WO2013016591A1 - High efficiency isoprene synthases produced by protein engineering - Google Patents

Abstract

The present invention provides new energy and chemical industry' solutions that are sustainable both environmentally and economically. The invention relates to the use of variant isoprene synthases designed to increase the rate of isoprene production in genetically engineered microorganisms or "biocatalysts" serving as a viable alternative to petroleum-dependent chemicals and energy.

Description

[0001] The invention relates, in pari, to high efficiency enzymes needed for the economic viability of enzyme driven processes for the production of renewable isoprene,

BACKGROUND OF INVENTION

f 0002] Isoprene is a versatile feedstock utilized in production of synthetic rubber (poly-isoprene), elastomers, lubricants, and specialty chemicals. The chemical value of isoprene, CH₂=C(CH₃)CH=CH₂, is provided by two double bonds and a branched methyl group, which enable it to react to form a wide range of valuable products, including poly- isoprene. Furthermore, isoprene has a boiling point of 34°C, a property that offers a number of production and purification advantages. Isoprene is currently produced by the petrochemical industry as a by-product of the thermal cracking of crude oil, The yield of isoprene from this process is small and requires the collection of 5-carbon molecule (C5) streams from several refineries and the separation of individual products from these streams, including isoprene. Approximately 800,000 metric tons of isoprene is produced by this route every year. Efficient polymerization of this product into poly-isoprene is handicapped by impurities that are difficult to separate from the C5 stream of thermally cracked oil.

[0003] The global market for poly-isoprene (rubber) is greater than $25 billion per year. Global demand for rabber (natural and synthetic) by the transportation industry_' is growing by more than 5% (-300,000 metric tons) per year. Demand is highest in China, which has overtaken the United States in car manufacturing. The global supply of natural rubber is constrained by the productivity of rubber trees, competing uses for irrigated acreage, and the spread of a fungal pathogen which devastated plantations in aibber's native South America. The supply of synthetic isoprene to make synthetic poly- isoprene (also known as Isoprene Rubber) is constrained by refinery capacity and the price of oil. As a result, prices for natural rubber and synthetic isoprene have risen steadily, doubling over the past 5 years to a present level of approximately $3 per kilogram, and are anticipated to increase at the same rate over the next decade,

[0004] In addition to the poly-isoprene content of rubber, many plants, primarily trees, produce and emit large quantities of monomelic isoprene, over 500,000 metric tons per year. Isoprene is the simplest member of a large (~40,000 members) family of plant isoprenoids including natural rubber. A number of reasons have been suggested for why plants produce isoprene, including its use as: a flowering signal (Terry, et al, (1995) J. Exp. Bot. 46: 1629-1631 ), a way of releasing phosphate inadvertently converted to dimethyl ally! diphosphate (safety valve) (Rosenstiel, et al., (2004) Plant Biol. 6: 12-21) and a role in defense against being eaten by insects, mediated through the attraction of natural enemies (Laothawornkitkul, et al, (2008) Plant Cell & Environment. 31: 1410- 1415). However, the most likely benefits to plants of producing and emitting isoprene are protection from oxidative damage by quenching reactive oxygen species (ROS) and thermo-protection against transient changes in temperature in the leaf canopy by strengthening hydrophobic interactions within membranes.

[0005] Plants produce isoprene from dimethylallyl pyrophosphate, also known as dimethylallyl diphosphate (DMADP), using the enzyme isoprene synthase, To date, confirmed isoprene synthases have been cloned and expressed from several species of Populus and from kudzu: (Miller, et al, (2001) Planta 213: 483-487; Sasaki, et al, (2005) FEBS Letters 579: 2514-2518; Sharkey, et al, (2005) Plant Physiology 137: 700- 712; Behnke, et al, (2007) The Plant Journal 51: 485-499; Laothawornkitkul, et al, (2008) Plant Cell & Environment. 31 : 1410-141 ), Expression of these isoprene synthase genes in microbial species that produce DMADP to make isoprenoids other than isoprene, but that lack an endogenous isoprene synthase, could provide a novel approach to isoprene production.

[0006] Combining the addition of isoprene synthase and genes to increase the production of DMADP and the removal or reduction of endogenous microbial genes that are not vital but divert carbon away from the production of isoprene, results in the production of microorganisms genetically engineered to produce high levels of isoprene, (Whited et al, (2010) Industrial Biotechnology 6: 152-163). [0007] A key factor determining economic feasibility of producing isoprene by enzyme driven routes, fermentative or otherwise biochemical, is the ability of the isoprene synthase used to efficiently produce a large quantity of isoprene. More specifically, the enzyme synthesizing isoprene preferably produces isoprene at a high rate, e.g., in a fermentative setting greater than 2,5g of isoprene per litre per hour of microbial culture, and, even more preferably, will be able to produce many times its own weight in isoprene. These goals can be realized by engineering an isoprene synthase with appropriate _m and k_cai.

J0008] Currently, all native isoprene synthases that have been studied have a high _rrj so require high concentrations of their substrate DMADP in order to reach maximal rates of isoprene production. For the plants that naturally express isoprene synthases, it may be evolutionarily advantageous for their isoprene synthases to have high _ms to ensure that carbon is first directed to essential functions for the plant and only secondarily to isoprene production through the activity of isoprene synthases. Not wishing to be bound by theory, it could be hypothesized that a very low K_m isoprene synthase may be disadvantageous to plants and therefore less likely to occur in nature. In addition to their high K_m, known isoprene synthases have a very low turnover constant, k_cat> or overall catalytic rate. Furthermore, plant isoprene synthases did not evolve to function in an extracellular or non-plant cellular environment such as that found inside microbes grown in a fermenter. Consequently, their performance in such settings will be sub-optimal. Therefore, what is needed to meet the increasing worldwide demand for isoprene and products derived from isoprene is an isoprene synthase with optimized K_m and K_cai values, which efficiently synthesizes isoprene in a fermentative mileau. Further, methods for engineering one or a combination of ._m and k_cai values of an isoprene synthase would represent a significant advancement o ver conventional methods of the art,

|0009] This invention provides isoprene synthases having properties necessary' for efficient fermentation of feedstocks to produce isoprene, and protein engineering approaches for producing enzymes with an enhanced ability, relative to wild type isoprene synthases, to catalyse the formation of isoprene. Broadly speaking, methods include modifying existing isoprene producing enzymes or converting related enzymes that do not natural ly produce isoprene into enzymes capable of catalyzing isoprene formation. In various embodiments, such enzymes are sufficiently efficient in the catalysis of isoprene formation to be useful in an industrial setting.

SUMMARY OF THE INVENTION

[0010] The present invention provides isoprene synthases able to catalyze the synthesis of isoprene with an activity greater than that found in natually occurring isoprene synthases. Among the factors contributing to the present invention are novel insights into the structure and activity of known isoprene synthases, and other closely related hemiterpenoid and terpenoid synthases. In various embodiments, the superior isoprene synthases of the in vention are produced by one or more of the following routes: (a) identification and, preferably, isolation, of a novel native isoprene synthase, (b) conversion of a methyl butenol synthase into an isoprene synthase by the addition, removal or substitution of one or more amino acids, (c) replacement of one or more unstructured arm of a characterized isoprene synthase with the arm of a snapdragon monoterpene synthase, e.g., one that makes the acyclic monoterpenes ocimene and myrcene, (d) conversion of an acyclic monoterpene synthase that removes diphosphate from an aliylic isoprenoid precursor into an isoprene synthase by substitution of one or amino acids in their active sites, e.g., with phenylalanine, tyrosine or tryptophan, (e) opening up the active site of a characterized isoprene synthase by replacing one phenylalanine in the active site with tyrosine or tryptophan and replacing another phenylalanine in the active site with an amino acid smaller tha phenylalanine, (1) conversion of an enzyme that uses dimethylallyl diphosphate (DMADP) and a cation intermediate into an isoprene synthase by reducing the size of its binding pocket by replacing amino acids that line the active site with phenylalanines, (g) conversion of pentalenene synthase into an isoprene producing enzyme through replacement of amino acids in its active site, (h) conversion of phellandene synthase into an isoprene producing enzyme through replacement of amino acids in its active site, and (g) other mutations of conserved amino acids in the active site through addition, deletion or substitution of amino acids. [0011 ] In various embodiments, one or more of the approaches listed abo ve are combined to produce a single novel isoprene synthase to further increase its efficiency and/or activity relative to a wild type isoprene synthase in the synthesis of isoprene. in exemplary embodiments, the active site of the novel isoprene synthases may be modified by opening up their active sites through replacement of the phenylalanines smaller amino acids such as leucine. In various embodiments, replacement of the unstructured arm of an isoprene synthase (as modeled in (Sharkey et al., (2005) Plant Physiology 137: 700- 712) and shown in (Koksal et al., (2010) Journal of Molecular Biology 402: 363-373) with the arm of snapdragon nionoterpene synthase that lacks a canonical double arginine is combined with a novel isoprene synthase or an isoprene synthase whose active site has been opened up throu gh replacement of the phenylalanines

[0012] In various embodiments of the invention, the thermostability of the novel isoprene synthase will be increased relative to the corresponding wild type. The native isoprene synthases have evolved to function in terrestrial plants at temperatures that vary from the culture conditions ideal for microbes in an industrial bioreactor or fermenter. The present invention provides novel isoprene synthases having optimal activity at the optimal temperature for microbes producing isoprene in a bioreactor or fermenter. f 0013] In other embodiments of the invention, it is provided variant isoprene synthases that are more resistant than the corresponding wild type enzymes to oxidative stress.

[0014] In various embodiments of the invention, the novel isoprene synthases will be modified relative to the corresponding wild type enzyme to facilitate greater activity in their new setting in either microbial hosts or combinations of isolated enzymes. These modifications include changing codon usage from that preferred by terrestrial plants to that preferred by the chosen microbial host. These modifications optionally further include removal of cell localization sequences that direct isoprene to compartments within a terrestrial plant cell, for example, the chloroplast that microbes do not have. These modifications optionally further include changes to increase the stability and solubility of the enzymes producing isoprene.

[0015] In another aspect of the in vention, it is pro vided an isolated nucleic acid sequence having a sequence encoding an isoprene synthase variant of a parent eucalyptus isoprene synthase, said sequence operably linked to a promoter, wherein the isoprene synthase variant is capable of catalyzing a reaction that synthesizes isoprene from DM ADP fdimethyiallyl disphosphate) and said isoprene synthase variant is truncated at the N-termimis as compared to the parent eucalyptus isoprene synthase, In some embodiments, the parent eucalyptus isoprene synthase is E. globulus of SEQ ID NO: 6. The isoprene synthase variant may have a sequence that has at least five, at least ten, at least fifteen, at least twenty, at least twenty five or at least thirty amino acids truncated from the N-terminus as compared to the parent eucalyptus isoprene synthase.

[0016] Alternatively, the isoprene synthase variant has a sequence that has five to thirty five, ten to thirty five, fifteen to thirty five, twenty to thirty five, twenty five to thirty five, or thirty to thirty five amino acids truncated from the N-terminus as compared to the parent eucalyptus isoprene synthase. In some embodiments, the isoprene synthase variant has a sequence that has thirty five amino acids tnincated from the N-terminus as compared to the parent eucalyptus isoprene synthase. In still other embodiments, the isoprene synthase variant has a sequence that has less than thirty five amino acids truncated from the N-terminus as compared to the parent eucalyptus isoprene synthase.

[0017] In still another aspect of the in vention, it is provided an isolated nucleic acid sequence having a sequence encoding a variant of a parent solanum pheilandrene synthase, said sequence operably linked to a promoter, wherein the variant is capable of catalyzing a reaction that synthesizes isoprene from DMADP (dimethylaliyl

disphosphate) and said variant is truncated at the N-terminus as compared to the parent solanum pheilandrene synthase. In some embodiments, the parent solanum pheilandrene synthase is S. lycopersicum of SEQ ID NO: 23. The isoprene synthase variant may have a sequence that has at least five, at least ten, at least fifteen, at least twenty, at least twenty five, at least thirty, or at least thirty five amino acids truncated from the N- terniinus as compared to the parent solanum pheilandrene synthas.

[0018] Alternatively, the variant has a sequence that has five to thirty six, ten to thirty- six, fifteen to thirty six, twenty to thirty six, twenty five to thirty six, or thirty to thirty six amino acids truncated from the N-terminus as compared to the parent solanum pheilandrene synthas. In some embodiments, the variant has a sequence that has thirty six amino acids truncated from the N-terminus as compared to the parent solanum phel!andrene synthas. In still other embodiments, the variant has a sequence that has less than thirty six amino acids truncated from the N-terminus as compared to the parent solanum phellandrene synthas.

[0019] The promoter is, in some embodiments, a prokaryotic promoter, e.g. pTrc promoter. Those of skill in the art would be able to select other suitable prokaryotic promoter for use in the present invention. In preferred embodiments, the promoter is not a strong promoter.

[0020] In another aspect of the invention, it is provided an expression vector ocomprising the nucleic acid sequence described abo ve. In still another aspect of the invention, it is provided an isolated host cell comprising the heterologous nucleic acid sequence or expression vector described above. The host cell may in some embodiments be a bacterial cell, e.g. Escherichia coli. In some embodiments, to avoid rate-limiting steps in the isoprene production process, the host cell may further comprise one or more recombinant nucleic acid sequence of a MEP pathway gene, such as one or more selected from dxs, ispD, ispF, and idi.

[0021 ] In other embodiments of the in vention, it is pro vided an isolated isoprene synthase variant of a parent eucalyptus isoprene synthase, wherein said variant comprises a truncation in the N-terminal portion of isoprene synthase as compared to the parent eucalyptus isoprene synthase and wherein said variant is capable of catalyzing a reaction that synthesizes isoprene from DMADP (dimethylailyl disphosphate). Also provided is a variant of a parent solanum phellandrene synthase, wherein said variant comprises a truncation in the N-terminal portion of isoprene synthase as compared to the parent eucalyptus isoprene synthase and wherein said variant is capable of catalyzing a reaction that synthesizes isoprene from DMADP (dimethylailyl disphosphate). Also provided are methods of producing isoprene comprising: ( a) providing a host cell comprising an expression vector including the nucleic acid sequence described above; and (b) culturing the host cell under conditions suitable for producing isoprene, e.g. optionally further comprising (c) recovering the isoprene, e.g. still optionally further comprising (d) polymerizing the isoprene. BRI EF DESCRIPTION OF DRAWINGS

[0022] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component is labeled in ever}' drawing. In the drawings:

[0023] FIGURE 1: Kinetic behavior of isoprene synthase proteins from poplar

(Sciinitzler et al, (2005) Planta 222: 777-786). Top panels show normal and Hanes- Wooif plots of native (circle) and un-tagged recombinant (triangle) isoprene synthase. Bottom plots show^r N-terminal (open triangle) and C-terminal (open square) isoprene synthase, These results suggest isoprene synthase may exhibit substrate inhibition and cooperative kinetics.

[0024] FIGURE 2: Mechanism of hemiterpene synthesis from dimethylallyl diphosphate (DMADP).

[0025] FIGURE 3: Reaction mechanisms leading to hemiterpenes and monoterpenes. Cyclic monoterpenes require a rotation around die 2-3 bond, which is facilitated by reattaching the diphosphate to carbon 3 of the linalyS cation. This step is not required for making isoprene or acyclic monoterpenes.

[0026] FIGURE 4: Isoprene is made from dimethylallyl diphosphate by elimination of the diphosphate to yield a carbocation intermediate. Abstraction of any one of six protons of the two methyl groups leads to the formation of isoprene. Similar chemistry beginning with geranyl diphosphate leads to acyclic monoterpenes. Proton abstraction of any of the methyl hydrogens leads to beta myrcene while either E (trans) or Z (cis) beta ocimene is made depending upon which proton of carbon 4 is abstracted.

[0027] FIGURE S: Structure of bornyl diphosphate synthase (PDB 1N1 Z).

The black line starting at the upper right is the RRXgW unstructured arm. Gray ribbons are the β-subunit helices with no direct role in catalysis. The orange ribbon is helix A. of the a subunit. The short sand colored stretch is the C-terminus. The unstructured blac N-terminal amino acids, the orange A-helix amino acids, and the C-terminal amino acids must fold together in the region that joins the two subunits. This may be the cause of frequent misfolding found in isoprene synthases expressed in bacteria. Blue = reaction product, green = Mg, orange spheres = diphosphate, red = DDXXD sequence.

Visualized with MacPyMOL.

[0028] FIGURE 6: N and C termini of horny] diphosphate synthase and modeled ocimene synthase of snapdragon. Black = N terminus of BPPS, Gray = modeled N terminus of ocimene synthase. Purple = identically located leucine residues that will serve as the cut over location for a chimeric enzyme. Green is BPPS structure while blue is ocimene synthase modeled structure. The sand-colored line is the C- terminus of 13PPS that does not occur in ocimene synthase and will be cleaved in the chimeric enzyme. Modeled with SWISS-MODEL (Arnold et al., (2006) Bioinformatics 22: 195-201; Guex and Peitseh, (1997) Electrophoresis 18: 2714-2723; Schwede et al,, (2003) Nucleic Acids Research 31: 3381-3385).

[0029] FIGURE 7: View of the binding pocket taken from 3I4X (PDB crystal structure, drawn in MacPy ol). The purple color is surface provided by tyrosines, green are carbons of the DMADP analog DMASP. Exemplary mutations to the active site include, either alone or in combination, converting the lower two tyrosines to phenylalanine and converting one or more amino acids on the facing wall to

phenylalanine.

[0030] FIGURE 8: The in vivo isoprene production by E. coli BL21 strains expressing either E. globulus or P. alba isoprene synthases, with or without heterologous expression of dxs, ispD, ispF, and idi, was measured in a FIS as described in Example

[0031 ] FIGURE 9: Specific activity was obtained at various DMADP concentrations for E. globulus isoprene synthase and indicated by the light grey diamonds. Data were fitted to Equation 1 using the constants shown in Table 1 and the fitted equation is shown by the dark grey line as described in Example 3.5.

[0032] FIGURE 10: A comparison of the relative expression and solubility of E. globulus, M. alterniforia, and R. pseudoacacia isoprene synthases is shown. Total, soluble, and insoluble protein fractions are shown after induction of the indicated plasmids described in Example 4.4 and the E. globulus isoprene synthase expressed at high levels in soluble form. [0033] FIGURE 11: Specific activity was obtained according to Example 5.2 at various DMADP concentrations for S. ivcopersicum pheliandrene synthase and indicated by the plotted line.

DETAILED DESCRIPTION OF THE INVENTION

Defintions

[0034] This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0035] "Sustainable energy," as used herein, refers broadly to energy other than fossil fuels. Exemplary sources of sustainable energy include, but are not limited to, solar energy, water power, wind power, geothermal energy, wave energy, and energy produced from other sources, such as wastes and renewables.

[0036] As used herein, the term "hydrocarbon compounds" includes hydrocarbons and hydrocarbon derivatives, e.g., alcohol, halide, thiol, ether, aldehyde, ketone, carboxylic acid, ester, amine, and amide, etc.

[0037] As used herein, the term "carbonaceous chemical," refers to any carbon- containing chemical that can be produced by a biocatalyst. In various embodiments, the carbonaceous chemical is a hydrocarbon, while in other embodimenis, the chemical includes one or more heteroatoms, e.g., O, S, N, P and the like. The heteroatoms can be joined to one or more carbon atoms or, when there is more than one heteroatom. they are optionally joined to each other, e.g., S0₃H. The carbonaceous chemical can include residues that are alkyl, heteroalkyl, aryl or heteroaryi residues.

[0038] In various embodiments, the method and system of the invention is of use to produce a carbonaceous chemical in an "essentially pure state," As used herein, the term "essentially pure state," refers to a purity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% at least 99,5%, at least 99.9% or at least 99.95%. [0039] The term "alkyl," by itself or as part of substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include mono-, di- and multivalent radicals, having the number of carbon atoms designated (i.e. Ci-Cio means one to ten carbons). In some embodiments, the term "alkyl" means a straight or branched chain, or combinations thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n- propyi, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cycloliexvl, (cyciohexyl)methyl, cyc!opropyimethyS, homologs and isomers of, for example, n-penty!, n-hexyl, n-heptyl, n-octyl, and the like, An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4- pentadienyl), ethynyl, 1~ and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term "alkyl," unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as "heteroalkyl" with the difference that the heteroalkyl group, in order to qualify as an alkyl group, is linked to the remainder of the molecule through a carbon atom. Alkyl groups that are limited to hydrocarbon groups are termed "homoalkyl".

[0040] The term "alkenyl" by itself or as part of another substituent is used in its conventional sense, and refers to a radical derived from an alkene, as exemplified, but not limited, by substituted or unsubstituted vinyl and substituted or unsubstituted propenyl. Typically, an alkenyl group will have from 1 to 24 carbon atoms, with those groups having from 1 to 10 carbon atoms being useful examplars.

[0041] The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkaiie, as exemplified, but not limited, by -CH₂CH₂CH₂CH₂-, and further includes those groups described below as "heteroalkylene." Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being useful exemplars in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. [0042] The terms "alkoxy," "alkyl amino" and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.

[0043 j The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated n umber of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si, S, B and P and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. in some embodiments, the term

"heteroalkyl," by itself or in combination with another term, means a stable straight or branched chain, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom. The heteroatom(s) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH₂-CH₂-O-CH₃, -CH₂-CH₂-NH-CH 3 , -CH₂-CH₂-N(CH₃)-CH₃, -CH₂-S-CH₂-CH₃, -CI I K-S{0 Ί 1 :. - CH₂-CH₂-S(0)₂-CH₃, -H i C! ! -O-i I . -Si(CH₃)₃, -CH₂-CH=N-OCH₃, and -CH=CH- N(CH₃)-CH₃. Up to two heteroatoms may be consecutive, such as, for example, -CH₂- NH-OCH₃ and -CH₂-0-Si(CH₃)₃. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH₂-CH₂-S-CH₂-CH₂- and ~^H₂-S-CH₂-CH₂-NH-CH₂- , For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termmi (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for aikyiene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C0₂R'- represents both -C(0)OR' and -OC(0)R'.

[0044] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in

combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycie is attached to the remainder of the molecule, A "cycloalkyl" or "heterocycloalkyl" substituent may be attached to the remainder of the molecule directly or through a linker, wherein the linker is preferably a!ky!ene. Examples of eyeloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, l -(l,2,5,6-tetrahydropyridyl), 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran- 2~yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2- piperazinyl, and the like.

[0045] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloaikyi. For example, the term "halo(C₁-C₄)a3kyl" is mean to include, but not be limited to, trifluoromethyi, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0046] The term "aryl" means, unless otherwise stated, a polyunsaturated, aromatic, substituent that can be a single ring or multiple rings (preferably from 1 to 3 rings), which are fused together or linked covalently. The term "heteroaryl" refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, S, Si and B, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3- pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-irnidazoryl, pyrazinyl, 2-oxazolyl, 4-oxazo y , 2- phenyl-4-oxazolyi, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4- thiazolyi, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyi, 3-pyridyl, 4- pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5- indolyl, 1 -isoquinoiyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6- quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

[0047] For brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) optionally includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alky! groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., pheiioxymethyl, 2- pyridyloxymethyS, 3-(l-naphthyloxy)propyl, and the like).

[0048] Each of the above terms (e.g., "alkyl," "heteroalkyl," "a yl" and "heteroaryl") are meant to include both substituted and unsubstituted forms of the indic ated radical. Exemplary substituents for each type of radical are provided belo w.

[0049] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocyc!oalkyl, cycloa!kenyl, and heterocyeloalkeny!) are genetically referred to as "alkyl group substituents," and they can be one or more of a variety of groups selected from, but not limited to: substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycioalkyl, - OR', =0, =NR', \ -OR^' . -NR'R", -SR\ -halogen, -ΗϋΠΠ-Γ^'. -OC(0)R', -C(0)R', - i O -lV. -CONR'R", -OC(0)NR'R", -NR"C(0)R', -NR'-C(Q)NR"R"\ -NR"C(0)₂R', - NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR"', -S(0)R', -S(0)₂R', -S(0)₂NR'R", ~NRSQ₂R\ -CN and -NO? in a number ranging from zero to (2m'+l), where m' is the total number of carbon atoms in such radical. R', R", R'" and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the present inventions includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but not be limited to, 1-pyrrolidinyl and 4- morpholiny!. From the above discussion of substituents, one of skill in the art wi ll understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF₃ and -CH₂CF₃) and acyl (e.g., -C(0)CH₃, -C(0)CF₃, -C(0)CH₂OCH₃, and the like). [0050] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are genetically referred to as "aiyl group substituents." The substituents are selected from, for example: substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, -OR', =0, =NR', =N-OR', -NR'R", -SR', -halogen, - SiR'R"R"\ -OC(0)R', -C(0)R', ~C0₂R', -CONR'R", -OC(0)NR'R", -NR"C(Q)R\ -NR'-C(0)NR"R"', -NR"C(0)₂R', -NR-C(NR'R"R'")=NR"", -\R-V{ \R 1V) XR^" \ - S(0)R', -S(0)₂R', -S(⁽»--NR^' R^" -NRSG₂R', -CN and -N0₂, -R', -N₃, -CH(Ph)₂, fluoro(Ci-C4)alkoxy, and fluoro(C₁-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R", R"' and R"" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound of the present invention incl udes more than one R group, for example, each of the R groups is independently selected as are each R', R", R' " and R"" groups when more than one of these groups is present. f 0051] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(0)-(CRR')_q-U-, wherein T and U are independently -NR-, -0-, -CRR'~ or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH₂)_r-B-, wherein A and B are independently --CRR'-, -0-, - R-, -S-, -S(0)-, ~S(0)₂~, -S(0)₂ ^'NR'- or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR')_s-X-(CR"R'")_ci-, where s and d are

independently integers of from 0 to 3, and X is --0-, -NR'-, -S-, -8(0)-, -8(0)?-, or - S(0)₂NR'-. The substituents R, R', R" and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (CrC₆)alkyl.

[0052] As used herein, the term "acyl" describes a substituent containing a carbonyl residue, C(Q)R. Exemplary species for R include H, halogen, substituted or unsubstituted a!kyl, substituted or unsubstituted ary!, substituted or unsubstituted heteroaryl, and substituted or unsubstituted iieterocycloaikyi,

[0053] As used herein, the term "fused ring system" means at least two rings, wherein each ring has at least 2 atoms in common with another ring. "Fused ring systems" may include aromatic as well as non-aromatic rings. Examples of "fused ring systems" are naphthalenes, indoles, quinoiines, chromenes and the like.

[0054] As used herein, the term "heteroatom" includes oxygen (O), nitrogen (N), sulfur (S), silicon (Si) and boron (B).

[0055] The symbol "R" is a general abbreviation that represents a substituent group. Exemplary substituent groups include substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted iieterocycloaikyi groups.

[0056] By "amino acid" and "amino acid identity" as used herein is meant one of the 20 naturally occurring amino acids or any non-natural analogues that may be present at a specific, defined position. By "protein" herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e. "analogs", such as peptoids (see, Simon et al., PNAS USA 89(20):9367 (1992)) particularly when LC peptides are to be administered to a patient. Thus "amino acid", or "peptide residue", as used herein means both naturally occurring and synthetic amino acids. For example, homophenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.

[0057] "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chain may be in either the (R) or the (S) configuration, in the preferred embodiment, the amino acids are in the (S) or L-configuration. If non- naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradation.

[0058] As used herein, the terms "starting gene" and "parent gene" refer to a nucleic acid which is a gene of interest that encodes a protein of interest that is to be improved and/or changed using the present invention. Likewise, the terms "starting protein" and "parent protein" refer to a protein of interest that is to be improved and/or changed using the present invention.

[0059] In various embodiments, the nucleic acid is a recombinant nucleic acid. For instance, in some embodiments, an isoprene synthase nucleic acid is operably linked to another nucleic acid encoding all or a portion of another polypeptide such that the recombinant nucleic acid encodes a fusion polypeptide that includes an isoprene synthase and all or part of another polypeptide (e.g., a peptide that facilitates purification or detection of the fusion polypeptide, such as a His-tag). In some embodiments, part or ail of a recombinant nucleic acid is chemically synthesized. In some aspects, the nucleic acid is a heterologous nucleic acid. By "heterologous nucleic acid" is meant a nucleic acid whose nucleic acid sequence is not identical to that of another nucleic acid naturally found in the same host cell.

[0060] In particular embodiments the nucleic acid includes a segment of or the entire nucleic acid sequence of any naturally-occurring isoprene synthase nucleic acid. In some embodiments, the nucleic acid includes at least or about 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, or more contiguous nucleotides from a naturally-occurring isoprene synthase nucleic acid. In some aspects, the nucleic acid has one or more mutations compared to the sequence of a wild-type (i.e., a sequence occurring in nature) isoprene synthase nucleic acid. In some embodiments, the nucleic acid has one or more mutations (e.g., a silent mutation) that increase the transcription or translation of isoprene synthase nucleic acid. In some embodiments, the nucleic acid is a degenerate variant of any nucleic acid encoding an isoprene synthase polypeptide.

[0061] An isoprene synthase nucleic acid can be incorporated into a vector, such as an expression vector, using standard techniques (Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 2001 , which is hereby incorporated by reference in its entirety, particularly with respect to the screening of appropriate DNA sequences and the construction of vectors), Methods used to ligate the DN A construct comprising a nucleic acid of interest such as isoprene synthase, a promoter, a terminator, and other sequences and to insert them into a suitable vector are well known in the art, Additionally, vectors can be constructed using known recombination techniques (e.g., Invitrogen Life Technologies, Gateway Technology), [0062] As used herein, "homologous genes" refers to a pair of genes from different, but usually related species, which correspond to each other and which are identical or very similar to each other. The term encompasses genes that are separated by speciation (i.e., the development of new species) (e.g., orthoiogous genes), as well as genes that have been separated by genetic duplication (e.g., paralogous genes),

[0063] As used herein, "ortholog" and "orthoiogous genes" refer to genes in different species that have evolved from a common ancestral gene (i.e., a homologous gene) by speciation. Typical ly, orthologs retain the same function during the course of evolution. Identification of orthologs finds use in the reliable prediction of gene function in newly sequenced genomes.

[0064] As used herein, "paralog" and "paralogous genes" refer to genes that are related by duplication within a genome. While orthologs retain the same function through the course of evolution, paraiogs evolve new functions, even though some functions are often related to the original one.

[0065] As used herein, "homology" refers to sequence similarity or identity, with identity being preferred. This homology is determined using standard techniques known in the art (See e.g., Smith and Waterman, Adv Appl Math, 2:482, 1981 ; Needleman and Wunsch, J Mol Biol, 48:443, 1970; Pearson and Lipman, Proc Natl Acad Sci USA, 85:2444, 1988; programs such as GAP, BESTFIT, FASTA, and TFASTA in the

Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis,; and Devereux et al, Nucl Acid Res, 12:387-395, 1984).

[0066] As used herein, an "analogous sequence" of an isoprene synthase is one wherein the function of the gene is essentially the same as the gene based on the kudzu isoprene synthase. Additionally, analogous genes include at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity with the sequence of the kudzu isoprene synthase, in additional embodiments more than one of the above properties applies to the sequence. Analogous sequences are determined by known methods of sequence alignment. A commonly used alignment method is BLAST, although as indicated above and below, there are other methods that also find use in aligning sequences. [0067] "Percent sequence identity," "percent amino acid sequence identity," "percent gene sequence identity." and/or "percent nucleic acid/polynucleotide sequence identity," with respect to two amino acids, polynucleotide and/or gene sequences (as appropriate), refer to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. Thus, 80% amino acid sequence identity means that 80% of the amino acids in two optimally aligned polypeptide sequences are identical,

[0068] The phrase "substantially identical" in the context of two nucleic acids or polypeptides thus refers to a polynucleotide or polypeptide that comprising at least 70% sequence identity, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, preferably at least 98% and preferably at least 99% sequence identity as compared to a reference sequence using the programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).

[0069] Various isoprene synthase polypeptides and nucleic acids can be used in the compositions and methods of the invention. As used herein, "polypeptides" includes polypeptides, protems, peptides, fragments of polypeptides, and fusion polypeptides that include part or all of a first polypeptide (e.g., an isoprene synthase) and part or all of a second polypeptide (e.g., a peptide that facilitates purification or detection of the fusion polypeptide, such as a His-tag). In various embodiments, a polypeptide has at least or about 50, 100, 150, 175, 200, 250, 300, 350, 400, or more amino acids. In some embodiments, the polypeptide fragment contains at least or about 25, 50, 75, 100, 150, 200, 300, or more contiguous amino acids from a full-length polypeptide and has at least or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,, 90%, 95%, 100% or greater than 100% of an activity of a corresponding full-length polypeptide. In particular embodiments, the polypeptide includes a segment of or the entire amino acid sequence of any naturally-occurring isoprene synthase. In some embodiments, the polypeptide has one or more mutations compared to the sequence of a wild-type (i.e., a sequence occurring in nature) isoprene synthase.

[0070] In some embodiments, the polypeptide is a heterologous polypeptide. By "heterologous polypeptide" is meant a polypeptide whose amino acid sequence is not identical to that of another polypeptide naturally expressed in the same host ceil.

[0071] By "variant polypeptide" as used herein is meant a polypeptide sequence that differs from that of a parent polypeptide sequence by addition, deletion or substitution of at least one amino acid modification. Variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the nucleic acid sequence that encodes it. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g. from about one to about ten amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent. The variant polypeptide sequence herein will preferably possess at least about 80% identity with a parent polypeptide sequence, and most preferably at least about 90% identity, more preferably at least about 95% identity, Accordingly, by "isoprene synthase variant" as used herein is meant an isoprene synthase sequence that differs from that of a parent isoprene synthase sequence by virtue of at least one amino acid modification.

[0072] As used herein, the terms "active site," "binding site" or "binding pocket" refer to a region of a polypeptide or a molecular complex comprising the polypeptide that, as a result of the primary amino acid sequence of the polypeptide and/or its three-dimensional shape, favorably associates with another chemical entity or compound including ligands or inhibitors. Thus, an active site may include or consist of features such as interfaces between domains. Chemical entities or compounds that may associate with an active site include, but are not limited to, compounds, ligands, cefaclors, substrates, inhibitors, agonists, antagonists, etc.

[0073] "Structural reaction residues" and "site-construction residues" refer to a three- dimensional collection of amino acids involved in an enzymatic reaction. For example, these would include those forming the active site, those coordinating metal ions and those forming the substrate bind region. In particular, those forming the flexible loops and N-terminus and the adjacent residues that stabilize the flexible segments when substrate is bound, in an exemplar}_' embodiment, the invention provides an enzyme with isoprene synthase activity that is a variant polypeptide modified by addition, deletion or substitution of at least one amino acid that is a structural reaction residue.

[0074] As used herein, "equivalent or homologous residues" refers to amino acid residues that are shared by certain proteins. Equivalent residues may be identified by determining homology at the level of tertiary stnicture for a terpene synthase (e.g., isoprene synthase) whose tertiary structure has been determined by x-ray

crystallography. Equivalent residues are defined as those for which the atomic coordinates of two (2) or more of the main chain atoms of a particular amino acid residue of the terpene synthase having putative equivalent residues and the substrate of interest (e.g., N on N, CA on CA, C on C and O on O) are within 0.2 nm and preferably 0.15 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein a toms of the terpene synthases and subs trates analyzed. The preferred model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available, determined using methods known to those skilled in the art of crystallography and protein characterization/analysis. For example, equivalent residues which are functionally analogous to a specific residue of isoprene synthase are defined as those amino acids at a structurally homologous synthase which may adopt a conformation such that they either alter, modify, or contribute to protein stnicture, substrate binding or catalysis in a manner defined or attributed to a specific residue of isoprene synthase.

"Biocatalyst", as used herein refers to a microbe that is converts a carbon source into a product, in this case isoprene, that is expelled from the microbe that produced it.

THE EMBODIMENTS

10075] Embodiments of the present in vention provide a key component of ne w energy and chemical industry solutions that are not dependent on fossil fuels and are

consequently sustainable both environmentally and economically. The present invention provides compositions for producing isoprene. Specifically, the composition includes no vel enzymes capable of producing isoprene, some of which are isoprene synthases, that have favorable characteristics in isolation or when expressed in a biocataiyst. In either case they are useful for the efficient and economical production of isoprene from a variety of carbonaceous feedstocks.

Isoprene Synthase Enzyme Kinetics

[0076] Isoprene synthase genes have been cloned and characterized from two plant genera: Populus (poplars and aspen) (Miller, et al., (2001) Planta 213: 483-487; Sharkey, et al, (2005) Plant Physiology 137: 700-712; Sasaki, et al, (2005) FEES Letters 579: 2514-2518 and Behnke, et al., (2007) The Plant Journal 51 : 485-499) and Pueraria montana (kudzu) (Sharkey, et al., (2005) Plant Physiology 137: 700-712).

[0077] The enzyme catalyzes the divalent cation-dependent, irreversible, and stoichiometric conversion of dimethyiallyi diphosphate (DMADP) to isoprene and pyrophosphate. The wild type protein, first purified from aspen leaves, was determined to be functional as a heterodimer (Sil ver and Fall, (1995 ) J. Biological Chemistry 270: 13010-13016). The associated monomers were found to be similar, but not identical (62 and 58 kDa). However, monomers and/or homodimers have activity since single gene products can make isoprene. The native enzyme is sensitive to denaturation due to dilution as well as oxidation, and therefore the samples were kept concentrated and in a high concentration of DTT. This suggests that a free thiol may be required for catalytic activity. The enzyme required a divalent cation for activity, either Mg ^r or Mn'⁺, Kinetic analysis of the native enzyme demonstrates maximal activity at pH 8.0. The apparent k_cat and K_m for the enzyme were estimated to be 1.7 s^"1 and 8 niM respectively, but the enzyme also exhibited significant substrate inhibition (Eq. 1). The highest activity of the native protein was estimated to be 900 nmol isoprene min ¹ mg-1 a t 10 raM DMADP.

Equation 1 . Enzymatic rate equation for enzyme with substrate inhibition.

[0078] Characterization of the recombinant hybrid poplar protein expressed in E. coli yielded additional insights into this enzyme, Activity was highest from protein obtained when a leader signal peptide was deleted from the gene (Miller, et al., (2001 ) Planta 213: 483-487). Recombinant proteins with N- and C-terminal polyhistidine tags were compared to the unmodified recombinant protein as well as native protein isolated from hybrid poplars (Schnitzier et aL (2005) Planta 222: 777-786). Unlike the protein from aspen, the hybrid poplar protein and its recombinant versions were all monomelic, although the purification conditions were different from the previous work with the aspen enzyme. This suggests that the heterodimer observed in the aspen leaves may be comprised of alternatively modified versions of the same gene product. The addition of the polyhistidine tags to the protein significantly affected the activity of the recombinant isoprene synthase protein. All of the proteins exhibited a broad pH optimum of 8.0 except for the protein with N-terminal tag, which exhibited a pH optimum of 9.0. All of the enzymes had temperature optima of 40 °C except for the protein with the (^"'-terminal tag, which was more thermostable (optimal activity at 45-50 °C). All of the proteins also displayed interesting kinetic behavior (Figure 1). When plotted in a standard ichaelis- Menten format, it appears that these enzymes also suffer from substrate inhibition above 10 mM DMADP. However, by examining Hanes-Woolf plots, it appears that the enzymes also exhibit a sigmoidal substrate dependency, indicati ve of positi ve

cooperativity (Eq. 2, Figure 1), and this is most pronounced in the absence of the polyhistidine tag. Positi ve eo-operativity is unusual but not unheard of in monomeric enzymes. Apparent K_m values obtained from the data were in the range of 2-3 mM for all of the poplar enzymes.

Equation 2. Enzymatic rate equation for positive co-operativity.

E,k S"

Rate =—^ί-^^ί

K_u + S"

[0079] Recently, a modified Michaelis-Menten equation allowing for both

cooperativity and substrate inhibition has been shown to fit methyl butenol and isoprene production of methyl butenol synthase another hemiterpene sythnase (Eq. 3) (Gray et al, (2011) http://www.jbc.org/content/early/2011/04/19/jbc.Ml 11.237438.short). In this equation H is the Hill coefficient describing cooperativity, K_ls is the binding affinity of the substrate to the enzyme-substrate complex and x is the number of substrate molecules that could bind to and inactivate the enzyme substrate complex. It is likely that this equation will most closely model the activity of isoprene synthases.

Equation 3. a modified Michaelis-Menten equation allowing for both

cooperativity and substrate inhibition

[0080] The recombinant enzyme from Populus alba was not expressed with a polyhistidine tag, and was found to be very similar to that of the hybrid poplar (Sasaki, et al, (2005) FEBS Letters 579: 2514-2518). This protein was also monomelic, had a pH optima of 8.0, a temperature optima of 40 °C, and an apparent ._m of 8.7 mM. No kc_at was determined.

[0081] The recombinant enzyme from kudzu was expressed without the leader sequence and with an N-terminal polyhistidine tag (Sharkey, et al., (2005) Plant

Physiology 137: 700-712). The kinetic beha vior of the enzyme was measured, and a clear sigmoidal dependence on the substrate concentration was observed (Eq. 2), again indicative of cooperative behavior. This results in an apparent K_m of 7.7 mM, Hill coefficient (n) of 4.1 , and a k_cat of 0.088 mol mol^"1 s^"1. The enzyme was not

characterized further, nor was the aspen isoprene synthase protein, which was also cloned in this work,

Isoprene Synthase Mechanism of Action

[0082] The mechanism of action of isoprene synthase can be hypothesized based on the reaction mechanisms of other terpene synthases (Figure 2). The most commonly used reaction model is the well-accepted mechanism for limonene synthase

(Rajaonarivony et al, (1992) Arch. Biochem. Biophys. 296: 49-57). A ten-carbon precursor (geranyl diphosphate, GDP) binds to the limonene synthase enzyme. Three magnesium atoms are coordinated around the diphosphate and a signature sequence of DDXXD interacts with some of these magnesiums. A long arm of the protein that has a highly conserved double arginme (RR) moves to close off the active site. This RR has a conserved glutamate (E) a few base pairs upstream and this constitutes the beginning (N- terminus) of the functional enzyme. The E-RRXgW beginning of the enzyme binds and closes the active site. The RR is required for the first steps of the reaction. The diphosphate is released from the end of GDP making a linalyi cation (Figure 3). The diphosphate is reattached at carbon 3, This reattachment ensures that the bond between carbons 2 and 3 is a single bond, and this allows rotation around that bond required to convert the linalyi cation to a neryl cation. The neryl cation is the immediate precursor of cyclic monoterpenes (e.g. limonene). Proton abstraction leads to quenching of the cation and typically bond formation between carbons 1 and 6 making a six-membered ring.

[0083] The complicated detachment and reattachment of the diphosphate is almost universal among monoterpene synthases but two notable exceptions exist. First, it was reported that snapdragon flo wers have a different class of monoterpene synthases that do not have the canonical RRXgW. These enzymes are able to make acyclic monoterpenes (ocimene and myrcene) but not cyclic monoterpenes (Dudareva et al., (2003) The Plant Cell 15: 1227-12 1). It was speculated that the lack of the double R prevents this enzyme from making the linalyi to neryl conversion and so cannot form cyclic rnonoterpenes. However, acyclic monoterpenes can be formed from the linalyi cation. Second, it was reported that trichomes of tomato have a enzyme (a phellandrene synthase) that uses neryl diphosphate (NDP), the cis isomer of GDP (Schilmiller et al, (2009) PNAS 106: 10865-10870). This molecule forms the neryl cation as soon as the diphosphate is removed and so does not require the rotation around the 2-3 bond required when G DP is the starting material. This simplifies the reaction mechanism. This enzyme can use GDP at a low rate. Like many terpene synthases, phellandrene synthase makes various products. When NDP is the substrate only cyclic products are made but when GDP is the substrate some acyclic monoterpenes are made. This indicates that the acyclic monoterpenes are made from the linalyi cation and when NDP is supplied the neryl cation is not converted to the linalyi cation.

[0084] Additional insight into terpene synthases come from a recent report on kaurene synthase. The bifunctional copalyl diphosphate synthase/kaurene synthase of

Physcomatrella patens has a similar mechanism in which a cation is quenched by proton abstraction. Most kaurene synthases make only kaurene but the P. patens enzyme will sometimes quench the cation with water resulting in kaurane (Kawaide et al, (2011) FEBS Journal. 278: 123-133). A similar reaction in a hemiterpene synthase would produce methyl butenol. Unpublished sequences of methyl butenol synthase show a similar amino acid substitution as the P, patens kaurane synthase and may explain why this enzyme makes methyl butenol instead of isoprene.

[0085] Finally, there are now additional insights into what makes an enzyme an isoprene synthase. Modeling (Sharkey et al., (2005) Plant Physiology 137: 700-712) and the crystal structure ( oksal et al., (2010) JAM Biol 402: 363-373) show that two phenylalanines reduce the relative size of the active site in isoprene synthase explaining why these enzymes may not function as monoterpene synthases. However, if this were the only explanation for what makes an isoprene synthase then ail monoterpene synthases should act as isoprene synthases. At the very least all acyclic monoterpene synthases should show isoprene synthase activity. This is because the acyclic monoterpene synthase reaction involves proton abstraction from either the methyl carbon to make myrcene of from carbon 4 to make the ocimenes (which ocimene is made depends upon which proton is abstracted) (Figure 4). While it has not been extensively tested, there are no reports of significant isoprene synthase activity in an ocimene or myrcene synthase. Extensive analyses of isoprene synthase active sites have shown that not only are there two phenylalanines making the site smaller, several other

phenylalanines line the active site. This makes the active site rich in pi electrons (from the double bonds in phenylalanine). A strong but often overlooked molecular interaction is cation-pi interactions (Dougherty, (1996) Science 271: 163-169; Gallivan and

Dougherty, (1999) PNAS 96: 9459-9464). The cation made upon elimination of the diphosphate from DMADP could be stabilized by cation-pi interactions with some of the phenylalanines of the active site. This could give time for the proton abstraction and proton abstraction of any of the six methyl protons would result in the formation of isoprene. Cation quenching by proton abstraction may occur by a number of different mechanisms and may account for the large variety of terpenes that are made by terpene synthases. [0086] Current published isoprene synthases (from the Poplar family and kudzu) have a low catalytic activity (k_cat) and high K_m. The present invention provides an isoprene synthase in which at least one of these parameters are improved. In an exemplar}' embodiment, one or both of these parameters is impro ved by a t least about 1, by at least about two or by at least about three orders of magnitude. In various embodiments, the improved k_cai provides higher yields, e.g., at maximum substrate availability. In various embodiments, the lower _m provides advantages in the synthesis of isoprene, e.g., the alleviation of probable feedback on substrate production. In an exemplary embodiment, the improved K_m enhances the ease of steering carbon into isoprene production. In various embodiments the invention provides enzymes with isoprene synthase activity with modified stability and/or solubility (e.g., in the fermentation medium or intracellular mileau), thereby providing an increased le vel of isoprene synthase that can be expressed in a cell or in vitro system. In an exemplary embodiment the alterations to stability and/or solubility increase isoprene synthase activity in a cell or in vitro systems by at least about an order of magnitude, at least about two orders of magnitude or at least about three orders of magnitude. It is likely that a total increase in isoprene synthase activity in a cell or in vitro system of approximately three orders of magnitude will be necessary to make bio-catalyst driven production of isoprene economically viable.

Accordingly, the present invention provides enzymes with an isoprene synthase activity that is at least about one order of magnitude greater, at least about two orders of magnitude greater and at least about three orders of magnitude greater in a cell or in vitro environment than the corresponding activity in the parent polypeptide or provide a platform for directed evolution or structure-based prediction of additional changes that will achieve such favourable kinetics..

[0087] The following sections set out how these large gains in isoprene synthase activity are attained.

identifying novel isoprene synthases with improved native characteristics

[0088] In an exemplary embodiment, the invention provides a novel polypeptide with isoprene synthase activity.

[0089] All efforts to date to impro ve isoprene synthase have relied on j ust two platforms, the group of highly similar Populus sequences (SEQ ID NOs: 1-4) and the kudzu sequence (SEQ ID NO: 5). These have been chosen on the basis of availability, not suitability. With many new sequences now available to us three more platforms are added, the Eucalyptus {Eucalyptus globulus) (SEQ ID NO: 6), the Tea Tree {Melaleuca alternifolia) (SEQ ID NO: 7) and the Black Locust {Robinia pseudoacacia) (SEQ ID NO: 8) isoprene synthases which are still somewhat related to the current genes, have a very different evolutionary history and some unique properties, isoprene synthase is similar enough to the angiosperm terpene synthases that structures can be reasonably predicted but different enough to provide significant novelty from which to select high value constructs.

Gymnosperm Hemiterpene Based Approach; Converting methyl butenol synthases into isoprene synthases

[0090] In an exemplary embodiment, the invention provides a variant polypeptide with isoprene synthase activity based on a parent methyl butenol synthase polypeptide or other hemiterpene synthase from gymnosperm species converted by addition, deletion or substitution of one or more amino acid into a polypeptide with isoprene synthase activity.

[0091 ] Currently a highly active methyl butenol synthase has been cloned and expressed from Pinus sabiniana (SEQ ID NO: 9) and an inactive isoprene synthase has been cloned from Picea pungens (SEQ ID NO: 10). Because of new insight into the control of cation quenching by water (Kawaide et ai, (2011) FEBS Journal 278:123- 133) the active methyl butenol synthase, which currently makes 3% of its product as isoprene, is converted to produce all isoprene with a single amino acid change (S440F or S440Y) (SEQ ID NOs: 1 1 and 12).

Acyclic-Monoterpene-Synthase-Based Approach One: Replacement of an

unstructured arm of characterized isoprene synthases with the structured arm of myrcene synthase

[0092] In various embodiments, the invention provides a variant polypeptide with isoprene synthase activity. In these embodiments, the unstructured arm of isoprene synthase is replaced with the structured arm of myrcene synthase in a variant

polypeptide.

[0093] The Tps-g group of monoterpene synthases (Dudareva et al., (2003) The Plant Cell 15: 1227-1241) are the seventh terpenoid synthase subfamily identified. The subfamilies identified earlier are designated Tps-a through 7 ? -/(Bohimami et al., (1998) PNAS 95: 4126-4133). The Tps-g subfamily makes acyclic monoterpenes, presumably because they have a ver different initial domain, in that they lack the RRxgW motif, which is a characteristic feature of the other families of monoterpene synthases: the Tsp- b family of angiosperm monoterpene synthases and the Tsp-d family of conifer monoterpene synthases. This initial domain closes off the active site and likely moves during catalysis. It is often unresolved in crystal structures but is resolved in PDB ΓΝ1Ζ (Figure 5). Lack of the RR in Tps-g enzymes likely prevents the linalyl to neryl cation conversion required for cyclic monoterpenes but this conversion is not required for acyclic synthases or hemiterpene synthases. This arm is likely to be a significant component of the difficulty in expressing isoprene synthases because of its flexibility and lack of secondary structure. Chimeric proteins based on the native isoprene synthases are generated with the long initial arm replaced by amino acids found in Tps-g genes. These genes have a very different initial sequence with amino acids that may help form secondary structure. Homology modeling is used to identify the exact location for switching from the isoprene synthase gene sequence to the Tps-g sequence.

Specifically, fusion should occur at a conserved leucine at the base of the long arm of all isoprene synthases. The arm substitution is designed to help solubility and has potential to improve the activity in other ways including but not limited to: not allowing the linalyl to neryl rotation that might slow down the enzyme.

[0094] By switching from one enzyme to another at exactly this leucine there is the best chance of making an enzyme that retains the desired activity. The following indicate the position of this leucine in the isoprene synthase identified to date. It is predicted that all future native isoprene synthases will also contain this conserved leucine. The position of this conserved leucine is LI 48 in the Populus sequence (SEQ ID NOs: 1, 2, 3 and 4), L153 in the kudzu (SEQ NO: 5), 1 .137 in the Eucalyptus sequence (SEQ ID NO: 6), L137 in the Melaleuca sequence (SEQ ID NO: 8) and L91 of the Robinia sequence (S EQ ID NO: 9). All amino acids -terminal of this leucine are replaced with amino acids D46 through L 100 of the Snapdragon gene AY 195608 (SEQ ID NO: 13) (Figure 6) to produce the hybrids (SEQ ID NOs: 14, 15, 16, 17, and 18). In addition, any changes are made that might be needed to accommodate the new arm sequence are made. Acyclic Monoterpene Synthase Based Approach Two; Conversion of acyclic monoterpene synthases into isoprene synthases by replacing amino acids in their active sites

[0095] In an exemplary embodiment, the invention provides a variant polypeptide with isoprene synthase activity. Enzymes according to this embodiment, are produced by converting a parent acyclic monoterpene synthase polypeptide into an isoprene synthase by adding, deleting or substituting one or more amino acid residues.

[0096] Given the hypothesis that cation-pi interactions may play a significant role, enzymes that start with diphosphate removal from an allylic isoprenoids precursor can be converted into a variant polypeptide with isoprene synthase activity, Suitable families of enzymes belong to the following named groups further identified by their Enzyme Commission (EC) Numbers. Enzymes that remove diphosphate from geranyl diphosphate including: myrcene synthases (EC 4.2.3.15), S-linalool synthases (EC 4.2.3.25) and R-linalool synthases (EC 4.2.3.26). Enzymes that remove diphosphate from farnesyl diphosphate including: a-farnesene synthase (EC 4.2.3.46} and β-farnesene synthase (EC 4.2.3.47), (3S, 6E) nerolidol synthase (EC 4.2.3.48) and (3R, 6E) nerolidol synthase (EC 4.2.3.49). Enzymes that remove diphosphate from neryi diphosphate including: β-phellandrene synthase (EC 4.2.3.52).

[0097] In order to convert the parent polypeptide to the variant polypeptide one or more amino acids are substituted that line the active site, at the bottom of the pocket that would accommodate DMADP, with phenylalanine, tyrosine, or tryptophan to increase the surface area of the active site that is accounted for by pi electrons. Homology modeling may further refine the predictions of which amino acids should be converted into which other amino acids. One specific example is provided to illustrate the broader point: conversion of Snapdragon myrcene synthase (SEQ ID NO: 13) into an isoprene synthase by conversion of V331 and/or V444 to phenylalanine (F shown), tyrosine (Y) or tryptophan (W) (not shown) (SEQ ID NOs: 20, 21 and 22).

Conversion of pheJIandrene synthase Into an isoprene producing enzyme

[0098] In various embodiments, the invention provides an enzyme with isoprene synthase activity by pro viding a variant polypeptide derived from parent pheilandrene synthase polypeptide. The phellandrene synthase is converted, by variation (addition, deletion or substitution) of one or more amino acid residues in the protein. In various embodiments, the amino acid is in the active site. Many monoterpenes are produced by monoterpene synthases from the prenyl diphosphate precursor geranyl diphosphate (G PP) in which two isoprene units are joined in the trans (E) configuration. The type VI glandular triehomes, small hair- like structures, on the surface of M82 variety of cultivated tomato (Solarium lycopersicum produce an unusual monoterpene synthase that uses neryl diphosphate (NPP) the cis-isomer of GPP as a substrate. This

monoterpene synthase is a β-phellandrene synthase (EC 4.2.3.52) called PHS1

(Schilmiller et al, (2009) PNAS 106: 10865-10870). PHS1 has a high k_cat producing more than 4 molecules of phellandrene per second using NPP as a substrate.

Phellandrene synthase is different from typical cyclic monoterpene synthases in that it does not have the RR at the beginning of the active site. The reason for this is that it does not have to do the linalyl to neryl cation chemical conversion that most cyclic monoterpene synthases have to do. Based on homology to the stracture of isoprene synthases, it is converted into an isoprene producing enzyme by either or both of the mutations V524F and M672F (SEQ ID NO: 23).

Pi-Electron Based Approaches

[0099] In an exemplary embodiment, the invention provides a variant polypeptide in which the pi-electron structure of the active site of the relevant parent polypeptide is varied to lower _m, increase k_cat or both, or to convert an enzyme without (or with minimal) isoprene synthase activity into an enzyme with isoprene synthase activity.

[0100] It is postulated that if pi electrons are important to stabilizing the cation intermediate, this could slow the reaction mechanism by making the reaction

intermediate too stable. This might be evolutionarily advantageous by keeping the K_m high (with an active site slightly smaller than optimal). This would ensure that isoprene synthase did not interfere with other metabolism needed by plants. However, in an industrial setting a low K_m form of the enzyme has significant advantages. One or the other of the two site-constructing pheny lalanines, shown in bold and underlined in Populus tremuloides isoprene synthase (IspS) F338 and F485 (SEQ ID NO: 2.4), Populus alba IspS F338 and F485 (SEQ ID NO: 25), Populus nigra IspS F338 and F485 (SEQ ID NO: 26), Populus trichocarpa IspS F301 and F448 (SEQ ID NO: 27), Pueraria Montana IspS F343 and F493 (SEQ ID NO: 28), Eucalyptus globulus IspS F326 and F473 (SEQ ID NO: 29), Melaleuca alternifolia 1326 and F473 (SEQ ID NO: 30) and Robinia pseudoaccacia IspS F281 and F428 (SEQ ID NO: 31), with a tyrosine or tryptophan and the other phenylalanine are replaced with a smaller amino acid, for example leucine, to provide sufficient pi-electron character to the active site without unduly restricting the binding of DMADP. The active site is examined to find other potential locations for a phenylalanine or other pi-electron contributing amino acids in ways that do not constrict the active site. Variation in the location of phenylalanines has been found in unpublished sequences of isoprene synthases from spruce F337 and F541 (Picea pungens) (SEQ ID NO: 10) and hops F354 and F530 (Humulus lupulus) (SEQ ID NO: 32) and indicating that it should be possible to vary the location of the pi-electron contributing amino acids without destroying enzyme activity.

Conversion of dimeth lalfyl tryptophan synthase into an isoprene producing enzyme

10101] In an exemplar}_' embodiment, the present invention provides a variant polypeptide of a parent dimethyiallyl tryptophan synthase with isoprene synthase activity. Exemplary enzymes according to this embodiment have one or more amino acid remo ved, added or substituted relative to the amino acid sequence of the parent.

[0102] A variety of enzymes that use dimethylally diphosphate (DMADP) and a cation intermediate are all good targets for conversion into isoprene synthases. One example is a gene unrelated to known isoprene synthases, dimethyiallyl tryptophan synthase. This protein uses DM ADP and has a high (poor) K_m (8 μΜ) and higher k_cat (0.37 per second). The structure of this enzyme is known (3I4X, PDB) (Figure 7) and it is known to use a carbocation intermediate of DMADP similar to that in the isoprene synthase reaction (Luk and Tanner, (2009) J Am Chem Soc 131: 13932-13933). Based on knowledge of isoprene synthase evolution it is predicted that adding phenylalanine residues to locations likely to reduce the size of the binding pocket or con verting some tyrosine residues that line the pocket with phenylalanine residues have potential for converting this enzyme into a novel isoprene synthase. The binding pocket residues on dimethylallyl tryptophan synthase are L81, T82, R83, Y191, Y345 and Y398 (SEQ ID NO: 33). One, two, three, four, five or al l six of these residues are converted to phenylalanine.

[0103] The families of enzymes that use dimethylaliy diphosphate (DMADP) and a cation intermediate and can be converted into isoprene synthases to form a variant polypeptide include the following identified by name and enzyme commission (EC) number include but are not limited to: dimethylallyltranstransferases (EC 2.5.1.1 ), geranyltranstransf erases (EC 2.5.1.10), trans-octaprenyltranstransferases (EC 2.5.1.1 1), adenylate dimethylallytranstransferases (EC 2.5.1.27), dimethylallylcistransferases (EC 2.5,1.28), famesyltranstransferase (EC 2.5,1.29), trihydroxypterocarpan

dimethylallyltransferases (EC 2.5.1.33), dimethylallytryptophan synthase (EC 2,5.1 ,34), chyrsanthemyl diphosphate synthase (EC 2.5.1.67) and lavandulyl diphosphate synthase (EC 2.5.1.11).

Conversion of pentalenene synthase into an isoprene producing enzyme

[0104] In an exemplary embodiment, the invention provides a variant polypeptide of a parent pentalenene synthase that produces isoprene, The variant is prepared by the addition, deletion or substitution of one or more amino acid relative to the parent polypeptide,

[0105] Pentalenene is a complex tricyclic sesquiterpene (a terpenoid containing 15 carbon atoms), that is produced from farnesyl diphosphate by the action of pentalenene synthase (EC. 4.3.2.7). Pentalenene, is the hydrocarbon precursor of the

pentaleno lactone family of antibiotics. The crystal structure of pentalene synthase has been resolved (Lesburg et al., (1997) Science 277: 1820-1824), revealing the fact that it is unusually a single domain terpenoid synthase. For this reason it may behave much better in bacteria and in other ways be more stable. Pentalenene synthase of Streptomyces sp. UC53 I9 (SEQ ID NO: 34) is converted into an isoprene synthase by converting V179 and/or N219 into a F, W or Y (SEQ ID NOs: 35, 36 and 37). Example 1: Isoprene synthase expression and purification

[0106] Utilizing theoretical amino acid protein sequences in this disclosure, bacterial expression constructs, N-terminal or C-terminal flag tagged, capable of regulated expression of recombinant proteins described in this disclosure are generated, The backbone vector used is the pET vector system (Novagen) or similar, The gene sequences are codon optimized for bacterial expression and synthetically generated to match the amino acid sequences in this disclosure with a Flag tag. The synthetically created gene sequences is then sub-cloned using unique, engineered restriction sites into a commercially-available expression construct (e.g. Novagen) for expression of the protein. Upon transformation into DH5a, six clones for each construct are tested for presence of the insert by restriction digest analysis.

[0107] The expression constructs generated are used to transform E. coli BL21(DE3) to generate clones for expression screening. Following the selection of the best expressing clones, induction experiments (3hrs and overnight, 37°C and 18°C) are performed on the selected clones. SDS-PAGE or immunoblot is run to compare protein expression (total and soluble) and the optimal parameters for soluble expression are chosen for production at the IE scale. The selected clones are grown in LB medium and the cultures are induced at an OD between 0.8 and 1.0. After induction, cell paste is harvested by centrifugation and stored at -20°C until purification. SDS-PAGE or Western blot is performed on an analytical sample to confirm the protein expression.

[0108] Recombinant protein is purified from cell paste using Flag affinity

chromatography. Briefly, cells will be iysed using a microfluidics homogeiiizer and clarified by centrifugation. Clarified lysate is loaded onto a chromatography column containing M2 anti-Flag resin. The resin is washed to remove contaminating proteins and bound protein is competitively eluted with flag peptide according to standard methods. Fractions containing the protein of interest are pooled, dialyzed into PBS + 5% glycerol, concentrated, aliquotted, and stored frozen at -8Q°C pending studies. All steps of the purification are monitored by SDS-PAGE-Coomassie.

[0109] For the un-tagged recombinant proteins, an alternative separation protocol is used based on ion exchange chromatography (Silver and Fall (1995) J Biol. Chem. 270: 13010-13016). The pi of the recombinant protein is 5.5, and this will be is exploited to separate the proteins on a Sepharose Q column. Active fractions from the ion exchange column are also concentrated and purified by gel filtration and handled the same as the tagged proteins.

Example 2: Isoprene synthase assays,

[0110] One of the products of the isoprene synthase reaction is pyrophosphate, and a continuous enzyme-coupled assay has been developed for assaying phosphate (Webb (1992) PNAS 89: 4884-4887), and this has been extended to monitor pyrophosphate for enzymatic kinetic parameter estimation (Miller et al. (2007) J. Bacteriology 189: 8196- 8205). There are a number of commercially available kits for measuring enzyme kinetics by monitoring pyrophosphate production. These include but are not limited to a

PPiLight (Lonza), PiPer Pyrophosphate Assay kit (Invitrogen), as well as EnzCheck (Molecular Probes). These assays are modified for the continuous measurement of isoprene synthase mutant kinetics.

[0111 j In order to determine kinetic parameters for the mutant and wild type enzymes, enzymatic reaction rates are needed at varying substrate concentrations. Since this enzyme may display substrate inhibition and/or substrate cooperativeness (Eq. 1, Eq. 2 and together Eq. 3), it will be important to collect appropriate data in order to distinguish between, and quantify the impacts of, these effects. Initial rates of the enzymes are determined with varying initial DMADP concentrations. In order to ensure that only initial rates are estimated, the rates are calculated only during the consumption of the first 10% of the initial DMADP substrate. At least 4 values above and below the K_m of the enzyme are used so that an accurate estimation of the kinetic parameters can be made. The experimental data is fit to the appropriate enzymatic rate equations (Eq. 1, Eq. 2 and together Eq. 3) using a least squares method. If the data matches predictions that both substrate inhibition and positive cooperativeness are present, then Equation 3 will be used.

[0112] The performance of the mutan t isoprene synthase proteins is assessed using kinetic analysis described above. In addition, the activity of the isoprene synthase proteins in a more oxidative environment (with a decreased concentration of DTT) is explored. Once beneficial mutations are identified from the analysis of the mutants, the mutations can be combined to determine if the beneficial impacts are additive . In addition, the mutants are examined with and without a tag, as this has previously been shown to affect the performance of the recombinant isoprene synthase enzymes. It is anticipated that this approach may be able to significantly improve the k_C3t, K_m, substrate inhibition, and coopertivity issues with the isoprene synthase enzyme. In addition, it may be possible to impact the susceptibility of the enzyme to oxidative stress,

10113] Further, the acti vity of the isoprene synthase proteins at a range of different temperatures is explored, Isoprene synthases produce bursts of isoprene in response to transient heat stress. Repeating the assay described above at a range of temperatures enables examination of this phenomenon on isolated proteins in vitro.

Confirming the production of isoprene and analysis of other products

[0114] Hemiterpene and other terpenoid synthases may produce more than one product. In addition to the pyrophosphate assay, samples of the product of these enzymes are adsorbed onto a Solid Phase Micro Extraction (SPME) fiber and then the fiber is heated in a GC MS.

Example 3

Example 3.1 : Plasmids and Strains pTrcHis2B-PHspS

[0115] The P, alba isoprene synthase (except the N terminal plastid targeting sequence, amino acids 1 to 36) was codon optimized for E. coli expression and synthesized by GenScript. A 1.6 kb Bsal to Xhol fragment was cloned into the Ncol and Xhol sites of pTrcHis2B to yield pTrcHis2B-Pl IspS (SEQ ID NO:38). p TrcHis2B~E2ispS

[0116] The E. globulus isoprene synthase (except the N terminal plastid targeting sequence, amino acids 1 to 35) was codon optimized for is. coli expression and synthesized by GenScript. A 1.6 kb Ncol to Xhol fragment was cloned into the Ncol and Xhol sites of pTrcHis2B to yield pTrcHis2B-E2]spS (SEQ ID NO:39). pJexpress401-E4lspS-His

[0117] The E. globulus isoprene synthase (except the N terminal plastid targeting sequence, amino acids 1 to 35) with a C terminal linker and 6 histidine motif was codon optimized for E. coli expression, synthesized, and cloned into pJexpress401 by DNA2.0 (SEQ ID NO:40, pJExpress401-E4IspS-His).

[0118] Four E. coli MEP pathway genes (dxs, ispD, ispF, and idi) were placed under control of the pTrc promoter and cloned into the Smal site of pCL1920 (SEQ ID NO:41, pCL-SDFi).

Example 3.2: isoprene quantification

J0119] In vivo isoprene production was measured by injecting flask headspace samples into a Tianmei GC9700 GC equipped with a photoionization detector. The column was a PLOT-Q (Tianmei), the column temperature was 140 C, the injector temperature was 150 C, and the detector temperature was 150 C. The injection volume was 1 ml, gas. Isoprene standards were used to quantify the signal.

|ΌΪ 20] Samples to measure in vitro production were assayed on a fast isoprene sensor (Hills Scientific) per the manufacturer instructions. Isoprene standards were used to quantify the signal.

Example 3.3: In vivo isoprene production

[0121] To make pre-seed cultures, single colonies from each strain to be tested were used to inoculate 4 mL LB medium plus appropriate antibiotics and grown overnight at

37 C with shaking. To make seed cultures, for each strain, 1 mL of pre-seed was inoculated into 75 mL mineral medium (37.9 mM (NH )₂SQ₄, 8.3 mM MgS0₄, 36.8 mM

KH₂PO4, 85.1 mM a₂P0₄, 1.4 mM CaCl₂, 0.007 mM thiamine, 0.476 mM citric acid monohydrate, 0.107 mM MnS0₄, 0.018 mM I eSO ,. 0.007 mM ZnS0₄, 0.002 mM

Q1SO4, 0.003 mM C0CI2, 0.003 mM NaMoO) plus 10 g/L glucose, 5 g/L yeast extract, and appropriate antibiotics and cultured at 37 C with shaking for 10 hours. For each strain, the seed culture w^ras diluted to OD600 of 0.1 in 30 mL mineral medium plus 5 g/L glucose, 1 g/L yeast extract, appropriate antibiotics, and 0.4 mM IPTG and incubated at

30 C with shaking, Isoprene was measured at each time point then the headspace of each flask was flushed with a mix of 50% oxygen and 50% air. At 24 and 34 hours, additional glucose was added to each flask to a final concentration of 2 g/L.

Results

(0122] The E. globulus isoprene synthase, when expressed in E. coli cells, produces more isoprene than the P. alba isoprene synthase (Figure 8). E. coli BL21 strains expressing either E. globulus or P. alba isoprene synthases, plus or minus heterologous expression of dxs, ispD, ispF, and idi, were grown in minimal medium under aerobic conditions and isoprene production measured. Cumulative culture productivity of isoprene is shown for E. coli BL21 expressing heterologous MEP genes and ii. globulus isoprene synthase (inverted triangles), heterologous MEP genes and P. alba isoprene synthase (circles), E. globulus isoprene synthase (triangles), and P. alba isoprene synthase (squares).

Example 3.4: E. globulus isoprene synthase protein purification

[0123] Overnight seed culture of FM5 cells containing the Jexpress401 ~E4IspS-His piasmid were diluted 1 : 100 into 500 mL LB cultures and grown to OD600 -0.9 at 37 C, IPTG was added to a final concentration of 1 mM and induction occurred for ~4 hours at room temperature. Cultures were centrifuged and cell pellets frozen.

[0124] Frozen pellets were resuspended in lysis buffer (50 mM NaH₂P0₄, 300 mM NaCl, 10 mM imidazole, pH 8.0 with Roche Mini EDTA-free tablets per the

manufacturer's instructions) and cells dismpted by sonication. Ceil lysates were treated with RNAse A and DNAse I (Qiagen) and cleared by centrifugation. Cleared lysates were incubated with nickel NT A resin for 1 hour at 4 C then loaded into a disposable chromatography column. The column was washed with 5 times the bed volume with wash buffer (50 mM Xai l -PO ;. 300 mM NaCl, 10 mM imidazole, pH 8.0) then eluted with elution buffer (50 mM NaH₂P0₄, 300 mM NaCl. 250 mM imidazole, pH 8.0). Glycerol was added to 15% and the samples flash frozen in liquid nitrogen and stored at -80 C.

Example 3.5: In vitro protein kinetics

[0125] Purified isoprene synthase was assayed for kinetic properties using in vitro conversion of DMADP to isoprene. For each of six different concentrations of DMADP (0, 0.25, 0.5, 1, 2, and 4 mM), 75 uL of appropriately diluted DMADP solution was mixed with 73 uL reaction buffer (100 mM HEPES, 40 mM KC1, 20 mM MgCl₂, 10% glycerol, pH 7.8), 1 uL 1 mM DTT, and 2.5 uL of the purified isoprene synthase at 0.3 ug/mL. The reaction was allowed to occur in a sealed tube at 37 C for 12 minutes, after which 1 mL of headspace was injected into a F1S for measurement of the isoprene produced. Controls with no added protein were used as appropriate to subtract nonspecific isoprene production. Specific activity measurements were fitted to Equation 1 to determine KM, k_cat, and Kjs.

Equation 1 : Enzymatic rate equation for enzyme with substrate inhibition.

[0126] The K_M tor E. globulus isoprene synthase is lower (more preferable for production of isoprene) than is reported for other isoprene synthases. Specific activity was obtained at various DMA DP concentrations for E. globulus isoprene synthase (Figure 9, light grey diamonds). The data were fitted to Equation 1 using the constants shown in Table 1 and the fitted equation is shown (Figure 9, dark grey line).

Table 1 : Kinetic parameters of purified E. globulus isoprene synthase.

|0127] The published K_M values for various non-Eucalyptus isoprene synthases range from 0.3 mM to 9 mM (Rasulov, et al., (2009) Plant Physiology 149: 1609-1618 and references therein and Sasaki, et al, (2005) FEBS Letters 579: 2514-2 18). The K_M value of 0.03 mM for E. globulus favorably compares to that of other published isoprene synthases, such as those from P, alba and other Poplar species, and thus represents a superior isoprene synthase for the commercial production of isoprene. Example 4

Example 4.1: Plasmids and Strains pTrc-E4-6His

[0128] The entire coding sequence of the E, globulus isoprene synthase (except the N terminal plastid targeting sequence, amino acids 1 to 35) in pjexpress401-E4IspS-His was transferred to the Ncol/Xhol site of pTrcHis2B using PGR to introduce an Ncol site at the N terminus.

E. globulus active site mutant IspS

[0129] Sequences identical to pJexpress401-E4IspS-His except the indicated amino acid mutations were synthesized and cloned into pJexpress404 by DNA2.0, p Tre~Ml~6His

[0130] The M. altemifolia isoprene synthase (SEQ ID NO 7, except the N terminal plastid targeting sequence, amino acids 1 to 32) with a C terminal linker and 6 histidine motif (ENLYFQSGSGSG SG HHHHHH) was codon optimized for E. coli expression and synthesized by DNA2.0, A BsmBI/XhoI fragment containing the isoprene synthase was then ligated into an Ncol/Xhol digest of pTrcHis2B to yield pTrc-Ml-6His. pTrc-Rl-6His

[0131] The R. pseudoaccacia isoprene synthase (SEQ ID NO 8) with a C terminal linker and 6 histidine motif (ENL YFQSGSGSGSGHHHHHH) was codon optimized for E. coli expression and synthesized by DNA2.0. An Ncol/Xhol fragment containing the isoprene synthase was then ligated into an Nco!/Xhol digest of pTrcHis2B to yield pTrc- R 1 -61 lis.

M rcene synthase

[0132] The A. majus myrcene synthase (SEQ ID NO 13, except the N terminal plastid targeting sequence, amino acids 1 to 45) with a C terminal linker and 6 histidine motif (ENLYFQSGSGSGSGHHHHHH) was codon optimized for E. coli expression, synthesized, and cloned into pJexpress404 by DNA2.0. Variants were made with the indicated amino acid substitutions. Dimeihylallyl tryptophan synthase

[0133] The A. f migatus dimeihylallyl tryptophan synthase (SEQ ID NO 33) with a C terminal linker and 6 histidme motif (ENLYFQSGSGSGSGHHHHHH) was codon optimized for E. coli expression, synthesized, and cloned into pJexpress404 by DNA2.0. Variants were made with the indicated amino acid substitutions, with "all" representing L81 F, T82F, R83F, Y191F, Y345F, and Y398F.

Phettandrene synthase

[0134] The S. lycopersicum phellandrene synthase (SEQ ID NO 23, except the N terminal plastid targetmg sequence, amino acids 1 to 36) with a C terminal linker and 6 histidme motif (ENLYFQSGSGSGSGHHHHHH) was codon optimized for E. coli expression, synthesized, and cloned into pJexpress4Q4 by DNA2.0. Variants were made with the indicated amino acid substitutions,

MBO synthase

[0135] The P. sabiniana methyl butanol synthase (SEQ ID NO 9, except the N terminal plastid targeting sequence, amino acids 1 to 43) with a C terminal linker and 6 histidine motif (ENLYFQSGSGSGSGHHHHHH) was codon optimized for E. coli expression, synthesized, and cloned into pJexpress404 by DNA2.0. Variants were made with the indicated amino acid substitutions,

Pentalenene synthase

[0136] The Strepiomyces sp. UC5319 pentalenene synthase (SEQ ID NO 34) with a C terminal linker and 6 histidine motif ( ENLYFQSGSGSGSGHHHHHH) was codon optimized for E. coli expression, synthesized, and cloned into pJexpress401 by DNA2.0. Variants were made with the indicated amino acid substitutions.

pAl

[0137] Five B. subtilis MEP pathway genes (dxs, dxr, ispD, ispF, and idi) were placed under the control of strong E, coli promoters and cloned into the Hindlll/SacI sites of pCL1920. Example 4,2: Isoprene quantification

[0138] In vivo isoprene production was measured by injecting flask headspace samples into a Tianmei GC9700 GC equipped with a photoionization detector. The column was a PLOT-Q (Tianmei), the column temperature was 140 C, the injector temperature was 150 C, and the detector temperature was 150 C. The injection volume was 1 mL gas. I soprene standards were used to quantify the signal.

Example 4,3: In vivo isoprene production

[0139] To make pre-seed cultures, single colonies from each strain to be tested were used to inoculate 4 mL LB medium plus appropriate antibiotics and grown overnight at 37 C with shaking. To make seed cultures, for each strain, 300 uL of pre-seed was inoculated into 30 mL MOPS mineral medium (37.9 mM (N i l iLS _:, 4,2 mM MgS() |. 2.9 mM H₂P0₄, 7.1 mM Na₂P0₄, 0.7 mM CaC^'k 100 mM 3-(N-morpholino) propanesulfoic acid, 0.2 mg/L thiamine, 0.476 mM citric acid monohydrate, 0.107 mM MnS0₄, 0.018 mM FeS0 , 0.007 mM ZnS0₄, 0.002 mM CuS0₄, 0.003 mM CoCl₂, 0.003 mM NaMoO) plus 10 g/L glucose, 5 g/L yeast extract, and appropriate antibiotics and cultured at 37 C with shaking for 6.5 hours. For each strain, 0.5 mL seed culture was diluted into 9.5 mL. fresh MOPS mineral medium plus 5 g/L glucose, 1 g/L yeast extract, appropriate antibiotics, and 1 mM IPTG and incubated at 37 C in an air tight flask with shaking. Isoprene in the headspace was measured after 16 hours.

Results

[0140] The E. globulus isoprene synthase, when expressed in E. coli cells, produces far more isoprene than the M. alterniforia or R. pseudoacacia isoprene synthase, or any of the non-isoprene synthases with variants predicted to increase isoprene production. E. coli FM5 strains expressing the indicated terpenoid synthase and heterologous expression of dxs, dxr, ispD, ispF, and idi, were grown in minimal medium under aerobic conditions and isoprene production measured (Table 2),

Table 2: E. globulus isoprene synthase is superior to other enzymes.

Average Culture Productivity of Standard Error of the Gene ame isoprene (mg/L) Mean E. globulus wild type ispS

pJexpress-E4-his 1.94 0.109 Average Culture Productivity of Standard Error of the

Gene ame Isoprene (m

pTrc-E4-his 1.54

E. globulus active site mutant IspS

EUC..F326L ..F473W 0.02

Euc_F326L_F473Y 0.14

Euc _F326W_F473L 0.08

Euc__F326Y_F473L 0.03

Hon E. globulus wild type IspS

pTrc-M l-6His 0.03

pTrc-Rl-6His 0.03

yrcene synthase

McS_WT 0.01

McS_V331F 0.01

McS_V444F 0.01

McS_V331F_V444F 0.01

Dimethylallyl tryptophan synthase

fgaPT2_WT 0.01

fgaPT2_„L81F 0.00

fgaPT2_T82F 0.01

fgaPT2_R83 F 0.01

fgaPT2_Y191F 0.01

fgaPT2_„Y345F 0.01

fgaPT2_Y398F 0.01

fgaPT2_a!l 0.02

Pheilandrene synthase

PhS_WT 0.02

PhS_„V524F 0.02

PhS_M672F 0.01

PhS_V524F_M672F 0.01

M BO synthase

M BO_WT 0.01

M BO_S440F 0.01

BO...S440Y 0.02

Pentalenene synthase

PS_„WT 0.01

PS J 177 F 0.02

PS_N219F 0.01

PS M73F F77V F78T I177F 0.02 Example 4,4: In vivo expression and solubility

[0141] Cells were grown and induced as above in MOPS mineral medium then 15 OD600 units of cells were pelleted by centrifugation. Cell extracts were prepared by cell lysis in a French press, and total protein samples saved. The remainder was subjected to centrifugation at 14,000 g for 10 minutes to separate soluble and insoluble proteins. Protein samples were electrophoresed on SDS-PAGE gels with Life Technologies Benchmark His-tagged protein standard and transferred to PVDF membranes. Proteins were visualized with anti-his antibodies and BQP/NBT per the manufacturer's instructions.

Results

[0142] The expression and/or solubility of the E. globulus isoprene synthase is far superior to that of M. alterniforia or R. pseuaoacacia isoprene synthases. Total, soluble, and insoluble protein fractions are shown after induction of the indicated plasmids, and the E. globulus isoprene synthase was shown to express at high level in soluble form, unlike the M, alterniforia or R, pseudoacacia isoprene synthases (Figure 10).

Example 5

Example 5.1: Protein Purification

[0134] Commercially obtained E. coli strain FM5-Alper (also called strain Q) were transformed with the pJexpress404 vector (DNA 2.0) containing sequence for a histidine- tagged phellandrene synthase (PhS) or a modified version (Mod A) of the His-tagged PhS. Both sequences were behind a T5 promoter. A resulting E. coli transformant containing each PhS sequence was grown in a 250 ml flask of LB overnight atroom temperature (OD ~ 1 ). Cultures were induced by adding enough IPTG to bring the culture concentration to 400 μΜ, Cultures were allowed to grow an additional eight hours until they reached an OD ~ 4. Cultures were then concentrated by centrifugation and cells were broken using a combination of freezing, sonication. and lysozyme.

Histidine-tagged protein was purified on Ni-agarose (Qiagen) according to

manufacture's directions. Protein elution from the columns was determined by image analysis of Coomassie-stained protein gels and protein concentration used in the assays were determined by a Lowry assay. Example 5.2: Enzyme Assays

[0134] Dimethylallyl diphosphate (DMADP) from either Echelon Biosciences or Isoprenoids LLC was assayed to determine the exact concentration of DMADP(a necessary step because DMADP is labile). Assays were carried out in a total volume of 200 LsL of assay buffer (100 niM Hepes pH 7.8, 40 mM KC1, 20 mM MgC12 10% glycerol). DM ADP stock or an equivalent amount of 2 mM NH4HC03 (the buffer used to dilute the DMADP) was used to establish different concentrations ofDADP in the assay . There was 20 ,ug protein in each sample. Three additional vials were assayed that had all of these components except the protein to determine the non-enzymatic conversion of DMADP in the system (1 to 3 %). The nonenzymatic signal was subtracted from the signal for the sample with protein. The vial was 2 niL. Vials were incubated for 1 hr at 37°C. At the end of 1 hr, 1 mL of head-space air was withdrawn by syringe while simultaneously adding 1 mL water to avoid creating a vacuum. The head- space air was injected into a chemiluminescent isoprene detection system (Fast Isoprene Sensor, Hills Scientific) that had a flowing gas stream, isoprene standards were drawn from a tank of compressed nitrogen with 3.25 ± 0.16 PPM isoprene (Airgas). The isoprene signal from each injection lasted about 15 seconds. Ail of the FlSsignal for 30 seconds centered on the injection peak were summed and 15 seconds of baseline signal before and after the peak were summed and subtracted from the injection signal. In all cases n=3 except in two cases where a large outlier datapoint was dropped (this did not change the value of the kineticparameters), Activities were calculated in terms of moles of isoprene per mole of enzyme per second (specific acti vity ). The Vmax estimated from Michaelis-Menton kinetics is the effective kcat when data are plotted this way. See Figure 1 1 .

[0143] While the present invention has been described with reference to the specific embodiments thereof! it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. All publications and patents cited herein are incorporated by reference in their entirety for all purposes.

SEQUENCES

Legend: The italicized amino acids represent leader sequences that are optionally completely or partially deleted or substituted. The bold amino acids indicate exemplar}' sites for modification, singly or in combination. Said modification encompasses substitution, deletion and insertion. Substitutions are indicated using the notation

[original residue in one-letter-code][position number] [substitution residue in one-letter- code]. For example, the notation S440F indicates that a serine residue at position 440 is being replaced with a phenylalanine residue. Position numbers are counted from the N- terminus of the sequences as represented in the instant specification.

SEQ ID NO 1

Populus iremuloides isoprene synthase

Adapted from Genbank: AY341431.1

MA TELLCLHR PISL THKLFR NPLPKVIQAT PL TLKLRCS V STENVSFSZT

STETRRSANY EPNSWDYDYL LSSDTDESIE VHKDKAKKLE AEYRREI ^'NE

KASFLTLLSL IDNVQRLGLG YRFESDIRRA LDRFVSSGGF DGVTKTSLHG

TALSFRLLRQ HGFSVSQSAF SGFKDQNGNF LSNLKSDIKA ILSLYEASFL ALEGENILDE AKVFAISHLK SLSESKIGKE LAEQVSHALE LPLHRRTQRL EAVWS IEAYR KKEDANQVLL ELAILDYNMI QSVYQRDLRE TSRWWRRVGL ATKLHFARDR LIESFYWAVG VAFEPQYSDC RNSVAKMFSF VTIIDDIYDV YGTLDELELF TDAVERWDVN AINDLPDYMK LCFLALYNTI NEIAYDNLKD KGENILPYLT KAWADLCNAF LQEAKWLYNK STPTFDDYFG NAWKSSSGPL QLIFAYFAVV QNIKKEEIEN LQKYRDI ISR PSHIFRLCND LASASAEIAR GETANSVSCY MRTKGISEEL ATESVMNLID ETWKKMNKEK LGGSLFAKPF VE AINLARQ SHCTYHN^'GDA HTSPDELTRK RVLSVITEPI LPFER

SEQ ID NO 2

Populus alba isoprene synthase

Adapted from Genbank: AB 198180.1

MATELLCLHR PISLTHKLFR NPLPKVIQAT PLTLKLRCSV STENVSFTET ETEARRSANY EPNSWDYDYL LSSDTDES IE VYKDKAKKLE AEVRREI NE KAEFLTLLEL IDNVQRLGLG YRFESDIRGA LDRFVSSGGF DAVTKTSLHG TALSFRLLRQ HGFEVSQEAF SGFKDQNGNF LENLKEDIKA ILSLYEASFL ALEGENILDE AKVFAISHLK ELSEEKIGKE LAEQVNHALE LPLHRRTQRL EAVWS IEAYR KKEDANQVLL ELAILDYNMI QSVYQRDLRE TSRWWRRVGL ATKLHFARDR LIESFYWAVG VAFEPQYSDC RNSVAKMFSF VTIIDDIYDV YGTLDELELF TDAVERWDVN AINDLPDYMK LCFLALYNTI NEIAYDNLKD KGENILPYLT KAWADLCNAF LQEAKWLYNK STPTFDDYFG NAWKSSSGPL QLVFAYFAVV QNIKKEEIEN LQKYHD ISR PSHIFRLCND LASASAEIAR GETANSVSCY MRTKGISEEL ATESVMNLID ETWKKMNKEK LGGSLFAKPF VETAINLARQ SHCTYHN^'GDA HTSPDELTRK RVLSVITEPI LPFER

SEQ ID NO 3

Populus nigra Isoprene synthase

Adapted from Genbank: HQ684728.1

MATELLCLHR PISLTHKLFR NPLPKVIQAT PLTLKLRCSV STENVSFTET ETETRRSANY EPNSWDYDYL LSSDTDES IE VYKDKAKKLE AEVRREINNE KAEFLTLPEL IDNVQRLGLG YRFESDIRRA LDRFVSSGGF DAVTKTSLHA TALSFRLLRQ HGFEVSQEAF SGFKDQNGNF LKNLKEDIKA ILSLYEASFL ALEGENILDE AKVFAISHLK ELSEEKIGKD LAEQVNHALE LPLHRRTQRL EAVWS IEAYR KKEDADOVLL ELAILDYNMI QSVYQRDLRE TSRWWRRVGL ATKLHFARDR LIESFYWAVG VAFEPQYSDC RNSVAKMFSF VTIIDDIYDV YGTLDELELF TDAVERWDVN AIDDLPDYMK LCFLALYNTI NEIAYDNLKD KGENILPYLT KAWADLCNAF LQEAKWLYNK STPTFDEYFG NAWKSSSGPL QLVF YFAVV QNIKKEEIDM LQKYHDI ISR PSHIFRLCND LASASAEIAR GETANSVSCY MRTKGISEEL ATESVMNLID ETWKKMNKEK LGGSLFAKPF VETAINLARQ SHCTYHNGDA HTSPDELTRK RVLSVITEPI LPFER

SEQ ID 4

Populus trichocarpa Isoprene synthase Adapted from Genbank: EU693027.1

CSVSTENVSF TETETETRRS ANYEPNS DY DYLLSSDTDE SIEVYKDKAK KLEAEVRREI NNEKAEFLTL LELIDNYQRL GLGYRFESDI RRALDRFVSS GGFDAVTKTS LHATALSFRL LRQHGFEVSQ EAFSGFKDQN GNFLENLKED I KAILS LYE A S FL ALE GEN I LDEAKVFAIS HLKELSEEKI GKDLAEQV H ALELPLHRRT QRLEAVLSIE AYRKKEDADQ VLLELAILDY NMTQSVYQRD LRETSRW RR VGLATKLHFA RDRLIESFY AVGVAFE PQY SDCRNSVAKM FSFV I TDDI YDVYGTLDEL ELFTNAVERW DVNAIDDLPD YMKLCFLALY NTINSIAYDN LKEKGENILP YLTKAWADLC NAFLQEAKWL YNKSTPTFDD YFGNAWKSSS GPLQLVFAYF AVVQ I KKE E IENLQKYRDI ISRPSHIFRL CNDLASASAE I ARGE ANS SCYMRTKGIS EELATES VMN LIDETWKKMN KEKLGGSLFA KPF VETAINL ARQSHCTYHN GDAHTSPDEL TRKRVLS VI T EPILPFER

SEQ ID NO 5

Pueraria Montana Isoprene synthase

Adapted from Genbank: A Y316691.1

MATNLLCLSN KLSSPTPTPS TRFPQSKNFI TQKTSLANPK PWRVICATSS

QFTQi ms RRSANYQPNL WNFEFLQSLE N^'DLKVEKLEE KATKLEEEVR

CM I NRVDT QP LSLLELIDDV QRLGLTYKFE KDI IKALENI VLLDENKKNK SDLHATALSF RLLRQHGFEV SQDVFERFKD KEGGFSGELK GDVQGLLSLY EASYLGFEGE ML LEE RT FS ITHLKNNLKE GTNTKVAEQV SHALELPYHQ RLHRLEARWF LDKYEPKEPR HOLLLELAKL DFNMVQTLHQ KELODLSRWW TEMGLASKLD FVRDRLMEVY FWALGMAPDP QFGECRKAVT KMFGLVT I I D DVYDVYGTLD ELQLFTDAVE R DVNAI TL PDYMKLCFLA LYNTVNDTSY SILKEKGHNN LSYLTKSWRE LCKAFLQEAK WSNNKIIPAF SKYLENASVS SSGVALLAPS YFSVCQQQED ISDHALRSLT DFHGLVRSSC VIFRLCNDLA TSAAELERGE TTNSIISY H ENDGTSEEQA REELRKLIDA EWKKMNRERV SDSTLLPKAF MEIAVNMARV SHCTYQYGDG LGRPDYATEN RIKLLLIDPF PINQLMYV

SEQ ID NO 6

Eucalyptus globulus "mts-1 mRNA for monoterpene synthase" actually an isoprene synthase

Adapted from Genbank: AB266390.1

MALRLLFTPH LPVLSSRRAN GRVRCSASTQ TSDPQEGRRS ANYQPSVWTY NYLQSIVAGE GRQSRREVEQ QKEKVQILEE EVRGALNDEK AETF IFA V DDIORLGLGD HFEE DISMAL RRCVSKGAVF MSLQKSLRGT ALGFRLLRQH GYEVSQDVFK IFLDESGSFV KTLGGDVQGV LSLYEASHLA FEEERILHKA RSFAIKHLEN LN S DVDKDLQ DQVKHELELP LHRRMPLLEA RRSIEAYSRR GYTNPQILEL ALTDFNVSQS YLQRDLQEML GWWNNTGLAK RLSFARDRLI ECFFWAVGIA HEPSLSICRK AVTKAFALIL VLDDVYDVFG TLEELELFTD A V RR W D L N AV EDLPVYMKLC YLALYN^'SVNE MAY Ξ T L LIE KG ENVIPYLAKA WYDLCKAFLQ EAKWSNSRII PGVESYLNNG WVSSSGS L IHAYFLASPS IRKESLSSLE HYHDLLRLPS LIFRLTNDIA SSSAELERGE TTNSIRCFMQ EKGISELEAR ECVKEEIDTA WKKMNKYMvD RSTFNQSFVR MTYNLARMAH CVYQDGDATG SPDDLSWNRV HSLIIKPISP ΑΆ

SEQ ID NO 7

Melaleuca alternifolia "putative monoterpene synthase mRNA", actually an isoprene synthase

Adapted from GenBank: AY279379.1

MALRLLSTPH LPQLCSRRVS GRVHCSASTQ V5DAQGGRRS A YQPSVWTY NYLQSLVADD IRRSRREVEQ EREKAQILEE DVRGALNDGN AEPMAIFALV DDIORLGLGR YFEEDISKAL RRCLSOYAVT GSLQKSLHGT ALSFRVLRQH GFEVSQDVFK IFMDESGSFM KTLGGDVQGM LSLYEASHLA FEEEDILHKA KTFAIKHLEN LNHDIDQDLQ DHVNHELELP LHRRMPLLEA RRFIEAYSRR SNVNPRILEL AVMKFNSSQL TLQRDLQDML GWWNNVGLAK RLSFARDRLM ECFFWAVGIA REPALSNCRK GVTKAFSLIL VLDDVYDVFG TLDELELFTD AVRRWHEDAV ENLPGYMKLC FLALYNSVND MAYETLKETG ENVTPYLTKV WYDLCKAFLQ EAKWSYNKIT PGVESYLNNG WVSS8GQVML THAYFLSSPS LRKEELESLE HYHDLLRLPS LIFRLTNDLA TSSAELGRGE TTNS TLCYMR EKGFSESEAR KQVIEQID A WRQMNKYMVD HSTFNRSFMQ MTYNLARMAH CVYQDGDATG APDDQSWNRV HSLIIKPVSL APC

SEQ ID NO 8

Rohinia pseudoaccacia isoprene synthase

Unpublished sequence

RRSANYQPNL WNFEFLQSQE YDL VETLQE RATKLEEEVR RLINRVDIEP LKLLELVDNV QRLGLTYKFE DDIN^'KALERI VSLDEREKSG LHATALIFRL LRQHGFEVSQ DVFESTROKE GRFKAEIKGD VQGLLSLYEA SYLGSEGENL LDEAREFSMT HLKNLNEGVV TPKLAEQI H. ALELPYHRRF QRLEARWFIE NYEVKEPHDR LLVELAKLDF NMVQSLQKKE VGESSRW KE IGLTSKLDFV RDRLVEVYFW ASGMAPDPQL SECRKAVTKM FGLV T IDDV YDVYGTLDEL ELFTNAVER.W DVNAVDTLPD YMKLCFFALY NTVNDTAYNL LKEKGDNNLP YLAKSWSDLC KAFLOEAKWS NNKI IPSFNK YIENASVSSS GGALLTPCYF SIRQDI N^'QA LDSLTNYHGP VRSSCAIFRL CNDLATSAAE LERGETTNS I TSCMQDNGIS EEQARDELRN LIDAEWKQMN RERVFDQTFP KAFIETAINM ARVSHCTYQY GDGLGRPDNT AENRIKLLLI DPLSN

SEQ ID NO 9

Pinus sabiniana Methyl bu tonal synthase

Unpublished sequence with S440 shown in bold and highlighted

MALLSVAPLA PRWCVHKSLV TSTKVKVVRR TISTSIRMCR I IESGEGVO RRIANHHSNL WDDNFIQSLS TPYGAISYHE SAQKLIGEVK EMINSISLKD GELITPSNDL LMRLSIVDSI ERLGIDRHFK SEIKSALDYV YSYWNEKGIG WGRDSVvADL NSTALGLRTL RLHGYPVSSD VLQHFKEQKG QFACSAIQTE GEIRSVLNLF RASQIAFPGE KVMEEAEVFS TIYLKEATLK LPVCGLSREI SYVLEYGWHI NLPRLEARNY IDVFGEDPIY LTPNMKTQKL LELAKLEFNM FHSLQOQELK LLSRWWKDSG FSQMTFPRRR HVEYYTLASC IDSEPOHSSF RLGFAKIFHL ATVLDDIYDT FGTMDELELF TAAVKRWHPS ATEWLPEYMK GVYMYLYETV NEMAGEAEKS QGRDTLNYGR ALEAYIDAl MEEAK IFSG FLPTFESYLD NGKVSFGYGI GTLQPILTLG IPFPHHILQE IDFPSRLNDV ASS ILRLKGD IHTYQAERSR GEKSSCISCY ΜΕΕΝΡΕΞΤΕΕ DATNHIN 3M.V DKLLKELN E YLRPDSNVPI TSKKHAFDTL RAFYHLYKYR DGFSVANYEI KNLVMTTVIE PVPL

S EC) ID

Picea pungens isoprene synthase (Spruce)

Unpublished sequence

MALLSVAPLV STWCVDKPLV GSSEAKALLR KIPTLEMCRL TKSVTPSISM CLTTTVSODG VQRRIANHHS MLWDDN^'FIQS LSTPYGATAY HERAQKLIGE VKVIINSILV EDGELITAPN DLLQRLS IVD SIERLGIDRH FK EIKSALD YVYSYWNEKG IGCGRDSVVN DLNTTALGLR TLRLHGYPVS SDVLEQFKDQ NGQFACSATQ TEGEIKKVLN LFRASLTAFP GEKVMEEAEI FS IYLKEAL LKIPVCSFSR EIAYVLEYGW HMNLPRLEAR NYIDVFGQDA IYL PNMRTQ KILELAKLEF NIFHSLQQKE LKHLSRWWKD SVFSQLTFPR HRHVEYYTLA SCIDIDPQHS SFRLGFAKIF HLATVLDDIY DTFGMMDELE LFTAAVKRWH PSAAEWLPEY MKGVYMMLYE TVYEMAREAE KSQGRDTLNY ARQALEAYID SYMKEAKWIS SGFLPTFSEY LDNGKVSFGY RIGTLQPILT LGIPFPHHIL QETDFPSRLN DLAGS ILRLK GDIHSYQAER SRGEESSGIS CYMKDNPEST EEDAVTYI A MVNRLLKELN WELLKPHSNV PTTSfvKHAFD ILRAFYHLYK DRDGFSVTRN EIRNLVMTTV IEPVPL

SEQ ID NO 1 1

Pimis sabiniana Methyl butonal synthase converted into an isoprene synthase

Route 1 : making the change S440F, shown in bold and highlighted

MALLSVAPLA PRWCVHKSLV TSTKVKVVRR TISTSIRMCR TT' ESGEGVQ RRIANHHSNL WDDNFIQSLS TPYGAISYHE SAQKLIGEVK EMINSISLKD GELITPSNDL LMRLSTVDST ERLGIDRHFK SEIKSALDYV YSYWNEKGTG WGRDSVVADL NSTALGLRTL RLHGYPVSSD VLQHFKEQKG QFACSAIQTE GEIRSVLNLF RASQIAFPGE KVMEEAEVFS TIYLKEATLK LPVCGLSREI SYVLEYGWHI NLPRLEARNY IDVFGEDPIY LTPNMKTQKL LELAKLEFNM FHSLQQQELK LLSRWWKDSG FSQMTFPRRR HVEYYTLASC IDSEPQHSSF RLGFAKIFHL ATVLDDIYDT FGTMDELELF TAAVKRWHPS ATEWLPEYMK GVYMVLYETV NEMAGEAEKS QGRDTLNYGR NALEAYIDAl MEEAKWIFSG FLPTFESYLD NGKVSFGYGI GTLQPILTLG IPFPHHILQE IDFPSRLNDV ASS ILRLKGD IHTYQAERSR GEKSSCISCY MEENPESTEE DAINHINSMV DKLLKELNWE YLRPDSNVPI TSKKHAFDTL RAFYHLYKYR DGFSVANYEI KNLVMTTVIE PVPL SEQ ID NO 12

Pinus sabiniana Methyl butonal synthase converted into an isoprene synthase

Route 1 : making the change S440Y. shown in bold and highlighted

MALLSVAPLA PRWCVHKSLV TSTKVKVVRR TISTSIRMCR ITTESGEGVQ RRIANHHSNL WDD FTQSLS TPYGAISYHE SAQKLIGEVK EMINSISLKD GELITPS DL LMRLSIVDSI ERLGIDRHFK SEIKSALDYV YSYWNEKGIG WGRDSVVADL NSTALGLRTL RLHGYPVSSD VLQHFKEQKG QFACSAIQTE GEIRSVLNLF RASQIAFPGE KVMEEAEVFS TIYLKEAILK LP CGLSREI SYVLSYGWHI NLPRLEARNY IDvFGEDPIY LTPNMKTQKL LELAKLEFNM FHSLQQQELK LLSRWWKDSG FSQMTFPRHR HVEYYTLASC IDSEPQHSSF RLGFA.KTFHL ATVLDDIYDT FGTMDELELF TAAVKRWHPS ATEWLPEYMK GVYMvLYETV NEMAGEAEKS QGRDTL YGR NALEAYIDAf MEEAKWIFSG FLPTFEEYLD NGKVSFGYGI GTLQPILTLG IPFPHHILQE IDFPSRLNDV ASS ILRLKGD IHTYQAERSR GEKSSCISCY MEENPESTEE DAINHINSMV DKLLKELNWE YLRPDSNVPI SKKHAFDIL RAFYHLYKYR DGFSVA YEI KNLVMTTVIE PVPL

SEQ ID NO 13

Snapdragon (Antirrhinum majus) myrcene synthase

Adapted from Genbank: AY195608.1

Amino acids to be transferred bold and highlighted

IFVDATLRLG V HHFQKEIE EILRKSYATM KSPIICEYHT LHEVSLFFRL MRQHGRYVSA DVFNNFKGES GRFKEELKRD TRGLVELYEA AQLSFEGERI LDEAENFSRQ ILHGNLAGME DNLRRSVGNK LRYPFHTS IA RFTGR.NYDDD LGGMYEWGKT LRELALMDLQ VERSVYQEEL LQVSKWWNEL GLYKKLNLAR NRPFEFYTWS MVILADYIML SEQRVELTKS VAFIYLIDDI FDVYGTLDEL I IFTEAVNK DYSATDTLPE NMKMCCMTLL DTINGTSQKI YEKHGYNPID SLKTTWKSLC SAFLVEAK S ASGSLPSANE YLENEKVSSG VYVVLVHLFC LMGLGGTSRG SIELNDTQEL MSSIAIIFRL WNDLGSAKNE HQ GKDGSYL NCYKKEHINL AAQAHEHAL ELVAIEWKRL NKESFNLNHD SVSSFKQAAL NLARMVPLMY SYDHNQRGPV LEEYVKFMLS D

SEQ ID NO 14 (the hybrid)

Hybrid between Populus alba Isoprene synthase (Genbank: AB198180.1) deleted up to L I 48 and the structured arm of Snapdragon (Antirrhinum majus) myrcene synthase

LYEASFLALE GENILDEAKV FAISHLKELS EEKIGKELAE QVNHALELPL HRRTQRLEAV WS IEAYRKKE DA QVLLELA ILDYNMIQSV YQRDLRETSR WWRRVGLATK LHFARDRLIS SFYWAVGVAF EPQYSDCRNS VAKMFSFV I TDDIYDVYGT LDELELE DA VERWDVNAIN DLPDYMKLCF LALYNTINEI AYD LKDKGE NILPYLTKAW ADLCNAFLQE AKWLYNKSTP TFDDYFGNA KSSSGPLQLV FAYFAVVQ I KKEEIENLQK YRDTISRPSH IFRLCNDLAS ASAE TARGET ANSVSCYMRT KGISEELATE SV NLIDETW KKMNKEKLGG SLFAKPFVST AINLARQSHC TYHNGDAHTS PDELTRKRVL SVITEPILPF ER

SEQ ID NO 15

Hybrid between Pueraria Montana Isoprene synthase (Genbank: A.Y316691.1) deleted up to LI 53 and the structured arm of Snapdragon (Antirrhinu majus) myrcene synthase

DVGSTPPPSK LHC iLCLHEK SLSCM&ELPM DYEGKI ETI

SFRLLRQHGF EVSQDVFERF KDKEGGFSGE LKGDVQGLLS

LYEA^'SYLGFE GENLLEEART FSITHLKNNL KEGI TKVAE QVSHALELPY HQRLHRLEAR WFLDKYEPKE PHHQLLLELA KLDFNMVQTL HQKELQDLSR WWTEMGLASK LDFVRDRLME VYFWALGMAP DPQFGECRKA VTKMFGLV I TDDVYDVYGT LDELQLFTDA VERWDVNAIN TLPDYMKLCF LALYNTVNDT SYS ILKEKGH NNLSYLTKSW RELCKAFLQE AKWSNNKIIP AFSKYLENAS VSSSGVALLA PSYFSVCQQQ EDISDHALRS LTDFHGLVRS SCVIFRLCND LATSAAELER GETTNSIISY MHENDGTSEE QAREELRKLI DAEWKKMNRE RVSDSTLLPK AFMEIAVNMA RVSHCTYQYG DGLGRPDYAT ENRIKLLLID PFPINQLMYV

SEQ ID NO 16

Hybrid between Eucalyptus globulus isoprene synthase (AB266390.1 ) deleted up to LI 37 and the structured arm of Snapdragon {Antirrhinum majus) myrcene synthase

DVGSTPPPSK LKQALCLNEK SLSCMAELPM DYEGKI ET HLLHLKG HGTAL GFRLLRQHGY EVSQDVFKTF LDESGSFVKT LGGDVQGVLS LYEASHLAFE EEHILHKARS FAIKHLENLN SDVDKDLQDQ VKHELELPLH RRMPLLEARR SIEAYSRRGY TNPQILELAL TDFNVSQSYL QRDLQEMLGW WN TGLAKRL SFARDRLIEC FFWAVGIAHE PSLSICRKAV TKAFALILVL DDVYDVFGTL EELELFTDAV RRWDLNAVED LPVYMKLCYL ALYNSVNEMA YETLKEKGEN VIPYLAKAWY DLCKAFLOEA KWSNSRIIPG VEEYLNNGWV SSSGSVMLIH AYFLASPS IR KEELESLERY HDLLRLPSLI FRLTNDIASS SAELERGETT NSIRCFMQEK GISELEAREC VKEEIDTAWK KMNKYMVDRS TFNQSFVRMT YNLARMAHCV YQDGDAIGSP DDLSWNRVHS LIIKPISPAA

SEQ ID NO 17

Hybrid between Melaleuca alternifolia isoprene synthase (GenBank: AY279379.1 ) del eted up to L I 37 and the structured arm of Snapdragon {Antirrhinum majus) myrcene synthase liHGTAL SFRVLRQHGF EVSQDVFKIF MDESGSFMKT LGGDVQGMLS LYSASHLAFE EEDILHKAKT FA I KRLENLN HDIDODLQDR VNRELELPLH RRMPLLEARR FIEAYSRRSN VNPRI LELAV MKFNSSQLTL QRDLQDMLGW WNN V G L AKRL SFARDRLMEC FFWAVGIARE PALSNCRKGV TKAFSLILVL DDVYDVFGTL DELELFTDAV RRWHEDAVEN LPGYMKLCFL A L Y N S VN DMA YETLKETGSN VTPYLTKVWY DLCKAFLQEA KWSYNKITPG VEEYLNNGWV SSSGQVMLTH AYFLSSPSLR KEELESLEHY HDLLRLPSLT FRLTNDLATS SAELGRGETT NSILCYMREK GFSESEARKQ VTEQIDTAWR QMNKYMVDHS TFNRSFMOMT YNLARMAHCV YODGDAIGAP DDOSWNRVHS LIIKPVSLAP C

Γ) ID

Hybrid between Robinia pseudoaccacia isoprene synthase deleted up to L91 and the structured arm of Snapdragon (Antirrhinum majus) myrcene synthase

LYEASYLGSE GENLLDEARE FSMTRLKNLN EGVVTPKLAE QINHALELPY HRRFQRLEAR WFIENYEVKE PHDRLLVELA KLDFNMVQSL QKKEVGESSR WWKEIGLTSK LDFVRDRLVE VYFWASGMAP DPQLSECRKA VTKMFGLVTI I DDVYDVYGT LDELELFT A VERWDVNAVD TLPDYMKLCF FALYNTVNDT AYNLLKEKGD NNLPYLAKSW SDLCKAFLQE AKWSNNKIIP SFNKYIE AS VSSSGGALLT PCYFSIRQDI TNQALDSLTN YHGPVRSSCA IFRLCNDLAT SAAELERGET TNSITSCMOD NGISEEQARD ELRNLIDAEW KQMNRERVFD QTFPKAFIET AI MARVSHC TYQYGDGLGR PDNTAENRIK LLLIDPLSN

SEQ ID NO 19

Physcomitrella patens PpCPS/KS niRNA for ent-kaurene synthase

Adapted from Genbank: AB302933

8709 is shown in bold and highlighted.

MASSTLIQNR SCGVTSSMSS FQIFRGQPLR FPGTRTPAAV QCLKKRRCLR PTESVLESSP GSGSYRIVTG PSGINPSSNG HLQEGSLTHR LPIPMEKSID NFQSTLYVSD IWSETLQRTE CLLQVTENVQ MNEWIEEIRM YFRNMTLGEI SMSPYDTAWV ARVPALDGSH GPQFHRSLQW I IDNQLPDGD WGEPSLFLGY DRVCNT L AC V IALKTWGVGA QNVERGIOFL OSNI YKMEED DANHMPIGFE I VFPAMME DA KALGLDLPYD ATILQQI SAE REKKMKKIPM AMVYKYPTTL LHSLEGLHRE VDWNKLLQLQ SENGSFLYSP ASTACALMYT KDVKCFDYLN QLL I KF'DH AC PNVYPVDLFE RLWMVDRLQR LGISRYFERE IRDCLQYVYR YWKDCG I GWA SNSSVQDVDD TAMAFRLLRT HGFDVKEDCF RQFFKDGEFF CFAGQSSQAV TGMFNLSRAS QTLFPGESLL KKARTFS NF LRTKHENNEC FDKWI ITKDL AGEVEYNLTF PWYASLPRLE HRTYLDOYGI DDIWIGKSLY KMPAVTNEVF LKLAKADFNM CQALHKKELE QVIKWNASCQ FRDLE FARQK SVECYFAGAA TMFEPEMVQA RLVWARCCVL TTVLDDYFDH GTPVEELRVF VQAVRTWN^'PE LI GLPEQAK ILFMGLYKTV NTIAEEAFMA QKRDVHHHLK HYWDKLIT!A LKEAEWAESG YVPTFDEYME VAEISYALEP IVCSTLFFAG HRLDEDVLDS YDYHLVMHLV NRVGRILNDI QGMKREASQG KISSVQIYME EHPSVPSEAM AIARLQELVD NSMOQLTYEV LRFTAVPKSC KRIHLNMAKI MHAFYKDTDG FSSLTAMTGF VKKVLFEPVPE

SEQ ID NO 20

Snapdragon (Antirrhinum majus) myrcene synthase converted into an isoprene synthase by making the change V331F (shown), V331 W or V331 Y (not shown).

Adapted from Genbank: AY195608.1

V33 IF is shown bold and highlighted

MAFCISYVGA LLPCSLSTRT KFAICHNTSK LHRAAYKTSR WNIPGOVGS PPPSKLHQAL CLNEHSLSCM AELPMDYEGK IKETRHLLHL KGENDPIESL IFVDATLRLG VNHHFQKEIE EILRKSYATM KSPIICEYHT LHEVSLFFRL MRQHGRYVSA DVFNFKGES GRFKEELKRD TRGLVELYEA AQLSFEGERI LDEAENFSRQ ILHGNLAGME DNLRRSVGNK LRYPFH SIA. RFTGRNYDDD LGGMYE GKT LRELALMDLQ VERSVYQEEL LQVSKWNEL GLYKKLNLAR NRPFEFYTWS MVILADYINL SEQRVELTKS IAFIYLIDDI FDVYGTLDEL TIFTEAVNKW DYSATDTLPE NMKMCCMTLL DTINGTSQKI YEKHGYNPID SLKTTWKSLC SAFLVEAKWS ASGSLPSANE YLENEKVSSG VYVVLVHLFC LMGLGGTSRG SIELNDTQEL MSSIAIIFRL WNDLGSAKNE HQNGKDGSYL CYKKEHI L TAAQAHEHAL ELVAIEWKRL NKESFNL HD SVSSFKQAAL NLARMVPLMY SYDHNQRGPV LEEYVKFMLS D

SEQ ID NO 21

Snapdragon (Antirrhinum majus) myrcene synthase converted into an isoprene synthase by making the change V444F (shown), V444W or V444Y (not shown).

Adapted from Genbank: A Yl 95608.1

V444F is shown bold and highlighted

MAFCISYVGA LLPCSLSTRT KFAICHNTSK LHRAAYKTSR WNIPGDVGSi: PPPSKLHQAL CLNEHSLSCM AELPMDYEGK IKETRHLLHL KGENDPIESL IFVDATLRLG VNHHFQKEIE EILRKSYATM KSPIICEYHT LHEVSLFFRL MRQHGRYVSA DVFNNFKGES GRFKEELKRD TRGLVELYEA AQLSFEGERI LDEAENFSRQ ILHGNLAGME DNLRRSVGNK LRYPFHTSIA RFTGRNYDDD LGGMYEWGKT LRELALMDLQ VERS YQEEL LQVSKWWNEL GLYKKLNLAR NRPFEFYTWS MVILADYINL SEQRVELTKS VAFIYLIDDI FDVYGTLDEL IIFTEAVNKW DYSATDTLPE NMKMCCMTLL DTINGTSQKI YEKHGYNPID SLKTTWKSLC SAFLVEAKWS ASGSLPSANE YLENEKVSSG VYVfLVHLFC LMGLGGTSRG SIELNDTQEL MSSIAIIFRL WNDLGSAKNE HQNGKDGSYL NCYKKEHINL TAAQAHEHAL ELVAIEWKRL NKESFNLNHD SVSSFKQAAL NLARMVPLMY SYDHNQRGPV LEEYVKFMLS D Snapdragon (Antirrhinum majus) myrcene synthase converted into an isoprene synthase by making the change V331F and V444F (shown), (V331F and V444W), (V331F and

V444Y), (V331W and V444F), (V331W and V444W), (V331W and V444Y), ( V331Y and V444F), (V331 Y and V444W) or (V331Y and V444Y) (not shown).

Adapted from Genbank: AY195608.1

V331F and V444F are shown bold and highlighted

MAFCISYVGA LLPCSLSTRT KFAICHNTSK LHRAAYKTSR WNIPGDVGST

PPPSKLHQAL CLNSHSLSCM AELPMDYEGK IKETRHLLHL KGENDPIESL

IFVDATLRLG V HHFQKSIE EILRKSYATM KSPIICEYHT LHEVSLFFRL

MRQHGRYVSA DVFNNFKGES GRFKEELKRD TRGLVELYEA AQLSFEGERI

LDEAENFSRQ ILHGNLAGME DNLRRSVGNK LRYPFHTSIA RFTGR.NYDDD

LGGMYEWGKT LRELALMDLQ VERSVYQEEL LQVSKWWNEL GLYKKLNLAR

NRPFEFYTWS MVILADYTNL SEQRVELTKS |AFIYLIDDT FDVYGTLDEL

I IFTEAYN^'KW DYSATDTLPE NMKMCCMTLL D ING SQKI YEKHGYNPID

SLKTTWKSLC SAFLVEAKWS ASGSLPSANE YLENEKVSSG VYViLVHLFC

LMGLGGTSRG SIELNDTQEL MSSIAITFRL WNDLGSAKNE HQNGKDGSYL

NCYKKEHI L AAQAHEHAL ELVAIEWKRL NKESFNLNHD SVSSFKQAAL LARMVPLMY SYDHNQRGPV LEEYVKFMLS D

SEQ ID NO 23

Solatium lycopersicum cultivar M82 p-phellandrene synthase (PHS1) Adapted from GenBank FJ 797957.1

This β-phellandrene synthase is converted into an isoprene producing enzyme using a combination of one or both of the changes V524F and M672F. V524 and M672 are shown bold, underlined and high-lighted.

MIVGYRSTII TLSHPKLGNG KTISSNAIFQ RSCRVRCSHS TTSSMNGFED

ARDRIRESFG KLELSPSSYD TAWVAMYPSR HSLMEPCFPQ CLD IIENQR

EDGSWGLNPT HPLLLKDSLS STLACLLALT KWRYGDEQIK RGLGFIETYG

WAVDNKDQIS PLGFEVIFSS MIKSAEKLDL NLPLNLHLVN LVKCKRDS I

KRNVEYMGEG VGELCDWKEM TKLHQRQNGS LFDSPATTAA ALTYHQHDQK

CYQYLNSIFQ QHKNWVPTMY PTKVHSLLCL VDTLQNLGVH RHFKSETKKA

LDEIYRLWQQ KNEQIFSNVT HCAMAFRLLR MSYYDVSSDE LAEFVDEERF

FATNGKYKSH VEILELHKAS QLAIDHEKDD ILDKIM^'WTR AFMEQKLLNN

GFIDRMSKKE VELALRKFYT TSHLAENRRY IKSYEENNFK ILKAAYRSPN

IN KDLLAFS IHDFELCQAQ HREELQQLKR FEDYRLDQL GLAERYIHAS

YLFGVTVIPE PELSDARLMY AKY MLLTIV DDHFESFASK DECFNIIELV

ERWDDYASVG YKSEKVKVFF SVFYKSTEEL A IAEIKQGR SVKNHLINLW

LELMKLMLME RYEWCSGKTI PSIEEYLYVT SITFCAKLIP LSTQYFLGIK

TSKDLLESDE ICGLWNCSGR vj§RILNDLQD SKREQKEVSI NLVTLLMKSM

SEEEAIMKIK EILEMNRREL LKMVLVQKKG SQLPQLCKDI FWRTSKWAHF TYSQTDGYRI ΑΞΕΜΚ ΗIDE VFYKPL H

SEQ ID NO 24 Populus tremuloides isoprene synthase

Adapted from Genbank: AY34143 L 1

The active site is opened up by making the mutation F338L and F485W (shown bold and underlined), F338L and F485Y, F338W and F485L, F338Y and F485L (not shown)

MATELLCLHR PISLTHKLFR NPLPKVIQAT PLTLKLRCSV STENVSFSET: ETETRRSANY EPNSWDYDYL LSSDTDES IE VRKDKAKKLE AEVRREI E KAEFLTLLEL IDNVQRLGLG YRFESDIRRA LDRFVSSGGF DGVTKTSLHG TALSFRLLRQ HGFEVSQEAF SGFKDQNGNF LENLKEDIKA ILSLYEASFL ALSGSNILDE AKVFAISHLK ELSEEKIGKE LAEQVSHALE LPLHRRTQRL EAVWSIEAYR KKEDA QVLL ELAILDYNMI QSVYQRDLRE TSRW RRVGL ATKLHFARDR LIESFYWAVG VAFEPQYSDC RNSVAKMLSF VTIIDDIYDV YGTLDELELF TDAVERWDVN AINDLPDYMK LCFLALYNTI NEIAYDNLKD KGENILPYLT KAWADLCNAF LOEAKWLYNK STPTFDDYFG NAWKSSSGPL QLIFAYFAVV ONIKKEEIEN LOKYHDI ISR PSHIWRLCND LASASAEIAR GETANSVSCY MRTKGISEEL ATESVMNLID ETWKKMNKEK LGGSLFAKPF VE AINLARQ SHCTYHNGDA HTSPDELTRK RVLSVITEPI LPFER

SEQ ID NO 25

Populus alba Isoprene synthase

Adapted from Genbank: AB 198180.1

The active site is opened up by making the mutation F338L and F485 W (shown bold and underlined), F338L and F485Y, F338W and F485L, F338Y and F485I, (not shown)

MATELLCLHR PISLTHKLFR NPLPKVIQAT PLTLKLRCSV STENVSFIET ETEARRSANY EPNSWDYDYL LSSDTDES IE VYKDKAKKLE AEVRREINNE KAEFLTLLEL IDNVQRLGLG YRFESDIRGA LDRFVSSGGF DAVTKTSLHG TALSFRLLRQ HGFEVSQEAF SGFKDQNGNF LENLKEDIKA ILSLYEASFL ALEGENILDE AKVFAISHLK ELSEEKIGKE LAEQV HALE LPLHRRTQRL EAVWSIEAYR KKEDA QVLL ELAILDYNMI QSVYQRDLRE TSRWWRRVGL ATKLHFARDR LIESFYWAVG VAFEPQYSDC RNSVAKMLSF VTIIDDIYDV YGTLDELELF TDAVERWDVN AINDLPDYMK LCFLALYNTI NEIAYDNLKD KGENILPYLT KAWADLCNAF LQEAKWLYNK STPTFDDYFG NAWKSSSGPL QLVFAYFAVV QNIKKEEIEN LQKYHDTISR PSHIWRLCND LASASAEIAR GETANSVSCY MRTKGISEEL ATESVMNLID ETWKKMNKEK LGGSLFAKPF VETAINLARQ SHCTYHNGDA HTSPDELTRK RVLSVITEPI LPFER

SEQ ID NO 26

Populus nigra Isoprene synthase

Adapted from Genbank: HQ684728.1

The active site is opened up by making the mutation F338L and F485W (shown bold and underlined), F338I, and F485Y, F338W and I 485! ., F338Y and F485L (not shown)

MATELLCLHR PISLTHKLFR NPLPKVIQAT PLTLKLRCSV STENVSFTF,T ETETRRSANY EPNSWDYDYL LSSDTDES IE VYKDKAKKLE AEVRREINNE KASFLTLPSL IDNVQRLGLG YRFESDIRRA LDRFVSSGGF DAVTKTSLRA TALSFRLLRQ HGFSVSQSAF SGFKDQ G F LKNLKEDTKA TLSLYEASFL ALEGENILDE AKVFAISHLK ELSEEKIGKD LAEQVNHALE LPLHRRTQRL EAVWSIEAYR KKEDADQVLL ELAILDYNMI OSVYORDLRE TSRWWRRVGL ATKLHFARDR LIESFYWAVG VAFEPQYSDC RNSVAKMLSF VTIIDDIYDV YGTLDELELF TDA ERWDVN AIDDLPDYMK LCFLALY TI EIAYDNLKD KGSNILPYLT KAWADLCNAF LQEAKWLYNK STPTFDEYFG AWKSSSGPL QLVFAYFAVV Q IKKEEIDM LQKYRDI ISR PSHIWRLCND LASASAEIAR GETANSVSCY MRTKGISEEL ATESVMNLTD ETWKKMNKEK LGGSLFAKPF VETAINLARQ SHCTYR GDA HTSPDELTRK RVLSVITEPI LPFER

Populus trichocarpa Isoprene synthase

Adapted from Genhank: EU693027.1

The active site is opened up by making the mutation F301L and F448W (shown bold underlined), F301L and F448Y, I 301 W and F448L, F301Y and F448L (not shown)

CSVSTENVSF TETETETRRS ANYEPNS DY DYLLSSDTDE S IEVYKDKAK KLEAEVRREI NNEKAEFLTL LELIDNYQRL GLGYRFESDI RRALDRFVSS GGFDAVTKTS LHATALSFRL LRQHGFEVSQ EAFSGFKDQN GN^'FLEN^'LKED IKAILSLYE SFLALEGENI LDEAKVFAIS RLKELSEEKI GKDLAEQV R ALELPLHRRT QRLEAvLSIE AYRKKEDADQ VLLELAILDY NMIQSVYQRD LRETSRWWRR VGLATKLHFA RDRLIESFYW AVGVAFEPQY SDCRNSVAKM LSFV IIDDI YDVYGTLDEL ELFTNAVERW DVNAIDDLPD YMKLCFLALY NTINEIAYDN LKEKGENILP YLTKAWADLC NAFLQEAKWL YNKSTPTFDD YFGNAWKSSS GPLQLVFAYF AVVQ IKKEE IENLQKYRDI ISRPSHIWRL CNDLASASAE IARGETANSV SCYMRTKGIS EELATESVMM LIDETWKKMN KEKLGGSLFA KPFVETAINL ARQSHCTYHN GDAHTSPDEL TRKRVLSVIT EPILPFER

SEQ ID NO 28

Pueraria Montana Isoprene synthase

Adapted from Genbank: A Y316691.1

The active site is opened up by making the mutation F343L and F493W (shown bold underlined), F343L and F493Y, F343W and F493L, F343Y and F493L (not shown)

MATNLLCLSN KLSSPTPTPS TRFPQSKNFI TQKTSLANPK PWRVICATSS QFTQi mS RRSANYQPNL WNFEFLQSLE NDLKVEKLEE KATKLEEEVR C INRVDTQP LSLLELIDDV QRLGLTYKFE KDI IKALE I VLLDENKKNK SDLHATALSF RLLRQHGFEV SQDVFERFKD KEGGFSGELK GDVQGLLSLY EASYLGFEGE N^'LLEEARTFS ITHLKNNLKE GINTKVAEQV SHALELPYHQ RLHRLEARWF LDKYEPKEPH HQLLLELAKL DFNMVQTLHQ KELQDLSRWW TEMGLASKLD FVRDRLMEVY FWALGMAPDP QFGECRKAVT KMLGLV I ID DVYDVYGTLD ELQLFTDAVE RWDVNAIN L PDYMKLCFLA LYNTVNDTSY SILKEKGH N LSYLTKSWRE LCKAFLQEAK SNNKIIPAF SKYLENASVS SSGVALLAPS YFSVCQQQED ISDHALRSLT DFHGLVRSSC VIWRLCNDLA TSAAELERGE TTNSIISYMH ENDGTSEEQA RSELRKLIDA SWKKMNRERV

SDSTLLPKAF MEIAVNMARV SHCTYQYGDG LGRPDYATEN RIKLLLIDPF PINQLMYV

SEQ ID NO 29

Eucalyptus globulus "mts-1 mRNA for nionoterpene synthase" actually an isoprene synthase

Adapted from Genbarik: AB266390.1

The active site is opened up by making the mutation F326L and F473W (shown bold and underlined), F326L and F473Y, F326W and F473L, F326Y and F473L (not shown)

MALRLLFTPH LPVLSSRRAN GRVRCSASTQ JSDPQEGRRS ANYQPSV TY

NYLQSIVAGE GRQSRREVEQ QKEKVQILEE EVRGALNDEK AETFTIFATV

DDIQRLGLGD HFEEDISMAL RRCVSKGAvF MSLQKSLHGT ALGFRLLRQH

GYEVSQDVFK IFLDESGSFV KTLGGDVQGV LSLYEASHLA FEEEHILHKA

RSFATKHLEN LNSDVDKDLQ DQVKHELELP LHRRMPLLEA RRS IEAYSRR

GYTNPOILEL ALTDF VSQS YLQRDLQEML GWWNNTGLAK RLSFARDRLI

ECFFWAVGTA HEPSLSICRK AVTKALALIL VLDDVYDVFG TLEELELFTD

AVR.RWDLNAV EDLPVYMKLC YLALYNSVNE MAYETLKEKG ENVIPYLAKA

WYDLCKAFLQ EAKWSNSRII PGVEEYLNNG VSSSGSV L IHAYFL SPS

IRKEELESLE HYHDLLRLPS LIWRLTNDIA SSSAELERGE TNS IRCFMQ

EKGISELEAR ECVKEEIDTA. WKKMNKYMVD RSTFNQSFVR MTYNLARMAH CVYODGDAIG SPDDLSWNRV HSLIIKPISP AA

SEQ ID NO 30

Melaleuca alternifolia "putative nionoterpene synthase mRNA", actually an isoprene synthase

Adapted from GenBank: AY279379.1

The active site is opened up by making the mutation F326L and F473W (shown bold and underlined), F326L and F473Y, F326W and F473L, F326Y and F473L (not shown)

MALRLLSTPH LPQLCSRRVS GRVRCSASTQ VSDAQGGRRS ANYQPSVWTY YLQSL ADD IRRSRREVEQ EREKAQILEE DVRGALNDGN AEPMAIFALV

DDIQRLGLGR YFEEDISKAL RRCLSQYAVT GSLQKSLHGT ALSFRVLRQH

GFEVSQDVFK IFMDESGSFM KTLGGDVQGM LSLYEASHLA FEEEDILHKA

KTFAIKHLEN LNHDIDQDLQ DHVNHELELP LHRRMPLLEA RRFIEAYSRR

SNVNPRILEL AVMKFNSSOL TLQRDLQDML GWWN VGLAK RLSFARDRLM

ECFFWAVGIA REPALSNCRK GVTKALSLIL VLDDVYDVFG TLDELELFTD

AVRRWHEDAV ENLPGYMKLC FLALYNSV D MAYETLKETG ENVTPYLTKV

WYDLCKAFLQ EAKWSYNKI PGVEEYLNNG WVSSSGQVML THAYFLSSPS

LRKEELESLE HYHDLLRLPS LIWRLTNDLA TSSAELGRGE TTNS ILCYMR

EKGFSESEAR KQVIEQID A WRQMNKYMVD HSTFNRSFMQ MTYNLARMAH CVYQDGDAIG APDDQS NRV HSLIIKPVSL APC

SEQ ID NO 31

Robinia pseudoaccacia isoprene synthase Unpublished sequence

The active site is opened up by making the mutation F281 L and F428W (shown bold underlined), F281L and F428Y, F281W and F428L, F281Y and F428L (not shown)

RRSANYQPNL WNFEFLQSQE YDLMVETLQE RATKLEEEVR RLTNR.VDIEP LKLLELVDNV ORLGLTYKFE DDINKALERI VSLDEREKSG LHATALIFRL LRQHGFSVSQ DVFESTROKE GRFKAEIKGD VQGLLSLYEA SYLGSEGENL LDEAREFSMT HLKNLNEGVV PKLAEQIMH ALELPYHRRF QRLEARWFIE NYSVKEPHDR LLVELAKLDF NMVQSLQKKE VGESSRWWKE IGLTSKLDFV RDRLVEYYFW ASGMAPDPQL SECRKAVTKM |GLVTIIDDV YDVYGTLDEL ELFTNAVERW DVN VDTLPD YMKLCFFALY NTVNDTAYNL LKEKGD NLP YLAKSWSDLC KAFLQEAKWS NNKIIPSFNK YIENASVSSS GGALLTPCYF SIRQDI NQA LDSLTNYHGP VRSSCAT|RL INDLA SAAE LERGET||SI TSCMQDNGIS EEQARDELRM LIDAEWKQMN RERYFDQTFP KAFIETAINM

:-I1 YQY GDGLGRPDNT AENRIKLLLI DPLSN

SEQ ID NO

Humul s lupul s (Hops) isoprene synthase

Unpublished sequence

F354 and F530 are indicated in bold, underlined and high-lighted

MQCMAVHQFA PLLSLLNCSR ISSDFGRLFT PKTSTKSRSS TCHPIQCTW A TDRRSANY EPSIWSFDYI QSLTSOYKGK SYSSRLNELK KEYKMMEDGT KECLAQLDLI DTLQRLGISY HFEDEINTIL KRKYINIQNN INHNYNLYST ALQFRLLRQH GYLYTQEYFN AFKDETGKFK TYLSDDIMGY LSLYEASFYA MKHENVLEEA RVFSTECLKE Y1MKMEQNK LLDHDLDHND NF VNHHVLT INHALELPLH WRTTRSEARW FIDVYEKKQD MDSTLLEFAK LDFN^'MYQSTH QEDLKHLSRW WRHSKLGEKL NFARDRLMEA FLWEYGLKFE PEFSYFKRTS ARL|VLI TI DD YDVYG L EELELFTKAY ER.WDVNATNE LPEYMKMPFL VLR TINEMA FDVLGDONFL NIEYLKKSLY DLCKCYLQEA KWYYSGYQPT LQEYIEMAWL SIGGPYILYH AYFCFTNPIT KESMKFFTEG YPNIIQQSCL IVRLADDFGT FSDEL RGDV PKS QCYMYD TGASEDEARE HIKFLICETW KDMNKN^'DEDN^' SCFSETFVEV CKNLARTAL| MYQYGDGHAS QNCLSKERIF ALTINPTNFH ERK

SEQ ID NO 33

Aspergillus fumigatus tryptophan dimethylallyltransferase

Adapted from Genbank AY775787

Resides that line the active site and can be converted into phenylalanines (F) are show^rn bold and highlighted.

MKAANASSAE AYRVLSRAFR FDNEDQKLWW HSTAPMFAKM LETANYTTPC QYQYLT YKE CVTPSLGCYP TNSAPRWLSI LTRYGTPFEL SL CSNSIVR YTFEPINQHT GTDKDPFNTH AIWESLQHLL PLEKSIDLEW FRRFKHDLTL NSEESAFLAH. NDRLYGGTIR TQNKLALDLK DGRFALKTYI fPALKAVYTG KTIHELVFGS VRRLAVREPR ILPPLNMLEE YIRSRGSKST ASPRLVSCDL TSPAKSR1K.I YLLEQMVSLE AMEDLWTLGG RRRDASTLEG LSLVRELWDL IQLSPGLKSY PAPYLPLGVI PDERLPLMAN FTLHQ DPVP EPQV§FTTFG MMDMAVADAL TTFFERRGWS EMARTYET L KSYYPHADHD KLNYLHA|IS FSYRDRTPYL SVYLQSFETG D AVANLSES KVKCODAACQ PTALPPDLSK TGVYYSGLH

SEQ ID NO 34

Streptomyces sp. UC5319 pentalenene synthase gene

Adapted from Genbank: U05213.1

MPQDVDFHIP LPGRQSPDHA RAEAEQLAWP RSLGLIRSDA AAERHLRGGY ADLASRFYPH ATGADLDLGV DLMSWFFLFD DLFDGPRGEN PEDTKQLTDQ VAAALDGPLP DTAPPIAHGF ADIWRRTCEG MTPAWCARSA RHWRNYFDGY VDEAESRFWN APCDSAAQYL AMRRHTIGVO PTVDLAERAG RFEVPHRVFD SAVMSAMLQI AVDVNLLLND lASLEKEEAR GEQNNMVMIL RREHGWSKSR SVSHMQNEVR ARLEQYLLLE SCLPKVGEIY QLDTAEREAL ERYRTDAVRT VIRGSYDWHR SSGRYDAEFA LAAGAQGYLE ELGSSAH

S EC) ID 35

Streptomyces sp, UC5319 pentalenene synthase gene converted into an isoprene synthase by making the change V179F (shown), V179W or V179Y (not shown)

Adapted from Genbank: U05213.1

V 179F is shown in bold and highlighted.

MPQDVDFHIP LPGRQSPDHA RAEAEQLAWP RSLGLIRSDA AAERHLRGGY ADLASRFYPH ATGADLDLGV DLMSWFFLFD DLFDGPRGEN PEDTKQLTDQ VAAALDGPLP DTAPPIAHGF ADIWRRTCEG MTPAWCARSA RHWRNYFDGY VDEAESRFWN^' APCDSAAQYL AMRRHTIG!Q PTVDLAERAG RFEVPHRVFD SAVMSAMLQI AVDVNLLLND lASLEKEEAR GEQNNMVMIL RREHGWSKSR SVSHMQNEVR ARLEQYLLLE SCLPKVGEIY QLDTAEREAL ERYRTDAVRT VIRGSYDWHR SSGRYDAEFA LAAGAQGYLE ELGSSAH

SEQ ID NO 36

Streptomyces sp, UC5319 pentalenene synthase gene converted into an isoprene synthase by making the change N219F (shown), N219W or N219Y (not shown)

Adapted from Genbank: U05213.1

N219F is shown in bold and highlighted.

MPQDVDFHIP LPGRQSPDHA RAEAEQLAWP RSLGLIRSDA AAERHLRGGY ADLASRFYPH ATGADLDLGV DLMSWFFLFD DLFDGPRGEN PEDTKQLTDQ VAAALDGPLP DTAPPIAHGF ADIWRRTCEG MTPAWCARSA RHWRNYFDGY VDEAESRFWN APCDSAAQYL AMRRHTIGVQ PTVDLAERAG RFEVPHRVFD SAVMSAMLQI AVDVNLLLiD lASLEKEEAR GEQNNMVMIL RREHGWSKSR SVSHMQNEVR ARLEQYLLLE SCLPKVGEIY QLDTAEREAL ERYRTDAVRT VIRGSYDWHR SSGRYDAEFA LAAGAQGYLE ELGSSAH. SEQ ID NO 37

Streptomyces sp, UC5319 pentalenene synthase gene converted into an isoprene synthase by making the change V179F and 219F (shown), (V179F and N219W), (V179F and N219Y), (V179W and 219F), (V179W and N2 I 9W). (V179W and N219Y), (V179Y and N219F), (V179Y and N219W) or (V 179Y and N219Y) (not shown)

Adapted from Genbank: U05213.1

V i ^' *- ! ·^' and N219F are shown in bold and highlighted.

MPQDVDFHIP LPGROSPDHA RAEAEOLAWP RSLGLiRSDA AAERRLRGGY ADLASRFYPH ATGADLDLGV DLMSWFFLFD DLFDGPRGEN PEDTKQLTDQ VAAALDGPLP DTAPPIAHGF ADIWRRTCEG MTPAWCARS RHWRNYFDGY VDSASSRFWN APCDSAAQYL AMRRHTIG§Q PTVDLAERAG RFEVPHRVFD SAvMSAMLQI AVDVNLLL|D lASLEKEEAR GEQNNMVMIL RREHGWSKSR SVSHMONEVR ARLEQYLLLE SCLPKVGE IY OLDTAEREAL ERYRTDAVRT VIRGSYDWHR SSGRYDAEFA LAAGAQGYLE ELGSSAH

SEQ ID NO:38 pTrcHis2B-PHspS gtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggca gccatcggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgc tcaaggcgcactcccgttctggataatgttttttgcgccgacatcataacggttctggc aaatattctgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaatt gtgagcggataacaa111cacacaggaaacagcgccgct.gagaaaaagcgaagcggcac tgctctttaacaatttatcagacaatctgtgtgggcactcgaccggaattatcgattaa ctttattattaaaaattaaagaggtatatattaatgtatcgattaaataaggaggaata aaccatgagatgtagcgtgtccaccgaaaatgtgtctttcaccgaaactgaaaccgaag ctcgtcgttctgcgaactacgaacctaacagctgggactatgattacctgctgtcctcc gacacggacgagtccatcgaagtatacaaagacaaagcgaaaaagctggaagccgaagt tcgtcgcgagattaataacgaaaaagcagaatttctgaccctgctggaactgattgaca acgtccagcgcct.gggcctggg11accg1.11cgagtctgat.atccgtggtgcgctggat cgcttcgtttcctccggcggcttcgatgcggtaaccaagacttccctgcacggtacggc actgtctttccgtctgctgcgtcaacacggttttgaggtttctcaggaagcgttcagcg gcttcaaagaccaaaacggcaacttcctggagaacctgaaggaagatatcaaagctatc ctgagcctgtacgaggccagcttcctggctctggaaggcgaaaacatcctggacgaggc gaaggttttcgcaatctctcatctgaaagaactgtctgaagaaaagatcggtaaagagc tggcagaacaggtgaaccatgcactggaact.gccactgcatcgccgt.actcagcgtctg gaagcagtatggtctatcgaggcctaccgtaaaaaggaggacgcgaatcaggttctgct ggagctggcaattctggattacaacatgatccagtctgtataccagcgtgatctgcgtg aaacgtcccgttggtggcgtcgtgtgggtctggcgaccaaactgcactttgctcgtgac cgcctgattgagagcttctactgggccgtgggtgtagcattcgaaccgcaatactccga ctgccgtaactccgtcgcaaaaatgttttctttcgtaacca11at.cgacgatatctacg atgtatacggcaccctggacgaact.ggagctg1.11actgatgcagttgagcg1.1gggac gtaaacgccatcaacgacctgccggattacatgaaactgtgctttctggctctgtataa cactattaacgaaatcgcctacgacaacctgaaagataaaggtgagaacatcctgccgt atctgaccaaagcctgggctgacctgtgcaacgctttcctgcaagaagccaagtggctg t.acaacaaatctactccgacc1t1gacgactac1tcggcaacgcatggaaatcctc1.1c tggcccgctgcaactggtgttcgcttacttcgctgtcgtgcagaacattaaaaaggaag agatcgaaaacctgcaaaaataccatgacaccatctctcgtccttcccatatcttccgt ctgtgcaatgacctggctagcgcgtctgcggaaattgcgcgtggtgaaaccgcaaatag cgtttcttgttacatgcgcactaaaggtatctccgaagaactggctaccgaaagcgtga tgaatctgatcgatgaaacctggaaaaagatgaacaaggaaaaactgggtggtagcctg t1cgcgaaaccgt1cgtggaaaccgcgatcaacct.ggcacgtcaatcteactgcac11a tcataacggcgacgcgcat.acct.ctccggat.gagctgacccgcaaacgcgttctgtctg taatcactgaaccgattctgccgtttgaacgctaactcgagatctgcagctggtaccat atgggaattcgaagctttctagaacaaaaactcatctcagaagaggatctgaatagcgc cgtcgaccatcatcatcatcatcattgagtttaaacggtctccagcttggctgttttgg cggatgagagaagattttcagcctgatacagattaaatcagaacgcagaagcggtctga t.aaaacagaa111.gcct.ggcggcagtagcgcggtggt.eecacctgaccccatgccgaac tcagaagtgaaacgccgtagcgccgatggtagt.gtggggtctccecatgegagagtagg gaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtttt atctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggattt gaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgcca ggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaact ctttttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccc tgataaatgc1.1caataat.at1gaaaaaggaagagtatgagta1tcaaca1.1tccgtgt. cgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgc tggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactg gatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgat gagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaag agcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtc acagaaaagcatcttacggat.ggcatgacagtaagagaa11.atgcagtgetgecataac catgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagc taaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccg gagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggc aacaacgttgegcaaactattaactggcgaactacttactctagcttcccggcaacaat taatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccg gct.ggct.gg11.1a11.gctgataaatctggagccggtgagcgtgggtctcgcggtatcat tgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacgggga gtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgatt aagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaact tcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaa tcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaagga t.c11c1tgagatcc111.1111.ctgcgcgt.aatctgct.gc11.gcaaacaaaaaaaccacc gctaccagcggtgg111.gt11.gccggatcaagagctaccaactct.ttttccgaaggt.aa ctggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggc caccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacc agtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagt taccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttg gagcgaacgacctacaccgaact.gagatacctacagcgt.gagctatgagaaagcgccac gc1.1cccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggag agcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggttt cgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatg gaaaaaegccagcaacgcggcctttttacggttcctggccttttgctggcettttgctc acatg1tc1.1tcctgcg11at.cccctgattctgtggataaccgtattaccgcc1t1gag tgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgagga agcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacacc gcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtataca ctccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgct gacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgt ctccgggagct.gcat.gtgtca.gagg11.1tcaccgtcatcaccgaaacgcgcgaggcagc agatcaattcgcgcgcgaaggcgaagcggcatgcatttacgttgacaccatcgaatggt gcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtg aatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagac cgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtgg aagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggc aaacagtcg1tgctga1.1ggcg11gccacctccagtctggccctgca.cgcgccgtcgca aattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcga tggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaa cgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtgga agctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatca acagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgca t1gggtcaccagcaaatcgcgct.g11agcgggcccattaagttctgt.ctcggcgcgtct. gcgtctggctggctggcat.aaat.atcteact.cgcaatcaaattcagccgat.agcggaac gggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgag ggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcg cgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacg ataccgaagacagctcatgttatatcccgccgtcaaccaccatcaaacaggattttcgc ctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaa gggcaatcagctgttgccegtetcactggtgaaaagaaaaaccaccctggcgcccaata cgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtt tcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagcgcgaattga tctg

SEQ ID NO:39

pTrcHis2B-E2IspS gtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggca gccatcggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgc tcaaggcgcactcccgttctggataatgttttttgcgccgacatcataacggttctggc aaatattctgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaatt gtgagcggataacaa1t1cacacaggaaacagcgccgct.gagaaaaagcgaagcggcac tgctc11.1aacaa11.1atcagacaatctgtgtgggca.ct.cgaceggaattatcgattaa ctttattattaaaaattaaagaggtatatattaatgtatcgattaaataaggaggaata aaccatggagggtcgccgttccgcgaactaccagccgtctgtgtggacctacaactacc tgcaatccatcgtcgccggcgaaggccgtcaatcccgccgtgaagttgaacagcagaaa gaaaaagtacagatcctggaagaagaggtgcgtggcgcactgaacgatgaaaaagcgga gacgttcaccatc11cgctactgtggatgacattcagcgtctgggcctgggcgatca11 tcgaggaagacatcagcaacgcgct.gcgt.cg11.gcgt.gagcaaaggtgcagtc1tcatg tctctgcaaaaaagcctgcacggcaccgcgctgggttttcgtctgctgcgccagcatgg ctatgaagtatctcaggacgtatttaagatctttctggatgaaagcggcagctttgtca aaaccctgggtggcgacgtgcagggcgtgctgtccct.gtacgaggct1ctcatctggcg tttgaagaagagcacattctgcacaaagcgcgttcttttgcgatcaaacacctggaaaa cctgaactctgacgtagataaagatctgcaagaccaggtgaaacacgaactggagctgc cgctgcaccgtcgcatgccgctgctggaagctcgtcgtagcattgaggcttactctcgc cgtggctacactaacccgcagattctggagctggctctgactgactttaacgtctctca gagetacctgcaacgtgatctgcaagaaatgctgggttggtggaacaacaccggtctgg ctaagcgcctgagc1.1tgcgcgcgaccgcct.gatcgaat.gc11.c11ctgggcegteggc a11.gctcatgaaccgagcctgagcatctgccgcaaggcagtgaccaaagc1.111gccct. gatcctggtactggacgatgtctacgatgttttcggtacgctggaagagctggagctgt tcaccgacgcggttcgccgttgggatctgaacgcggttgaagatctgccggtgtatatg aaactgtgctatctggctctgtacaactctgtaaatgagatggcttacgaaaccctgaa agaaaaaggcgaaaatgtcattccgtacctggcgaaagcgtggtacgacctgtgtaaag cat1cctgcaagaagccaaat.ggagcaactctcgta1.1atcccgggtgt.ggaagagt.ac ctgaacaacgg11.ggg1.1tcttcttccggctccgtaatgct.ga11.cacgcatatttcct ggcttccccgagcattcgtaaagaggagctggaatctctggagcactatcacgatctgc tgcgcctgccgtccctgatcttccgcctgaccaacgacattgcatcttccagcgctgaa ctggaacgcggcgaaaccaccaacagcatccgttgtttcatgcaggaaaaaggtatctc cgaactggaagcacgcgagtgtgtcaaagaagagatcgacactgcatggaaaaagatga acaagtacatggtagatcgctccactttcaaccagtcc1.1tgt.eegcatgacttacaac ctggcacgtat.ggcgcact.gcgt.ataccaggacggtgacgcgattgg11ccceggacga tctgtcctggaaccgtgtacactctctgatcattaaaccgatttctccggcggcttaac tcgagatctgcagctggtaccatatgggaattcgaagctttctagaacaaaaactcatc tcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttaaac ggtctccagcttggctgttttggcggatgagagaagattttcagcctgatacagattaa atcagaacgcagaagcggtctgataaaacagaatttgcctggcggcagtagcgcggtgg teccacctgaccccatgccgaactcagaagtgaaacgecgt.agcgecgatggt.agtgtg gggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagt cgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtagg acaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggc aggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatg gectttttgcgtttctacaaactctttttgtttatttttctaaatacattcaaatatgt atccgct.catgagacaataaccctgat.aaat.gc11.caat.aata11gaaaaaggaagagt. atgagtattcaacatttccgtgtcgcccttattcccttttttgeggcattttgccttcc tgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtg cacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgc cccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtatt atcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatg acttggttgagtactcaccagtcacagaaaagcatc1.1acggatggcatgacagtaaga gaa11.atgcagtgctgccataaccatgagtgataa.cactgcggccaac1.1ac1.1ctgac aacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaa ctcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgac accacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactact tactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggac cac1tct.gcgcteggeec1.1ccggctggctggtttattgetgataaatetggagccggt. gagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtat. cgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcg ctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatat atactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcct ttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcag accccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgc tgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagct accaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtcc ttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatac ctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttac cgggt1ggact.caagacgatag1.1accggataaggcgcagcggtcgggctgaacggggg gttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagat.acct.acag cgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggt aagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggt atctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgc tcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcct ggcc1.1ttgctggcct1.1tgcteacatg1.1c11.1cct.gcg1.1atcccct.ga11.ctgt.gg ataaccgtattaccgcc1t1gagtgagct.gataccgctcgccgcagccgaacgaccgag cgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttac gcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatg ccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgc cccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatcc gc1.1acagacaagct.gtgaccgtctccgggagctgcatgtgtcagag-g111.1caccgtc atcaccgaaacgcgcgaggcagcagat.caa1.1cgcgcgcgaaggcgaagcggcat.gcat. ttacgttgacaccatcgaatggtgcaaaacctttcgcggtatggcatgatagcgcccgg aagagagtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagag tatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttc tgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaa11aca11cccaacc gcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagt ctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatct.cgcgccgatcaact gggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcgg cggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggat gaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttga tgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgac tgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggccca ttaagttctgt.ctcggcgcgtct.gcgtctggctggctggcataaatatctcactcgcaa tcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaac aaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgecaacgat cagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcgga tatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgtcaa ccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaa ctctctcagggccaggcggtgaagggcaatcagctg1.1gcccgtcteactggt.gaaaag aaaaaccaccctggcgcccaatacgcaaaccgcctctecccgcgcgt1ggccgat1cat taatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaat taatgtgagttagcgcgaattgatctg

SEQ ID NO:40 p Jexpress401 -E4IspS-His ctcatgaccaaaatcccttaacgtgagttacgcgcgcgtcgttccactgagcgt cagaccccgtagaaaagatcaaaggatc1.1c11.gagatec1.1t11.1tct.gcgcgtaatc tgetgettgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaaga gctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactg ttcttctagtgtagccgtagttagcccaccacttcaagaactctgtagcaccgcctaca tacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtct tacegggttggactcaagacgatagttaccggataaggcgcagcggtegggetgaaegg gggg11cgtgcacacagcccagc1tggagcgaacgacct.acaccgaactgagataccta cagegtgagct.atgagaaagcgccacgc11cccgaagggagaaaggcggacaggt.atcc ggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcct ggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtga tgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggtt cctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctg t.ggat.aaccgta1.1accgcc1.1tgagtgagctgataccgct.cgccgcagccgaacgacc gagcgcagcgagt.cagt.gagcgaggaagcggaaggcgagagtagggaactgccaggcat caaactaagcagaaggcccctgacggatggcctttttgcgtttctacaaactctttctg tgttgtaaaacgacggccagtcttaagctcgggccccctgggcggttctgataacgagt aatcgttaatccgcaaataacgtaaaaacccgcttcggcgggtttttttatggggggag tttagggaaagagcatttgtcagaatatttaagggcgcctgtcactttgcttgatatat gagaa11.at11.aacc1tat.aaat.gagaaaaaagcaacgcactttaaataagatacgt1g ctttttcgattgatgaacacctataattaaactattcatctattatttatgattttttg tatatacaatatttctagtttgttaaagagaattaagaaaataaatctcgaaaataata aagggaaaatcagtttttgatatcaaaattatacatgtcaacgataatacaaaatataa tacaaactataagatgttatcagtatttattatgcatttagaataaattttgtgtcgcc cttaattgtgagcggataacaattacgagcttcatgcacagtgaaatcatgaaaaattt atttgctttgtgagcggataacaattataatatgtggaattgtgagcgctcacaattcc acaacggtttccctctagaaataattttgtttaacttttctagaaataattttgtttaa. ctttaagaggaggtaaaacatatggagggccgtcgcagcgcaaattaccaaccgtctgt ttggacttataactacttgcagtcgatcgttgcgggtgagggtcgtcaaagccgccgtg aggttgagcaacagaaagaaaaggttcagattctggaagaagaagtccgtggcgcactg aatgacgaaaaggcggaaacgttcaccatctttgcaaccgttgacgatattcagcgcct gggcctgggcgateatttcgaagaggatattagcaacgcgctgcgtegttgcgtgteta agggtgcggtctttatgagcttgcagaaaagcctgcacggtactgct.ctgggctttcgt. ctgctgcgccagcacggttatgaagtcagccaagacgtgttcaaaatcttcttggacga gagcggttcttttgtgaaaacgctgggtggtgacgtgcagggtgttctgtctttgtacg aggccagccatctggccttcgaagaagagcacatcctgcataaagcgcgtagcttcgcc atcaagcatttggaaaatctgaacagcgatgttgataaggacctgcaagaccaagttaa gcacgagctggagctgccgctgcaccgtcgtatgccgttgctggaagcgcgtcgctcta 1.1gaggcgt.actcccgccgtggctata.ccaatccgcagatcctggag11.ggccctgacc gac11.1aat.gtgagccagagctac1.1gcaacgt.gac1.1gcaagagatgctgggctggtg gaacaacacgggtctggcgaaacgcttgagctttgcgcgtgatcgtctgattgagtgtt tcttttgggcagtgggtatcgcgcacgagccgagcctgagcatttgtcgtaaagctgtc acgaaggcattcgctttgatcctggttctggatgacgtttacgatgtctttggcacgct ggaagagctggaactgtttaccgacgcagtgcgccgctgggatctgaatgccgtcgaag acctgccgg11.1acatgaaactgtgct.acct.ggccctgt.ataactccgtcaacgagatg gcgtatgaaaccctgaaagagaaaggcgagaatgt.ga11.ccgt.acct.ggcaaaagcctg gtacgatctgtgcaaggcattcctgcaagaggcgaagtggagcaacagccgtatcatcc ctggtgttgaagaatatctgaataacggttgggtcagcagcagcggctccgttatgctg atccatgcgtactttctggcgagcccgagcatccgtaaagaagaactggagtctctgga gcact.atcacgatctgctgcgtctgccttctct.gat1.1tccgcct.gaccaatgaca1.1g cgagcagctccgcagaactggaacgcggtgaaacgaccaactccattcgttgtttcatg caggagaaaggtatttccgaactggaggctcgtgaatgcgtgaaagaagagatcgacac cgcttggaaaaagatgaataagtatatggtcgatcgcagcaccttcaatcagagcttcg tccgtatgacctacaacctggcgcgtatggcgcattgcgtgtatcaggacggtgacgcg attggtagcccggacgatctgtcctggaaccgcgtccacagcctgatcattaagccgat tagcccagcggcagaaaacctgt.attttcagagcggcagcggcagcggcagcggccatc accatcaccacca11.aactcgagtcgagccccaagggcgacaccccctaa1.1agcccgg gcgaaaggcccagtctttcgactgagcctttcgttttatttgatgcctggcagttccct actctcgcatggggagtccccacactaccatcggcgctacggcgtttcacttctgagtt cggcatggggtcaggtgggaccaccgcgctactgccgccaggcaaacaaggggtgttat gagccatattcaggtataaatgggctcgcgataatgttcagaattggttaattggttgt aacactgacccct.at11.gt11.at11.1tct.aaat.acattcaaatat.gtat.ccgcteatga gacaataaccctgataaatgc1tcaataata11.gaaaaaggaagaatat.gagccata1t caacgggaaacgtcgaggccgcgattaaattccaacatggatgctgatttatatgggta taaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgcttgtatggga agcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgtt acagatgagatggtcagactaaactggctgacggaatttatgccacttccgaccatcaa gca1111.atccgtactcct.gatgatgcatgg1tacteaccact.gcgatccccggaaaaa cagcgttccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctg gcagtgttcctgcgccggttgcactcgattcctgtttgtaattgtccttttaacagega tegegtatttcgcctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcga gtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcat aaacttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataa ccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcg cagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttca. ttacagaaaeggetttttcaaaaatatggtattgataatcctgatatgaataaattgea gttteatttgatgctcgatgagtttttctaagcggcgcgccatcgaatggcgcaaaacc tttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaatatgaa accagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttccc gcgtggtgaaccaggccagccacg111ctgcgaaaacgcgggaaaaagtggaagcggcg atggcggagct.gaa1.1aca1tcccaaccgcgtggcacaacaactggcgggcaaacagtc gttgetgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcg cggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaa cgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcag tgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcct gcactaatgtteeggegttatttcttgatgtctctgaccagacacccatcaacagtatt a1111ctcccatgaggacggtacgcgact.gggcgtggagcatctggtcgca11.gggt.ca ccagcaaat.cgcgctg1.1agcgggcccattaagttct.gtct.cggcgcgt.ctgcgtct.gg ctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggc gactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgt tcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccatta ccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaa gat.agct.catg11at.atcccgccgttaaccaccat.ca.aaca.ggat11.1cgcctgctggg gcaaaccagcgtggaccgc11gctgcaactctctcagggccaggcggtgaagggcaatc agctgttgccagtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaacc gcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgact ggaaagcgggcagtga

SEQ ID NO:41 pCL-SDFi cccgtcttactgtcgggaattcgcgttggccgattcattaatgcagctggcacg acaggtttcccgactggaaagcgggcagtgagcgcaacgcaa11aatgt.gag1.1agctc actcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaat tgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttg catgcctgcaggtcgactctagaggatccccgcacccaactgatcttcagcatctttta ctttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaaggga ataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaag catttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaata aacaaaaagagtttgtagaaacgcaaaaaggccatccgtcaggatggccttctgcttaa tttgatgcctggcagtttatggcgggcgtcctgcccgccaccctccgggccgttgcttc gcaacgttcaaatccgctcccggcggatttgtcctactcaggagagcgttcaccgacaa acaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttgatgcc tggcagttccctactctcgcatggggagaccccacactaccatcggcgctacggcgttt cac11.ctgagt1cggcatggggtcaggtgggaccaccgcgctact.gccgccaggcaaat t.ctgttttatcagaccgc11ctgcg1tct.ga11.1aat.ctgt.atcaggct.gaaaatc1.1c tctcatccgccaaaacagccaagctggagaccgtttaaactcaatgatgatgatgatga tggtcgacggcgctattcagatcctcttctgagatgagtttttgttctagaaagcttcg aattcttatttaagctgggtaaatgcagataatcgttttctggcttcgcgatttgtcgc ctgcatcaecatccacggactgaacgcccacggcgtggcatcaataccgtgtaatacat ctgctaaatcacaccattgataatccatcactteatcatcattgatctgtaacgcacta gtggtgcgtgcggcaaataccggacacac11.ca11.1tccacaatgccactcggat.cggt. ggcgcggtagcgaaagtcaggatagatagattcaggaggcgtaatttccacgccaagct cataacggcaacggcggatcactgcgtcttegttgctttctcccagttgtgggtgccca caaaccgagttagtccacacgccaggccatgcttttttgctcagtgcgcggcgggtaac taataattgtcctttggcattaaacagccaactggagaacgcgagatgtaagcgggtgt ctgccgtgtgtgcggcatacttttccagcgtacccgtgggaactccctgtgcattcaat aaaatgacgtgttccgtttgcatgcccatggtatatctccttcttaaagttaaacaaaa. ttatttctagtteattttgttgccttaatgagtagcgccaccgcttcacaggcaatccc ttccccacgtccggtaaatcccagtttttccgtagtagtggctttcacgttaacatcat ccatatggcagecgagatctteggcaataaacacgcgcatttgtggaatgtgcggcaac atcttcggtgcctgagcgatgatagtgacatcgacgttgccaagggtataacccttcgc ctgaatacgacgccaggcttcgcgtagcagctcgcggctatcggcacctttaaatgccg gat.cggt.atccgggaacagcttgccgatatcccccagcgecgccgcgecaagcaatgca tcggtcaacgcatggagcgecacgtcgecatcagaatgcgccagcaatcctttttcgta aggaatgcgtacgccaccaatgataattgggccttcaccgccaaaggcatgtacgtcaa aaccgtgtccaattcgcatgcccatggtatatctccttcttaaagttaaacaaaattat ttctagtttatgtattctcctgatggatggttcgggtgaggtaaaactcggccagtgcc aaatcttccgggcgcgtgactttaatgttatccgcacggccttcgaccaactgaggatg gaatccgcaatattccagcgcegaggcttegtcggtaatagtcgcgccttca1.1tagag cgcgcgtcagacagtcatgtaacagctcacgagggaaaaattgcggcgtcagcgcgt.gc cataagccgttgcgatcaacggtatgagcaatggcatttttgcccggttcggcacgttt catagtatcgcgcactggtgcggcgaggatcccccccgtgcggctggtttcgctcaacg ccaacaatcgcgcgaggtcatcctgatgcaaacaaggacgagcggcgtcatgcaccaat acccactgcgcgtcgccagcggctttcagacctgccagcacggaatcggcacgctcatc accgccatctacaacggtgatttgcggatgattcgccagaggaagttgtgcaaaacggc tatcgccaggacttatggcaatgacgacacgtttcacccggggatgcgccagcagcgca tgcaccgagtgttcaagaatggtttgattaccgattgagagatattgcttaggacattc cgtttgcattcgacggccaaatccggccgccggaaccacggcgcaaacatccaaatgag tgg11gccatgcccatggt.atat.ctcc11cttaaagttaaacaaaa1.1a11.1ctag111. atgccagccaggccttgattttggcttccataccagcggcatcgaggccgagttcggcg cgcatttcttcctgagttccttgcggaataaagaagtccggcaggccaatgttcagcac gggtactggtttacgatgggccatcagcacttcgttcacgccgctgcctgcgccgccca taatggcgttttcttctacggtgaccagcgcttcatggctggcggccatttccagaatt aacgcttcatcaagcggtttcacaaaacgcatatcgaccagcgtggcgttcagcgattc ggcgactttcgccgcttctggcatcagcgtaccaaagttaaggatcgccag11.1ctcgc cacgacgc1.1cacaatgcctttgccaa11.ggtag111.11ccagcggcgt.cag1.1ccacg ccgaccgcgttgccacgcgggtagcgcaccgctgacgggccatcgttatagtgatagcc ggtatagagcatctggcgacattcgttttcatcgctcggggtcataatgaccatttccg gtatgcagcgcaggtaagagagatcaaaagcaccctgatgggtttgaccgtcagcacca acaatgcccgcgcggtcgatggcgaacaggaccggaagcttttgaatcgccacgtcatg cagcacctgatcataggcgcgttgcaggaaagtggagtaaatcgcgacaatgggtttgt acccaccaatcgccagacccgcagcaaaggt.caccgcgt.g11gctcggcaa11gccacg tcgaagtagcgatccgggaatttacgtgaaaactcgaccatgccggaaccttcacgcat cgccggagtaatcgccatcagcttgttgtctttcgctgccgtttcgcacaaccagtcgc caaagatttttgaatagctcggcaaaccgccgctacttttcggcaaacaaccgctggag ggatcaaatttaggcacggcgtggaaagtgatcgggtctttttctgccggttcataacc acgaccttttttggtcatgatatgcaggaactgcgggcctttcaggtcgcgcatgttct 1.1agcgtggtgat.aagccccagcacatcgtgaccgtccaccgggccgat.gtag11aaag cccagctcttcaaacaacgtgccaggcactaccatgcctttaatatgttcttcggtgcg tttgagcagctctttaattggcggcacgccagagaaaacttttttcccgccttcgcgca gtgaagagtaaagcttaccggaaagcagctgtgccagatggttgttgagcgcgccgaca ttttcggaaatcgacatttcattgtcgttgagaatcaccagcatatcaggacggatatc gcccgcgtgattcatcgcttcaaacgccatgcctgcggtaatcgcgccatcgccaatga cacagacggtgcggcgatttttgccttctttttcggcagcaaccgcaataccaattccg gcactgatggaggttgatgaatgcccgacgcttaatacgtcatattcgctttcgccgcg ccacgggaacgggtgcagaccgcctttctgacggatggtgccgattttgtcgcggcgtc cggtcaaaattttatgcggataagcctgatgccccacatcccaaatcaattggtcaaac ggggtgttgtagacatagtgcagcgccacggtcagttcgaccgtgcccagcccggaggc gaagtgcccgctggaacggctcacgctgtcgagtaaatagcggcgcag11cgtcgcaga g111cggtaaact.ctc1.11cggcaacagtcgtaactcctgggtggagtcgaccagtgcc agggtcgggtatttggcaatatcaaaactcatgcccatggtttattcctccttatttaa. tcgatacattaatatatacctctttaatttttaataataaagttaatcgataattccgg tcgagtgcccacacagattgtctgataaattgttaaagagcagtgccgcttcgcttttt ctcagcggcgctgtttcctgtgtgaaattgttatccgctcacaattccacacattatac gagccggatgattaattgtcaacagctcatttcagaatatttgccagaaccgttatgat gtcggcgcaaaaaaca11atccagaacgggagtgcgccttgagcgacacgaa11atgca gtgatttacgacctgca.cagccataccacagcttccgat.ggct.gcct.gacgccagaagc attggtgcagggtaccgagctcgaattcactggccgtcgttttacaacgtcgtgactgg gaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctg gcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatg gcgaatggcgcctgatgcggt.attttctccttacgcatctgtgcggta1.11cacaccgc atatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgaca cccgccaacacccgctgacgagcttagtaaagccctcgctagattttaatgcggatgtt gcgattacttcgccaactattgcgataacaagaaaaagccagcctttcatgatatatct cccaatttgtgtagggcttattatgcacgcttaaaaataataaaagcagacttgacctg atagtttggctgtgagcaattatgtgcttagtgcatctaacgcttgagttaagccgcgc cgcgaagcggcgtcggcttgaacgaattgttagacattatttgccgactaccttggtga tct.cgcc111cacgt.agtggacaaattcttccaactgat.ctgcgcgcgaggccaagcga tcttcttcttgtccaagataagcctgtctagcttcaagtatgacgggctgatactgggc cggcaggcgctccattgcccagtcggcagcgacatccttcggcgcgattttgccggtta ctgcgctgtaccaaatgcgggacaacgtaagcactacatttcgctcatcgccagcccag tcgggcggcgagttccatagcgttaaggtttcatttagcgcctcaaatagatcctgttc aggaaccggatcaaagag11cctccgccgctggacct.accaaggcaacgctat.g11ctc ttgcttttgtcagcaagatagccagatcaatgtcgatcgtggctggctcgaagatacct gcaagaatgtcattgcgctgccattctccaaattgcagttcgcgcttagctggataacg ccacggaatgatgtcgtcgtgcacaacaatggtgacttctacagcgcggagaatctcgc tctctccaggggaagccgaagtttccaaaaggtcgttgatcaaagctcgccgcgttgtt tcatcaagccttacggtcaccgtaaccagcaaatcaatatcactgtgtggcttcaggcc gccatccactgcggagccgtacaaatgtacggccagcaacgtcggttcgagatggcgct. cgatgacgccaactacctctgatagttgagtcgatacttcggcgatcaccgcttccctc atgatgtttaactttgttttagggcgactgccctgctgcgtaacatcgttgctgctcca taacatcaaacatcgacccacggcgtaacgcgcttgctgcttggatgcccgaggcatag actgtaccccaaaaaaacagtcataacaagccatgaaaaccgccactgcgccgttacca ccgctgcgttcggtcaaggttctggaccagttgcgtgagcgcatacgctacttgcatta cagcttacgaaccgaacaggcttatgtccactgggttcgtgccttcatccgtttccacg gtgtgcgtcacccggcaacc1.1gggcagcagcgaagt.cgaggca1.11ct.gtcctggctg gcgaacgagcgcaaggtttcggtctccacgcatcgtcaggcattggcggccttgctgtt cttctacggcaaggtgctgtgcacggatctgccctggcttcaggagatcggaagacctc ggccgtcgcggcgcttgccggtggtgctgaccccggatgaagtggttcgcatcctcggt tttctggaaggcgagcatcgtttgttcgcccagcttctgtatggaacgggcatgcggat cagtgagggtttgcaactgcgggtcaaggatctggatttcgatcacggcacgatcatcg tgcgggagggcaagggctccaaggatcgggccttg-atg1.1acccgagagc1.1ggcaccc agcctgcgcgagcaggggaattaattcccacgggttttgctgcccgcaaacgggctgtt ctggtgttgctagtttgttatcagaatcgcagatccggcttcagccggtttgccggctg aaagcgctatttcttccagaattgccatgattttttccccacgggaggcgtcactggct cccgtgttgtcggcagctttgattcgataagcagcatcgcctgtttcaggctgtctatg tgtgactgttgagctgtaacaagttgtctcaggtgttcaatttcatgttctagttgctt t.g111.1act.gg11.1cacctgttcta11aggtg1.1acatgct.g11catct.g11aca11.gt cgatctg11.catggtgaacagcttt.gaat.gcaccaaaaactcgtaaaagctct.gatgta tctatcttttttacaccgttttcatctgtgcatatggacagttttccctttgatatgta acggtgaacagttgttctacttttgtttgttagtcttgatgcttcactgatagatacaa gagccataagaacctcagatccttccgtatttagccagtatgttctctagtgtggttcg ttgtttttgcgtgagccatgagaacgaaccattgagatcatacttactttgcatgtcac tcaaaaattttgcctcaaaactggtgagctgaatttttgcagttaaagcatcgtgtagt g11.111c11agtccg11at.gtaggtaggaatctgatgtaatgg11g1.1ggt.a111.1gtc accatteatttttatctggttgttctcaagttcggttacgagatccatttgtctatcta gttcaacttggaaaatcaacgtatcagtcgggcggcctcgcttatcaaccaccaatttc atattgetgtaagtgtttaaatetttacttattggtttcaaaacccattggttaagcct 1.11aaactcatggtag1.1a11.1tcaagcattaacatgaa.c1.1aaa11catcaaggct.aa tctctatatttgccttgtgagttttcttttgtgttagttcttttaataaccactcataa atcctcatagagtatttgttttcaaaagacttaacatgttccagattatattttatgaa tttttttaactggaaaagataaggcaatatctcttcactaaaaactaattctaattttt cgcttgagaacttggcatagtttgtccactggaaaatctcaaagcctttaaccaaagga ttcctgatttccacagttctcgtcatcagctctctggttgctttagctaatacaccata agcat11.1ccctact.gatg1tcatcatctgagcgt.attggttataagtgaacgat.accg tccgt1c1t1cct1gtagggttttcaatcgt.gggg1tgagtagtgccacacagcataaa attagcttggtttcatgctccgttaagtcatagcgactaatcgctagttcatttgcttt gaaaacaactaattcagacatacatctcaattggtctaggtgattttaatcactatacc aattgagatgggctagtcaatgataattactagtccttttcctttgagttgtgggtatc tgtaaattctgctagacctttgctggaaaacttgtaaattctgctagaccctctgtaaa ttccgctagacctttgtgtgttttttttgtttatattcaagtggttataatttatagaa. t.aaagaaagaataaaaaaagataaaaagaatagatcccagccctgtgtataacteacta ctttagtcagttccgcagtattacaaaaggatgtcgcaaacgctgtttgctcctctaca aaacagaccttaaaaccctaaaggcttaagtagcaccctcgcaagctcgggcaaatcgc tgaatattccttttgtctccgaccatcaggcacctgagtcgctgtctttttcgtgacat tcagttcgctgcgctcacggctctggcagtgaatgggggtaaatggcactacaggcgcc ttttatggattcatgcaaggaaactacccataatacaagaaaagcccgtcacgggcttc tcagggcg1111a.tggcgggtctgctatgtggtgctatctgac1111.1gct.g11cagca gttcctgccctctgattttccagtctgaccactteggattatcccgtgacaggteattc agactggctaatgcacccagtaaggcagcggtatcatcaacaggctta

Claims

We claim:

1. An isolated nucleic acid sequence comprising a sequence encoding an isoprene synthase variant of a parent eucalyptus isoprene synthase, said sequence operably linked to a promoter, wherein the isoprene synthase variant is capable of catalyzing a reaction that synthesizes isoprene from DMADP (dimethylallyl disphosphate) and said isoprene synthase variant is truncated at the N-terminus as compared to the parent eucalyptus isoprene synthase.

2. The nucleic acid sequence of claim 1 , wherein the parent eucalyptus isoprene synthase is E, globulus of SEQ ID NO: 6,

3. The nucleic acid sequence of any of claims 1-2, wherein said isoprene synthase variant has a sequence that has at least five amino acids truncated from the N- termiiius as compared to the parent eucalyptus isoprene synthase,

4. The nucleic acid sequence of any of claims 1-3, wherein said isoprene synthase variant has a sequence that has five to thirty five amino acids truncated from the N-terminus as compared to the parent eucalyptus isoprene synthase.

5. The nucleic acid sequence of any of claims 1 -4, wherein said isoprene synthase variant has a sequence that has ten to thirty five amino acids truncated from the N-terminus as compared to the parent eucalyptus isoprene synthase.

6. The nucleic acid sequence of any of claims 1 -5, wherein said isoprene synthase variant has a sequence that has fifteen to thirty five amino acids truncated from the N-terminus as compared to the parent eucalyptus isoprene synthase.

7. The nucleic acid sequence of any of claims 1-6, wherein said isoprene synthase variant has a sequence that has twenty to thirty five amino acids truncated from the N-terminus as compared to the parent eucalyptus isoprene synthase.

8. The nucleic acid sequence of any of claims 1 -7, wherein said isoprene synthase variant has a sequence that has twenty five to thirty five amino acids truncated from the N-terminus as compared to the parent eucalyptus isoprene synthase.

9. The nucleic acid sequence of any of claims 1 -8, wherein said isoprene synthase variant has a sequence that has thirty to thirty five amino acids tamcated from the N-terminus as compared to the parent eucalyptus isoprene synthase,

10. The nucleic acid sequence of any of claims 1-9, wherein said isoprene synthase variant has a sequence that has thirty five amino acids truncated from the - terminus as compared to the parent eucalyptus isoprene synthase.

1 1 . The nucleic acid sequence of any of claims 1-2, wherein said isoprene synthase variant has a sequence that has less than thirty five amino acids truncated from the N-terminus as compared to the parent eucalyptus isoprene synthase.

12. The nucleic acid sequence of any of claims 1-1 1 , wherein the promoter is a

prokaryotic promoter.

13. The nucleic acid sequence of any of claims 1-12, wherein the promoter is a pTrc promoter.

14. An expression vector comprising the nucleic acid sequence of any of claims 1-13.

15. An isolated host cell comprising a heterologous nucleic acid sequence according to any of claims 1 -13.

16. An isolated host cell comprising a vector according to claim 14.

17. The isolated host cell of claim 15 or 16 being a bacterial cell.

18. The isolated host ceil of claim 17, wherein the bacterial cell is Escherichia coli,

19. The isolated host cell of any of claims 15-18, wherein the host cell further comprises one or more recombinant nucleic acid sequence of a MEP pathway gene.

20. The isolated host ceil of claim 19, wherein the MEP pathway gene is selected from dxs, ispD, ispF, and idi.

21. An isolated isoprene synthase variant of a parent eucalyptus isoprene synthase, wherem said variant comprises a truncation in the N-terminal portion of isoprene synthase as compared to the parent eucalyptus isoprene synthase and wherein said variant is capable of catalyzing a reaction that synthesizes isoprene from DMADP (dimethylallyl disphosphate) .

22. A method of producing isoprene comprising: (a) providing a host cell comprising an expression vector according to claim 14; and (b) culturing the host cell under conditions suitable for producing isoprene.

23. The method of claim 22, further comprising (c) recovering the isoprene.

24. The method of claim 23, further comprising fd) polymerizing the isoprene.

25. An isolated nucleic acid sequence comprising a sequence encoding a variant of a parent solarium phellandrene synthase, said sequence operably linked to a promoter, wherein the isoprene synthase variant is capable of catalyzing a reaction that synthesizes isoprene from DMADP (dimethylallyl disphosphate) and said isoprene synthase variant is truncated at the N-terminus as compared to the parent solanum phellandrene synthase.

26. The nucleic acid sequence of claim 25, wherein the parent eucalyptus isoprene synthase is S. fycopersic m of SEQ ID NO: 23.

The nucleic acid sequence of any of claims 25-26, wherein said variant has a sequence that has at least five amino acids truncated from the N-termimis as compared to the parent so!anum phel!andrene synthase.

The nucleic acid sequence of any of claims 25-27, wherein said variant has a sequence that has five to thirty six amino acids truncated from the N-terminus as compared to the parent solatium phellandrene synthase.

The nucleic acid sequence of any of claims 25-28, wherein said variant has a sequence that has ten to thirty six amino acids truncated from the N-terminus compared to the parent soianum phellandrene synthase.

30. The nucleic acid sequence of any of claims 25-29, wherein said variant has a sequence that has fifteen to thirty six amino acids truncated from the N-terminus as compared to the parent soianum phellandrene synthase.

31. The nucleic acid sequence of any of claims 25-30, wherein said variant has a sequence that has twenty to thirty six amino acids truncated from the N-terminus as compared to the parent soianum phellandrene synthase.

The nucleic acid sequence of any of claims 25-31 , wherein said variant has sequence that has twenty five to thirty six amino acids truncated from the N terminus as compared to the parent soianum phellandrene synthase.

33. The nucleic acid sequence of any of claims 25-32, wherein said variant has a sequence that has thirty to thirty six amino acids truncated from the N-terminus as compared to the parent soianum phellandrene synthase.

34. The nucleic acid sequence of any of claims 25-33, wherein said variant has a sequence that has thirty six amino acids truncated from the N-terminus as compared to the parent soianum phellandrene synthase.

35. The nucleic acid sequence of any of claims 25-26, wherein said variant has a sequence tha t has less than thirty six amino acids truncated from the N-termmus as compared to the parent eucalyptus isoprene synthase.

36. The nucleic acid sequence of any of claims 25-35, wherein the promoter is a prokaryotic promoter.

37. The nucleic acid sequence of any of claims 25-36, wherein the promoter is a pTrc promoter.

38. An expression vector comprising the nucleic acid sequence of any of claims 25- 37.

39. An isolated host cell comprising a heterologous nucleic acid sequence according to any of claims 25-37.

40. An isolated host cell comprising a vector according to claim 38.

41. The isolated host cell of claim 39 or 40 being a bacterial cell.

42. The isolated host ceil of claim 41, wherein the bacterial cell is Escherichia coli.

43. The isolated host ceil of any of claims 39-42, wherein the host cell further

comprises one or more recombinant nucleic acid sequence of a MEP pathway gene.

44. The isolated host ceil of claim 43, wherein the MEP pathway gene is selected from dxs, ispD, ispF, and idi.

45. A variant of a parent solarium pbellandrene synthase, wherein said variant

comprises a truncation in the N-terminal portion of isoprene synthase as compared to the parent eucalyptus isoprene synthase and wherein said variant is capable of catalyzing a reaction that synthesizes isoprene from DMADP

(dimethylallyl disphosphate) ,

46. A method of producing isoprene comprising: (a) providing a host cell comprising an expression vector accordmg to claim 38; and (b) culturmg the host ceil under conditions suitable for producing isoprene.

47. The method of claim 46, further comprising (c) recovering the isoprene.

48. The method of claim 47, further comprising (d) polymerizing the isoprene.