 7: Limonene production in a fed-batch process

[0151] Building on the optimized conditions established in shake flasks, the process was scaled up to a 5 L bioreactor using the MG1655ispA-N12Q8K strain. The initial media consisted of R-media with 20 g / L glycerol and a culture volume of 1.8 L, supplemented with 68 pg / mL chloramphenicol and 200 pg / mL ampicillin. After reaching an optical density (OD) of 10, induction was carried out with 200 pM IPTG. To facilitate the extraction of limonene, 1 L of dodecane was added, and glycerol feeding commenced. The feed solution comprised 800 g / L glycerol and 8 g / L MgSCL, with a feeding rate of 12.7 mL / h. Following induction, thetemperature was adjusted to 30°C, while the dissolved oxygen (DO) level was maintained at 30% and the pH at 7.

[0152] The bioreactor run was conducted for 70 hours, during which the maximum limonene titer reached 4.9 g / L, with a peak yield of 3.2% achieved at 65 hours (FIG. 11). The production rate was 114 mg / L / h. The enzyme remained active throughout production, as limonene levels continued to increase up to ~65 hours, indicating sustained activity during the culture. The optimized conditions, including specific IPTG induction, glycerol feeding, and dodecane addition, were crucial in maximizing yield and production rate. The observed accumulation of glycerol and acetate indicates that there is potential for further strain and process improvement. Nonetheless, this represents the highest titer achieved to date, demonstrating the effectiveness of both our engineered limonene synthase and the optimized conditions in the bioreactor setting, and its potential for industrial-scale production.

[0153] Table 5: Limonene production yields of Citrus sinensis limonene synthase (CITSI) mutants*WT: N-terminal (2-52) truncated wild-type CITSI, included as a benchmark.

[0154] Table 6: Limonene production yields of Agastache rugosa limonene synthase(AGARU) mutants*WT: N-terminal (2-69) truncated wild-type AGARU, included as a benchmark

[0155] Table 7: Limonene production yields of Nepeta tenuifolia limonene synthase (NEPTE) mutants*WT: N-terminal (2-72) truncated wild-type NEPTE, included as a benchmark.

[0156] Limonene synthase reference sequencesRSDMNPVVLELAILDANIVQAQLQLELKESLRWWRNTCFVEKLPFARDRLIESYFWS TGMVEPRQHANARIIMAKVIALITVMDDIYDVYGTLEELEQFTEAFRRWDVSSIDQL PTYMQLCFLAINNFVDDTAYNVLKESGVNVMTYLRKSWVDQAENYLMESKWYYS GHKPSLEEYLENSWISVSGPCVLTHEFFGVTDSLAKDTLDSLYEYHDFVRWSSYLLRL ADDLGTSVEEVSRGDVPKSIQCYMHDNDASEEEARQHIKGLIREMWKKMNVERVSE DSPFCRDF1RCCEDLGRMAQFMYHYGDGHGTQHPK1HQQ1AACLFQPFA* (SEQ ID NO: 2)

[0162] Amino acid sequence of full-length wild-type Nepeta tenuifolia limonene synthase

[0163] MALKMTSAVMQMAIPTKLANFVNNSDTHKQSLKLLRNVSTISTSAAAATP RHRLPVCCSASSSSSSQLPTTERRSGNYKPSRWDVDFMQSLNSDYQEERHRTKASELI TQVKNLLEKETSDDPIRQLELIDDLQRLGLSDHFEHEFKEVLNSIYLDNKYYNINIMK ETTSSRDLYSTALAFRLLREHGFQVAQEVFDCFKNEEGEFKASLSDDPRGLLQLYEA SFLFKEGENTLEIAREFATKLLQEKVNSSDEIDDNLLSSIRYSLEIPTYWSVIRPNVSV WIDAYRKRPDMNPVVLELAILDANIMQAQLQQELKEALGWWRNTWFVEKLPFARD RLVESYFWSTGMVPRRQHKTARQLMAKVIALITVMDDIYDVYGTLEELELFTDAFR RWDVSSIDHLPTYMQLCFLSINNFVVDTAYNILKETGVNVTTYLEKSWVDQAENYL MESKWFYSGHKPSLDEYLENSWISVSGPCVLTHEFFGVTDSLAKDTLDSLYEYHDIVRWSSYLLRLADDLGTSVEEVSRGDVPKSIQCYMNDNNASEEEAREHVKGLIRVMW KKMNAERVSEDSPFCKDF1RCCEDLGRMAQFMYHYGDGHGTQHAK1HQQ1TDCLFQ PF A* (SEQ ID NO: 3)

[0164] Amino acid sequence of full-length wild-type Mentha spicata limonene synthase

[0165] MALKVLS VATQMAIP SNLTTCLQP SHFKS SPKLL S S TNS S SRSRLRVYC S S S QLTTERRSGNYNPSRWDVNFIQSLLSDYKEDKHVIRASELVTLVKMELEKETDQIRQ LELIDDLQRMGLSDHFQNEFKEILSSIYLDHHYYKNPFPKEERDLYSTSLAFRLLREH GFQVAQEVFDSFKNEEGEFKESLSDDTRGLLQLYEASFLLTEGETTLESAREFATKFL EEKVNEGGVDGDLLTR1AYSLDIPLHWRIKRPNAPVWIEWYRKRPDMNPVVLELA1L DLNIVQAQFQEELKESFRWWRNTGFVEKLPFARDRLVECYFWNTGIIEPRQHASARI MMGKVNALITVIDDIYDVYGTLEELEQFTDLIRRWDINSIDQLPDYMQLCFLALNNF VDDTSYDVMKEKGVNVIPYLRQSWVDLADKYMVEARWFYGGHKPSLEEYLENSWQSISGPCMLTHIFFRVTDSFTKETVDSLYKYHDLVRWSSFVLRLADDLGTSVEEVSRG DVPKSLQCYMSDYNASEAEARKHVKWLIAEVWKKMNAERVSKDSPFGKDFTGCAV DLGRMAQLMYHNGDGHGTQHPIIHQQMTRTLFEPFA* (SEQ ID NO: 4)

[0166] Amino acid sequence of full-length wild-type Abies grandis limonene synthase

[0167] MALLSIVSLQVPKSCGLKSLISSSNVQKALCISTAVPTLRMRRRQKALVINMKLTT VSHRDDNGGG VLQRRI ADHHPNL WEDDF IQ SLS SPYGGS S YSERAET VVEEVK EMFNSIPNNRELFGSQNDLLTRLWMVDSIERLGIDRHFQNEIRVALDYVYSYWKEKE GIGC GRD STFPDLNST ALALRTLRLHGYNVS SD VLEYFKDEKGHFACP AILTEGQITR SVLNLYRASLVAFPGEKVMEEAE1FSASYLKKVLQK1PVSNLSGEIEYVLEYGWHTN LPRLEARNYIEVYEQSGYESLNEMPYMNMKKLLQLAKLEFNIFHSLQLRELQSISRW WKESGSSQLTFTRHRHVEYYTMASCISMLPKHSAFRMEFVKVCHLVTVLDDIYDTF GTMNELQLFTDAIKRWDLSTTRWLPEYMKGVYMDLYQCINEMVEEAEKTQGRDMLNYIQNAWEALFDTFMQEAKWISSSYLPTFEEYLKNAKVSSGSRIATLQPILTLDVPLP DYILQEIDYPSRFNELASSILRLRGDTRCYKADRARGEEASAISCYMKDHPGSIEEDAL NHINAMISDAIRELNWELLRPDSKSPISSKKHAFDITRAFHHVYKYRDGYTVSNNETKNLVMKTVLEPLAL* (SEQ ID NO: 5)

[0168] Amino acid sequence of full-length wild-type Picea sitchensis limonene synthase

[0169] MSPVSAIPLAYKLCLPRSLISSSRELNPLHITIPNLGMCRRGKSMAPASMSMI LTAAVSDDDRVQRRRGNYHSNLWDDDFIQSLSTPYGEPSYRESAERLKGEIKKMFRS MSKEDEELITPLNDLIQRLWMVDSVERLGIDRHFKNEIKSALDYVYSYWNEKGIGCGRDSWADLNSTALGFRTLRLHGYNVSSEVLKVFEDQNGQFACSPSKTEGEIRSALNL YRASLIAFPGEKVMEDAEIFSSRYLKEAVQKIPDCSLSQEIAYALEYGWHTNMPRLEA RNYMDVFGHPSSPWLKKNKTQYMDGEKLLELAKLEFN1FHSLQQEELQY1SRWWK DSGLPKLAFSRHRHVEYYTLGSCIATDPKHRAFRLGFVKTCHLNTVLDDIYDTFGTMDEIELFTEAVRRWDPSETESLPDYMKGVYMVLYEALTEMAQEAEKTQGRDTLNYA RKAWEIYLD S YIQEAKWIASGYLPTFQEYFENGKIS S AYRAAALTPILTLD VPLPEYIL KGIDFPSRFNDLASSFLRLRGDTRCYKADRARGEEASCISCYMKDNPGSTEEDALNHINSMINETIKELNWELLRPDSNIPMPARKHAFDITRALHHLYKYRDGFSVATKETKSLV SRMVLEPVTL* (SEQ ID NO: 6)

[0170] Amino acid sequence of full-length wild-type Cannabis sativa limonene synthase

[0171] MQCIAFHQFASSSSLPIWSSIDNRFTPKTSITSISKPKPKLKSKSNLKSRSRSS TCYSIQCTVVDNPSSTITNNSDRRSANYGPPIWSFDFVQSLPIQYKGESYTSRLNKLEKDVKRMLIGVENSLAQLELIDTIQRLGISYRFENEIISILKEKFTNNNDNPNPNYDLYAT ALQFRLLRQYGFEVPQEIFNNFKNHKTGEFKANISNDIMGALGLYEASFHGKKGESIL EEARIFTTKCLKKYKLMSSSNNNNMTLISLLVNHALEMPLQWRITRSEAKWFIEEIYE RKQDMNPTLLEFAKLDFNMLQSTYQEELKVLSRWWKDSKLGEKLPFVRDRLVECFL WQVGVRFEPQFSYFR1MDTKLYVLLT11DDMHDIYGTLEELQLFTNALQRWDLKELDKLPDYMKTAFYFTYNFTNELAFDVLQEHGFVHIEYFKKLMVELCKHHLQEAKWFYS GYKPTLQEYVENGWLSVGGQVILMHAYFAFTNPVTKEALECLKDGHPNIVRHASIIL RLADDLGTLSDELKRGDVPKSIQCYMHDTGASEDEAREHIKYLISESWKEMNNEDG NINSFFSNEFVQVCQNLGRASQFIYQYGDGHASQNNLSKERVLGLIITPIPM* (SEQ ID NO: 7)

[0172] Amino acid sequence of full-length wild-type Lavandula angustifolia limonene synthase

[0173] MSIISMHVGILNRPAAYNHLRNLDRRASKPRHVSSTAAATRLRVSCATQLE IKSVDETRRSGNYNPTAWDFNYIQSLDNQYKKERYSTRHAELTVQVKKLLEEEMEA VQKLELIEDLKNLGISYPFKDNIQQILNQIYNEHKCCHNSEVEEKDLYFTALRFRLLR QQGFEVSQEVFDHFKNEKGTDFKPNLADDTKGLLQLYEASFLLREAEDTLELARQFS TKLLQKKVDENGDDKIEDNLLLWIRRSLELPLHWRVQRLEARGFLDAYVRRPDMNPIVFELAKLDFNITQATQQEELKDLSRWWNSTGLAEKLPFARDRWESYFWAMGTFEPHQYGYQRELVAKIIALATVVDDVYDVYGTLEELELFTDAIRRWDRESIDQLPYYMQ LCFLTVNNFVFELAHDVLKDKSFNCLPHLQRSWLDLAEAYLVEAKWYHSRYTPSLE EYLNIARVSVTCPTIVSQMYFALPIPIEKPVIEIMYKYHDILYLSGMLLRLPDDLGTASF ELKRGDVQKAVQCYMKERNVPENEAREHVKFLIREASKQINTAMATDCPFTEDFAV AAANLGRVANFVYVDGDGFGVQHSKIYEQIGTLMFEPYP* (SEQ ID NO: 8)

[0174] Amino acid sequence of full-length wild-type Coffea arabica limonene synthase

[0175] MAI1NLPVPTNSSSEVNKHNHLRSCLPSGRATFTTLSAAAMRSATMAAANV REQSGQKQQLINRRSGNYEAPLWEFDYIQSLKNEYAGDIYVSRANELKEQVKMMLD EEDMKLLDCMELVDGLERLGLAYHFEGRINRLLSSDYKAIHEGNHQRNKEDLYAAA LEFRIFRQNGFNVPQDIFNDFITEDGEFDESLSEDTMGLLSLYEASFLSLEGEATLDLAREFTTKHLNNYLGKENTDQNLRILVYHALELPLRWRAPRIEARWYIDAYERSPNVNP TLLELAKIDFNTVQAIHQQDLKHVSWWWKNIRIAEKLTFIRDRIVENFFWAIGAVFEP QYGSCRRMLTKVFALITMIDDIYDVYGTLEELELFTDAVDRWDVKAIDQLPDYMRV GYLGFFNS1NEMAYDALKEQGVHIVEYLRKVWADLCKAYLQEAKWYYAGYTPTVE EYLENAW VSMS VP VMLMH A Y AG VTNPMNK E AMD VLDTHDI VRC S S YLLRF ADDLGTSPGEMKRGDVPKLVQCYMKEAGCSEEESREHVWFLLRETWKKMNKDSEWAESP FSKTFVTAAKNFGRVALVMYQYGDGHGLHSNPEAKDRILASLFSPVPPA* (SEQ ID NO: 9)

[0176] Amino acid sequence of full-length wild-type Perilla frutescens limonene synthase

[0177] MYTGVIMHMAIPIKPAHYLHNSGRSYASQLCGFSSTSTRAAIARLPLCLRFRCSLQASDQRRSGNYSPSFWN DYILSLNNHYKEESRHMKRAGELIVQVKMVMGKETDPVVQLELIDDLHKLALSHHFEKEIKEILFNISIYDHKIMVERDLYSTALAFRLLRQYGFKVPQEVFDCFKNDNGEFKRSLSSDTKGLLQLYEASFLLTEGEMTLELAREFAT1FLQEKLNDKTIDDDDDADTNLISCVRHSLDIPIHWRIQRPNASWWIDAYKRRSHMNPLV LELAKLDLNIFQAQFQQELKQDLGWWKNTCLAEKLPFTRDRLVECYFWCTGIIQPLQ HENARVTLAKVNALITTLDDIYDVYGTLEELELFTEAIRRWDVSSIDHLPNYMQLCFL ALNNFVDDTAYDVMKEKDINIIPYLRKSWLDLAETYLVEAKWFYSGHKPNMEEYLNNAWISISGPVMLCHVFFRVTDSITRETVESLFKYHDLIRYSSTILRLADDLGTSLEEVSRGDVPKSIQCYMNDNNASEEEARRHVRWLIAETWKKINEEVWSADSPFCKDFIACAADMGRMAQFMYHNGDGHGIQNPQIHQQMTDILFEQWL* (SEQ ID NO: 10)

[0178] Amino acid sequence of N-terminal (2-52) truncated wild-type Citrus sinensis limonene synthase

[0179] MRRSANYQPSIWDHDFLQSLNSNYTDETYKRRAEELKGKVKTAIKDVTEPLDQLELIDNLQRLGLAYHFEPEIRNILRNIHNHNKDYNWRKENLYATSLEFRLLRQHGYPVSQEVFSGFKDDKVGFICDDFKGILSLHEASYYSLEGESIMEEAWQFTSKHLKEMMITSNSKEEDVFVAEQAKRALELPLHWKAPMLEARWFIHVYEKREDKNHLLLELAKLEFNTLQA1YQEELKD1SGWWKDTGLGEKLSFARNRLVASFLWSMGIAFEPQFAYCRRVLTISIALITVIDDIYDVYGTLDELEIFTDAVARWDINYALKHLPGYMKMCFLALYNFVNEFAYYVLKQQDFDMLLSIKHAWLGLIQAYLVEAKWYHSKYTPKLEEYLENGL VSITGPLIITISYLSGTNPIIKKELEFLESNPDIVHWSSKIFRLQDDLGTSSDEIQRGDVPK SIQCYMHETGASEEVAREHIKDMMRQMWKKVNAYTADKDSPLTRTTAEFLLNLVRMSHFMYLHGDGHGVQNQETIDVGFTLLFQPIPLEDKDMAFTASPGTKG* (SEQ IDNO: 11)

[0180] Amino acid sequence of N-terminal (2-69) truncated wild-type Agastache rugosa limonene synthase

[0181] MRRSGNYSPSRWDVDFIQSLNSDYQEERHTRRASELITQVKMLMEKETTDPIRQLELIDDLQRLGLSDHFQNEFKEILNTIYLDNKYYNINIMREESRDLYSTALAFRLLREHGFQVAQEVFECFKNEEGDFKASLIDDTRGLLQLYEASFLFKEGENTLEIAREFTTKILQEKLKGDEIDDNLLSSIRYSLEIPNYWSVVRPNVSVWIDEYRKRSDMNPVVLELAILDANIVQAQLQLELKESLRWWRNTCFVEKLPFARDRLIESYFWSTGMVEPRQHANAR11MAKV1AL1TVMDD1YDVYGTLEELEQFTEAFRRWDVSS1DQLPTYMQLCFLA1NNFVDDTAYNVLKESGVNVMTYLRKSWVDQAENYLMESKWYYSGHKPSLEEYLE NSWISVSGPCVLTHEFFGVTDSLAKDTLDSLYEYHDIVRWSSYLLRLADDLGTSVEE VSRGDVPKSIQCYMHDNDASEEEARQHIKGLIREMWKKMNVERVSEDSPFCRDFIRC CEDLGRMAQFMYHYGDGHGTQHPKIHQQIAACLFQPFA* (SEQ ID NO: 12)

[0182] Amino acid sequence of N-terminal (2-72) truncated wild-type Nepeta tenuifolia limonene synthase

[0183] MRRSGNYKPSRWDVDFMQSLNSDYQEERHRTKASELITQVKNLLEKETSD DPIRQLELIDDLQRLGLSDHFEHEFKEVLNSIYLDNKYYNINIMKETTSSRDLYSTALA FRLLREHGFQVAQEVFDCFKNEEGEFKASLSDDPRGLLQLYEASFLFKEGENTLEIAR EFATKLLQEKVNSSDEIDDNLLSSIRYSLEIPTYWSVIRPNVSVWIDAYRKRPDMNPV VLELAILDANIMQAQLQQELKEALGWWRNTWFVEKLPFARDRLVESYFWSTGMVP RRQHKTARQLMAKVIALITVMDDIYDVYGTLEELELFTDAFRRWDVSSIDHLPTYMQLCFLSINNFVVDTAYNILKETGVNVTTYLEKSWVDQAENYLMESKWFYSGHKPSL DEYLENSWISVSGPCVLTHEFFGVTDSLAKDTLDSLYEYHDIVRWSSYLLRLADDLG TS VEEVSRGDVPKSIQCYMNDNNASEEEAREHVKGLIRVMWKKMNAERVSEDSPFC KDFIRCCEDLGRMAQFMYHYGDGHGTQHAKIHQQITDCLFQPFA* (SEQ ID NO: 13)

[0184] Table 8: Plasmids used in the invention

[0185] Table 9: Strains used in the invention

[0186] Equivalents

[0187] The foregoing examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will readily be apparent that numerous modifications and alterations may be made to the specific embodiments of the invention described above and illustrated in the examples without departing from the principles underlying the invention. All such modifications and alterations are intended to be embraced by this application.

Claims

ClaimsWhat is claimed is:

1. An engineered limonene synthase with increased limonene production relative to a reference limonene synthase, wherein the engineered limonene synthase comprises one or more mutations relative to the reference limonene synthase.

2. The engineered limonene synthase of claim 1, wherein the engineered limonene synthase is a Citrus sinensis limonene synthase, an Agastache rugosa limonene synthase, a Nepeta tenuifolia limonene synthase, a Mentha spicata limonene synthase, an Abies grandis limonene synthase, a Picea sitchensis limonene synthase, a Cannabis sativa limonene synthase, a Lavandula angustifolia limonene synthase, a Coffee arabica limonene synthase, or a Perilla frutescens limonene synthase3. The engineered limonene synthase of claims 1 or 2, wherein the reference limonene synthase is a wild-type Citrus sinensis limonene synthase (SEQ ID NO: 1), a wild-type Agastache rugosa limonene synthase (SEQ ID NO: 2), a wild-type Nepeta tenuifolia limonene synthase (SEQ ID NO: 3), a wild-type Mentha spicata limonene synthase (SEQ ID NO: 4), a wild-type Abies grandis limonene synthase (SEQ ID NO: 5), a wild-type Picea sitchensis limonene synthase (SEQ ID NO: 6), a wild-type Cannabis sativa limonene synthase (SEQ ID NO: 7), a wild-type Lavandula angustifolia limonene synthase (SEQ ID NO: 8), a wild-type Coffee arabica limonene synthase (SEQ ID NO: 9), or a wild-type Perill frutescens limonene synthase (SEQ ID NO: 10).

4. The engineered limonene synthase of any one of claims 1 to 3, wherein the engineered limonene synthase is a Citrus sinensis limonene synthase comprising a substitution at one or more amino acid positions equivalent to Q59, Q576, 1451, N307, E322, L449, 1450, S454, T578, 1336, or combinations thereof of a wild-type Citrus sinensis limonene synthase (SEQ ID NO: 1).

5. The engineered limonene synthase of claim 4, wherein the substitution at one or more amino acid positions equivalent to Q59, 1451, Q576, N307, E322, L449, 1450, S454, T578, 1336, or combinations thereof is Q59A, Q59E, Q59K, Q59L, Q59N, Q59S, Q59P, Q59T, I451A, I451C, I451F, I451L, I451M, I451S, I451T, I451V, I451W, I451Y, Q576A, Q576D, Q576G, Q576I, Q576L, Q576M, Q576N, Q576P, Q576S, Q576T, Q576Y, N307D, E322R, L449A, L449C, I450A, S454C, T578V, I336V, or combinations thereof.

6. The engineered limonene synthase of claim 4, wherein the substitution is at amino acid positions equivalent to Q59 and 1451.

7. The engineered limonene synthase of claim 6, wherein the substitution at amino acid positions equivalent to Q59 and 1451 is selected from the group consisting of:(i). Q59K and 1451 A;(ii). Q59K and I451T;(iii). Q59A and 1451 A; and(iv). Q59A and 145 IT.

8. The engineered limonene synthase of any one of claims 1 to 3, wherein the engineered limonene synthase is a Agastache rugosa limonene synthase comprising a substitution at one or more amino acid positions equivalent to S76, 1333, E344, P345, A349, N350, 1353, 1354, 185, Q380, E383, P598, or combinations thereof of a wild-type Agastache rugosa limonene synthase (SEQ ID NO: 2).

9. The engineered limonene synthase of claim 8, wherein the substitution at one or more amino acid positions equivalent to S76, 1333, E344, P345, A349, N350, 1353, 1354, 185, Q380, E383, P598, or combinations thereof is S76K, I333V, E344P, P345R, A349K, N350T, I353Q, I354L, I85M, Q380L, E383D, P598A, or combinations thereof.

10. The engineered limonene synthase of claim 8, wherein the substitution is at amino acid positions equivalent to:(i). E344, P345, A349, N350, 1353, and 1354;(ii). S76, E344, P345, A349, N350, 1353 and 1354;(iii). S76, 1333, E344, P345, A349, N350, 1353 and 1354;(iv). 185 and 1333; or(v). Q380 and E383.

11. The engineered limonene synthase of claim 10, wherein the substitution at amino acid positions equivalent to (i) - (v) is selected from the group consisting of:(i). E344P, P345R, A349K, N350T, I353Q, and I354L;(ii). S76K, E344P, P345R, A349K, N350T, I353Q, and I354L,(iii). S76K, 1333 V, E344P, P345R, A349K, N350T, 1353Q, and 1354L;(iv). I85M and I333V; and(v). Q380L and E383D.

12. The engineered limonene synthase of any one of claims 1 to 3, wherein the engineered limonene synthase is a Nepeta tenuifolia limonene synthase comprising a substitution at one or more amino acid positions equivalent to A605, K79, M88, V340, L387, D390, or combinations thereof of a wild-type Nepeta tenuifolia limonene synthase (SEQ ID NO: 3).

13. The engineered enzyme of claim 12, wherein the substitution at one or more amino acid positions equivalent to A605, K79, M88, V340, L387, D390, or combinations thereof of a wild-type Nepeta tenuifolia is A605P, K79S, M88I, V340I, L387Q, D390E, or combinations thereof.

14. The engineered limonene synthase of claim 12, wherein the substitution is at amino acid positions equivalent to:(i). L387 and D390; or(ii). M88 and V340.

15. The engineered limonene synthase of claim 14, wherein the substitution at amino acid positions equivalent to (i) or (ii) is selected from the group consisting of:(i). L387Q and D390E; and(ii). M88I and V340I.

16. An engineered limonene synthase with switched enantioselectivity relative to a reference limonene synthase, wherein the engineered limonene synthase comprises one or more mutations relative to the reference limonene synthase.

17. The engineered limonene synthase of claim 16, wherein the engineered limonene synthase is a Citrus sinensis limonene synthase, a Agastache rugosa limonene synthase, a Nepeta tenuifolia limonene synthase, a Mentha spicata limonene synthase, a Abies grandis limonene synthase, a Picea sitchensis, limonene synthase a Cannabis sativa limonene synthase, a Lavandula angustifolia limonene synthase, a Coffee arabica limonene synthase, or aPerilla frutescens limonene synthase.

18. The engineered limonene synthase of claim 16 or 17, wherein the reference limonene synthase is a wild-type limonene synthase from the same organism.

19. The engineered limonene synthase of any one of claims 16 to 18, wherein the engineered limonene synthase is a Citrus sinensis limonene synthase comprising a substitution at one or more amino acid positions equivalent to S312, 1336, T446, 1450, F484 and combinations thereof of a wild-type Citrus sinensis limonene synthase (SEQ ID NO: 1).

20. The engineered limonene synthase of claim 19, wherein the substitution at one or more amino acid positions equivalent to S312, 1336, T446, 1450, F484 and combinations thereof is S312C, I336N, T446S, I450M and F484L.

21. The engineered limonene synthase of claim 19, wherein the substitution is at amino acid positions equivalent to:(i). S312 and 1336;(ii). S312 and 1450;(iii). 1336 and 1450;(iv). 1450 and T446;(v). S312, 1336 and 1450;(vi). S312, 1450 and T446;(vii). 1336, 1450 and T446,(viii).1336, 1450 and F484;(ix). 1450, T446 and F484;(x). S312, 1450, T446 and F484;(xi). 1336, 1450, T446 and F484; or(xii). 1336, 1450, T446, F484 and S312.

22. The engineered limonene synthase of claim 21 , wherein the substitution at amino acid positions equivalent to (i) - (xii) is selected from the group consisting of:(i). S312C and I336N;(ii). S312C and I450M;(iii). I336N and I450M;(iv). T446S and I450M;(v). S312C, I336N and I450M;(vi). S312C, T446S, and I450M;(vii). I336N, T446S, and I450M;(viii). 1336N, 1450M and F484L;(ix). T446S, I450M and F484L;(x). S312C, T446S, I450M and F484L;(xi). I336N, T446S, I450M and F484L; or(xii). S312C, I336N, T446S, I450M and F484L.

23. The engineered limonene synthase of any one of claims 1 - 22, wherein limonene yield is increased when geranyl pyrophosphate (GPP) or neryl pyrophosphate (NPP) is the substrate for the engineered limonene synthase.

24. The engineered limonene synthase of any one of claims 1 - 23, wherein the engineered limonene synthase is truncated at the N-terminal or the C-terminal relative to a wild-type limonene synthase25. The engineered enzyme of any one of claims 1 - 24, wherein the truncation is an N- terminal truncation.

26. The engineered limonene synthase of claim 24 or 25, wherein the engineered limonene synthase enzyme is:(a) a truncated Citrus sinensis limonene synthase comprising a deletion of residues 2 - 52 at the N-terminal;(b) a truncated Agastache rugosa limonene synthase comprising a deletion of residues 2 - 69 at the N-terminal;(c) a truncated Nepeta tenuifolia limonene synthase comprising a deletion of residues 2 - 72 at the N-terminal;(d) a truncated Citrus sinensis, Agastache rugosa, or Nepeta tenuifolia limonene synthase comprising a deletion of the amino acids upstream of the position equivalent to R58 in a wild-type Mentha spicata limonene synthase except for the first methionine residue at the start of the N-terminal polypeptide sequence; or(e) a truncated Citrus sinensis, Agastache rugosa, or Nepeta tenuifolia limonene synthase comprising a deletion of the N-terminal plastid targeting sequence except for the first methionine residue at the start of the N-terminal polypeptide sequence.

27. A polypeptide sequence encoding the engineered limonene synthase according to any one of claims 1 - 26.

28. A polynucleotide sequence encoding the polypeptide sequence according to claim 27.

29. A vector comprising the polynucleotide sequence according to claim 28.

30. A host cell comprising the vector according to claim 29.

31. A method of determining a characteristic of a limonene synthase comprising the steps of:(a) expressing in a host cell:(i). a first expression vector comprising the genes encoding the enzymes of the mevalonate pathway and a geranyl pyrophosphate synthase (GPPS) gene or a neryl pyrophosphate synthase (NPPS) gene; and(ii). a second expression vector encoding a limonene synthase gene, wherein the genes encoding the enzymes of the mevalonate pathway are acetyl-CoA acetyltransferase (atoB), 3 -hydroxy-3 -methylglutaryl-CoA synthase HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), mevalonate diphosphate decarboxylase PMD) and isopentenyl-diphosphate isomerase (idi),(b) culturing the host cell to produce limonene; and(c) analyzing the limonene product to determine the characteristic of the limonene synthase.

32. The method of claim 31, wherein the genes encoding the enzymes of the mevalonate pathway and the GPPS or NPPS gene are in separate operons in the first expression vector.

33. The method of claim 32, wherein the operon containing the GPPS or NPPS gene is located between the origin of replication and the antibiotic resistance marker in the first expression vector.

34. The method of any one of claims 31 - 33, wherein the characteristic of the limonene synthase is limonene production, enantioselectivity, substrate specificity or combinations thereof.

35. The method of any one of claims 31 - 34, wherein the limonene synthase is a wild-type limonene synthase or a engineered limonene synthase.

36. The method of any one of claims 31 - 35, wherein the host cell is an Escherichia coli cell.

37. A method of producing limonene comprising the steps of:(a) expressing in a host cell :(i). genes encoding the enzymes of the mevalonate pathway,(ii). a gene encoding a geranyl pyrophosphate synthase (GPPS) or a gene encoding a neryl pyrophosphate synthase (NPPS); and(iii). a polynucleotide sequence encoding an engineered limonene synthase of any one of claims 1 to 26;(b) culturing the host cell in a culture medium under conditions suitable for producing limonene; and(c) extracting the limonene from the culture medium.

38. The method according to claim 37, wherein the genes encoding the enzymes of the mevalonate pathway are acetyl-CoA acetyltransferase (atoB), 3-hydroxy-3- methylglutaryl-CoA synthase HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), mevalonate diphosphate decarboxylase (PMD) and isopentenyl-diphosphate isomerase (idi).

39. The method according to claim 37, wherein the gene coding a GPPS is a truncated GPPS (trGPPS) and the gene coding a NPPS is a truncated NPPS (trNPPS), optionally wherein the trGPPS comprises the sequence set forth in SEQ ID NO: 21 and wherein the trNPPS comprises the sequence set forth in SEQ ID NO: 22.

40. The method according to any one of claims 37 to 39, wherein the host cell is mEscherichia coli (E. coli) host cell; optionally wherein the E. coli host cell is an E. coli MG1655 host cell.

41. The method according to claim 40, wherein the A. coli host cell comprises a mutated farnesyl diphosphate synthase (ispA) gene; optionally wherein the mutated ispA gene comprises the sequence set forth in SEQ ID NO: 24.

42. The method according to any one of claims 37 to 41, wherein the culture medium is R- rnedia or a feed solution.

43. The method according to claim 42, wherein the culture medium is a feed solution comprising glycerol and MgS04; optionally wherein the concentration of glycerol in the feed solution is 800 g / L and the concentration of MgSC in the feed solution is 8 g / L.

44. The method according to any one of claims 37 to 43, wherein the dissolved oxygen (DO) of the culture medium is maintained at about 30%.

45. The method according to any one of claims 37 to 44, wherein the genes encoding the enzymes of the mevalonate pathway, the GPPS or NPPS gene, the polynucleotide sequence encoding the engineered limonene synthase, and combinations thereof, are operably linked to an inducible promoter46. The method according to any one of claims 37 to 45, wherein the culture medium is supplemented with an inducer, optionally wherein the inducer is supplemented at the beginning of the culture process or the inducer is supplemented to the culture medium when the cells are at a predetermined cell density (OD600), optionally wherein the cells are at an OD600 of between about 0.05 to 1047. The method according to claim 46, wherein the inducer is isopropyl P-D-l- thiogalactopyranoside (IPTG) or lactose.

48. The method according to claim 47, wherein the inducer is IPTG.

49. The method according to claim 48, wherein the culture medium is supplemented with IPTG to a concentration of IPTG in the medium of about 0.5 pM to 500 pM, optionally wherein the concentration of IPTG in the medium is about 200 pM.

50. The method according to any one of claims 37 to 49, further comprising culturing the host cell in a culture vessel, optionally wherein the culture vessel is a tube, a flask or a bioreactor.

51. The method according claim 50, wherein the culture vessel is shaken rotationally during the culturing; optionally wherein the rotational shaking is at about 50 rpm to 500 rpm; optionally wherein the rotational shaking is at about 300 rpm.

52. The method according to claim 51, wherein the culture vessel is a bioreactor, and the culture medium is fed at a rate of 12.7 mL / h.

53. The method according to claim 37, wherein the limonene is extracted from the culture medium using dodecane

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