FIELD OF THE INVENTION

The invention relates to improved methods for the production of alkenes useful in the production of

biofuels and/or biochemicals , and expression vectors and host cells useful in such methods.

BACKGROUND TO THE INVENTION

With the diminishing supply of crude mineral oil, use of renewable energy sources is becoming increasingly important for the production of liquid fuels and/or chemicals. These fuels and/or chemicals from renewable energy sources are often referred to as biofuels.

Biofuels and/or biochemicals derived from non-edible renewable energy sources are preferred as these do not compete with food production.

Hydrocarbons such as alkenes are important

constituents in the production of fuels and/or chemicals. It would therefore be desirable to produce alkenes

(sometimes also referred to as bio-alkenes) from non- edible renewable energy sources.

SUMMARY OF THE INVENTION

A first aspect of the invention provides a method for preparing a mono-unsaturated alkene, comprising

contacting an aliphatic mono-unsaturated carboxylic acid with an Fdcl polypeptide (herein also referred to as ferulic acid decarboxylase polypeptide or ferulic acid decarboxylase enzyme) comprising an amino acid sequence with at least 21% sequence identity to SEQ ID NO : 1 and a Padl polypeptide (herein also referred to as phenacrylate decarboxylase polypeptide or phenacrylate decarboxylase enzyme) comprising an amino acid sequence with at least

17% sequence identity to SEQ ID NO : 2. In a preferred embodiment the invention provides a method for preparing a terminal mono-unsaturated alkene, comprising contacting an aliphatic a, β-mono-unsaturated carboxylic acid with a first polypeptide having ferulic acid decarboxylase activity, which first polypeptide comprises a first amino acid sequence with at least 21%, preferably at least 30% sequence identity, more

preferably at least 50% sequence identity, to the amino acid sequence set out in SEQ ID NO:l, and with a second polypeptide having phenacrylate decarboxylase activity, which second polypeptide comprises a second amino acid sequence with at least 17%, preferably at least 37% sequence identity, more preferably at least 60% sequence identity, to the amino acid sequence set out in SEQ ID NO: 2.

Without wishing to be bound by any kind of theory it is believed that decarboxylation of carboxylic acids by biological catalysts working under ambient conditions often requires the use of co-factors (for example thiamine; biotin; metals) that act as transient electron acceptors. It is believed that the heterolytic bond breakage of the C-C bond that gives rise to CO ₂ may result in carbanion species that are highly unstable, unless the electron pair can be delocalised. There have been reports of enzymes that do not require cofactors, but such enzymes often have very strict substrate requirements .

The so-called Fdcl/Padl enzyme system belongs to a family of enzymes that broadly catalyses the reversible decarboxylation of aromatic groups. The co-factor requirement, mechanism or individual properties of the family members have not been reported in any detail to date . The yeast Fdcl and Padl enzymes have been implicated in decarboxylation of aromatic substrates (see the article of Mukai N et al . (2010) J. Biosci. Bioeng. 109, 564-569) . Padl enzyme also has been singly implicated in decarboxylation of sorbic acid (see the article of

Stratford M et al. (2007) Appl . Environ. Microbiol .73 , 6534-6542) . All these substrates have a minimum of two double bonds adjacent to the carboxylic group in common, which would have been expected to be key to the enzyme mechanism. Surprisingly, it has now been found that

generation of terminal alkenes by decarboxylation of an aliphatic mono-unsaturated carboxylic acid with a carbon double bond between the a and β positions is possible using a combination of Fdcl and Padl.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used herein will have the meanings that are commonly understood by those of ordinary skill in the art .

Generally, nomenclatures used in connection with techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization, described herein, are those well known and commonly used in the art .

Conventional methods and techniques mentioned herein are explained in more detail, for example, in Sambrook et al . (Molecular Cloning, a laboratory manual [second edition] Sambrook et al. Cold Spring Harbor Laboratory, 1989) .

The identity of amino acid sequences and nucleotide sequences referred to in this specification is as set out in Table 3 at the end of the description. The terms "polynucleotide", "polynucleotide sequence" and "nucleic acid sequence" are used interchangeably herein. The terms "polypeptide", "polypeptide sequence" and "amino acid sequence" are, likewise, used interchangeably herein. Other sequences encompassed by the invention are provided in the Sequence Listing and listed in Tables 4 and 5 with reference to GenBank accession numbers.

Enzyme Commission (EC) numbers (also called "classes" herein) , referred to throughout this specification, are according to the Nomenclature Committee of the

International Union of Biochemistry and Molecular Biology (NC-IUBMB) in its resource "Enzyme Nomenclature" (1992, including Supplements 6-17) available, for example, at http://www.chem.qmul.ac.uk/ iubmb/enzyme/ . This is a numerical classification scheme based on the chemical reactions catalysed by each enzyme class (Enzyme

nomenclature 1992: recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes. Webb, E.C. (1992) . San Diego: Published for the International Union of Biochemistry and Molecular Biology by Academic Press. ISBN 0-12-227164-5). The skilled person can readily determine whether an enzyme falls within any particular enzyme class as referred to herein, for example using methods obtainable from the

International Union of Biochemistry and Molecular Biology (IUBMB) .

The term "Fdcl polypeptide" indicates a ferulic acid decarboxylase enzyme in class EC No . 4.1.1. The term "Fdcl polypeptide" is used interchangeably herein with the terms "Fdcl enzyme" and "Fdcl protein". The "first polypeptide having ferulic acid decarboxylase activity" is preferably a "Fdcl polypeptide". The "Fdcl polypeptide" or "first polypeptide having ferulic acid decarboxylase activity" may comprise the amino acid sequence SEQ ID NO : 1 ( Saccharomyces cerevisiae Fdcl protein) or a variant amino acid sequence, which is at least 21% identical, more preferably at least 30%, even more preferably at least50% or at least 55% identical, to SEQ ID NO : 1 (for example, a sequence listed in Table 4, such as SEQ ID NOs : 1 and 16-26) . More preferably the "Fdcl polypeptide" or "first polypeptide having ferulic acid decarboxylase activity" consists essentially of the amino acid sequence SEQ ID NO : 1 or a variant amino acid sequence, which is at least 21% identical, more

preferably at least 30%, even more preferably at least 50% or at least 55% identical, to SEQ ID NO : 1. Most preferably the "Fdcl polypeptide" or "first polypeptide having ferulic acid decarboxylase activity" consists of the amino acid sequence SEQ ID NO : 1 or a variant amino acid sequence, which is at least 21% identical, more preferably at least 30%, even more preferably at least50% or at least 55% identical, to SEQ ID NO : 1.

The term "Padl polypeptide" indicates a phenacrylate decarboxylase enzyme in class EC No . 4.1.1. The term "Padl polypeptide" is used interchangeably herein with the terms "Padl enzyme" and "Padl protein". The "second polypeptide having phenacrylate decarboxylase activity" is preferably a "Padl polypeptide". The "Padl

polypeptide" or "second polypeptide having phenacrylate decarboxylase activity" may comprise the amino acid sequence SEQ ID NO : 2 {Saccharomyces cerevisiae Padl protein) or a variant amino acid sequence, which is at least 17% identical, more preferably at least 37%, even more preferably at least 60% or at least 65% identical, to SEQ ID NO : 2 (for example, a sequence listed in Table 5, such as SEQ ID NOs : 2 and 27-37) . More preferably the "Padl polypeptide" or "second polypeptide having

phenacrylate decarboxylase activity" consists essentially of the amino acid sequence SEQ ID NO : 2 or a variant amino acid sequence, which is at least 17% identical, more preferably at least 37%, even more preferably at least 60% or at least 65% identical, to SEQ ID NO : 2. Most preferably the "Padl polypeptide" or "second polypeptide having phenacrylate decarboxylase activity" consists of the amino acid sequence SEQ ID NO : 2 or a variant amino acid sequence, which is at least 17% identical, more preferably at least 37%, even more preferably at least 60% or at least 65% identical, to SEQ ID NO : 2.

When an aliphatic mono-unsaturated carboxylic acid is contacted by both the Fdcl polypeptide and the Padl polypeptide, respectively by both the first polypeptide having ferulic acid decarboxylase activity and the second polypeptide having phenacrylate decarboxylase activity, it is possible to generate an alkene, which alkene comprises only one unsaturated carbon-to-carbon bond.

Advantageously, generation of a terminal alkene is possible from an aliphatic carboxylic acid with a carbon double bond between the a and β positions. That is, the invention advantageously allows for the generation of a mono-unsaturated terminal alkene, by contacting an aliphatic mono-unsaturated carboxylic acid, with a carbon double bond between the a and β positions, by both the Fdcl polypeptide and the Padl polypeptide, respectively by both the first polypeptide having ferulic acid

decarboxylase activity and the second polypeptide having phenacrylate decarboxylase activity.

By an "alkene" is herein understood an unsaturated aliphatic hydrocarbon compound comprising at least one carbon-to-carbon double bond. "Hydrocarbon compounds" are herein also referred to as "hydrocarbons". By a

"hydrocarbon compound" is herein understood a compound consisting of hydrogen and carbon. Examples of suitable alkenes that can be prepared (i.e. produced) using the method of the invention have in the range from equal to or more than 4 to equal to or less than 30 carbon atoms in linear or branched formation and comprise one or more double bonds. Particular examples of alkenes that can be prepared using the method of the invention include straight- or branched-chain alkenes having up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or up to 20 carbon atoms.

The one or more alkenes prepared using the method of the invention are mono-unsaturated alkenes. By a "mono- unsaturated alkene" is herein understood an alkene comprising only one unsaturated carbon-to-carbon bond, which one unsaturated carbon-to-carbon bond is a carbon- to-carbon double bond.

In a preferred embodiment the one or more alkenes prepared (i.e. produced) using the method of the

invention are terminal alkenes. Such a "terminal alkene" may herein also be referred to as for example an "1- alkene", an "alpha-alkene" , an " -alkene" or an " - olefin". By a "terminal alkene" is herein understood an alkene comprising a carbon-to-carbon double bond between a terminal carbon atom and its adjacent carbon atom in the alkene. That is, in a preferred embodiment the one or more alkenes prepared using the method of the invention are terminal mono-unsaturated alkenes.By an "aliphatic unsaturated carboxylic acid" is herein understood an aliphatic carboxylic acid comprising an unsaturated carbon-to-carbon bond. By an "aliphatic mono-unsaturated carboxylic acid" is herein understood an aliphatic unsaturated carboxylic acid having only one unsaturated carbon-to-carbon bond. Preferably the unsaturated carbon- to-carbon bond is a so-called carbon-to-carbon double bond. Hence, preferably the aliphatic unsaturated

carboxylic acid is an aliphatic unsaturated carboxylic acid comprising a single carbon-to-carbon double bond.

Thus, preferably the aliphatic mono-unsaturated carboxylic acid is an alkenoic acid. Alkenoic acids are herein also referred to as "enoic acids". By an alkenoic acid is herein understood an unsaturated aliphatic carboxylic acid comprising a carbon-to-carbon double bond. More preferably the aliphatic mono-unsaturated carboxylic acid is an alpha-alkenoic acid (also sometimes referred to as for example -alkenoic acid or 2-alkenoic acid) . By an alpha-alkenoic acid is herein understood an unsaturated aliphatic carboxylic acid that comprises a carbon-to-carbon double bond between the a and β

positions of the carbon chain (relative to the carboxyl group) . When the aliphatic mono-unsaturated carboxylic acid is such an alpha-alkenoic acid, the alkene prepared in the method according to the invention may

advantageously be a terminal alkene.

In an especially preferred embodiment, the aliphatic mono-unsaturated carboxylic acid is an aliphatic , β- mono-unsaturated carboxylic acid. By such an " aliphatic a, β-mono-unsaturated carboxylic acid" is herein

understood an aliphatic carboxylic acid, comprising ony one single unsaturated carbon-to-carbon bond, which single unsaturated carbon-to-carbon bond is located between the -carbon and the β-carbon of the carboxylic acid. Preferably the aliphatic a, β-mono-unsaturated carboxylic acid, is an aliphatic a, β-mono-unsaturated carboxylic acid with a single carbon-to-carbon double bond. Such an aliphatic a, β-mono-unsaturated carboxylic acid with a single carbon-to-carbon double bond may herein also be referred to as an " a, β-mono-unsaturated alkenoic acid". Hence, preferably the aliphatic mono- unsaturated carboxylic acid is an aliphatic a, β-mono- unsaturated alkenoic acid. As explained above, the alkene prepared when using such an aliphatic a, β-mono- unsaturated alkenoic acid may advantageously be a

terminal mono-unsaturated alkene.

More than one type of carboxylic acid may be

contacted with the Fdcl and Padl polypeptides in a single step, resulting in the production of a mixture of

alkenes, dependent on the carboxylic acids initially present, before the method is carried out. In a preferred embodiment a mixture of alkenes, comprising at least one or more terminal mono-unsaturated alkenes, is prepared by contacting a mixture of aliphatic unsaturated carboxylic acids comprising at least one or more a, β-mono- unsaturated alkenoic acids.

The method may subsequently comprise isolating the alkene and/or mixture of alkenes. The term "isolating the alkene" indicates that the alkene, or mixture of alkenes, is separated from other non-hydrocarbon components. This may indicate that, for example, at least about 50% by weight of a sample after separation is composed of the alkene (s), for example, at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99% or 100%. The alkene produced during the working of the invention can be separated (i.e., isolated) by any known technique. One exemplary process is a two-phase (bi-phasic) separation process, involving conducting the method for a period and/or under conditions sufficient to allow the alkene (s) to collect in an organic phase and separating the organic phase from an aqueous phase. This may be especially relevant when, for example, the method is conducted within a host cell such as a micro-organism, as described below. Bi-phasic separation uses the relative

immiscibility of hydrocarbons to facilitate separation. "Immiscible" refers to the relative inability of a compound to dissolve in water and is defined by the compound's partition coefficient, as will be well

understood by the skilled person.

In an embodiment of the invention, the Fdcl and/or Padl polypeptides (respectively the "first polypeptide having ferulic acid decarboxylase activity" and/or

"second polypeptide having phenacrylate decarboxylase activity") are expressed by a recombinant host cell, such as a recombinant micro-organism. Therefore, the method of the first aspect of the invention may take place within a host cell, i.e., the method may be at least partially an in vivo method. The host cell may be recombinant and may, for example, be a genetically modified microorganism.

Therefore, a micro-organism may be genetically modified, i.e., artificially altered from its natural state, to express at least one of the Fdcl and/or Padl polypeptides (respectively the "first polypeptide having ferulic acid decarboxylase activity" and/or "second polypeptide having phenacrylate decarboxylase activity") and, preferably, both of these. Other enzymes described herein may also be expressed by a micro-organism. Preferably, the enzymes are exogenous, i.e., not present in the cell prior to modification, having been introduced using

microbiological methods such as are described herein. Furthermore, in the method of the invention, the enzymes may each be expressed by a recombinant host cell, either within the same host cell or in separate host cells. The hydrocarbon may be secreted from the host cell in which it is formed.

The host cell may be genetically modified by any manner known to be suitable for this purpose by the person skilled in the art. This includes the introduction of the genes of interest, such as one or more genes encoding the Fdcl and/or Padl polypeptides (respectively the "first polypeptide having ferulic acid decarboxylase activity" and/or "second polypeptide having phenacrylate decarboxylase activity"), on a plasmid or cosmid or other expression vector which reproduces within the host cell. Alternatively, the plasmid or cosmid DNA or part of the plasmid or cosmid DNA or a linear DNA sequence may integrate into the host genome, for example by homologous recombination. To carry out genetic modification, DNA can be introduced or transformed into cells by natural uptake or mediated by processes such as electroporation . Genetic modification can involve expression of a gene under control of an introduced promoter. The introduced DNA may encode a protein which could act as an enzyme or could regulate the expression of further genes.

Such a host cell may comprise a nucleic acid sequence encoding a Fdcl and/or Padl polypeptide (respectively the "first polypeptide having ferulic acid decarboxylase activity" and/or "second polypeptide having phenacrylate decarboxylase activity") . For example, the cell may comprise at least one nucleic acid sequence comprising at least one of the polynucleotide sequences SEQ ID NOs:3-7 or 38-59 or a complement thereof, or a fragment of such a polynucleotide encoding a functional variant or fragment of either of the enzymes Fdcl and/or Padl, for example enzymes as described herein. The nucleic acid sequences encoding the enzymes may be exogenous, i.e., not

naturally occurring in the host cell. In a second aspect of the invention the recombinant host cell, such as a micro-organism, preferably comprises at least one

polypeptide which is a Fdcl enzyme, for example, having an amino acid sequence which is at least 21% identical to SEQ ID NO : 1 and in EC class 4.1.1.- (for example any of SEQ ID NOs:16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and/or 26); and/or preferably comprises at least one polypeptide which is a Padl enzyme, for example, having an amino acid sequence which is at least 17% identical to SEQ ID NO : 2 and in EC class 4.1.1.- (for example, any of SEQ ID

NOs:27, 28, 29, 30, 31, 32, 33, 34, 35, 36, and/or 37), or a functional variant or fragment of any of these sequences. For example, the recombinant host cell may comprise a polynucleotide encoding a polypeptide which is at least 21% identical to SEQ ID NO : 1 or encoding a polypeptide which is at least 17% identical to SEQ ID NO: 2, or a functional variant or fragment of either of these. The polynucleotide may, therefore, comprise at least one of the polynucleotide sequences SEQ ID NOs:3, 4, 5, 6 and/or 7 and/or 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 and/or 59. In one embodiment, the recombinant host cell

comprises a polypeptide comprising an amino acid sequence which is at least 21% identical to SEQ ID NO : 1 (for example, selected from the sequences specified in Table 4 herein, such as SEQ ID NOs : 1 and 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26) and a polypeptide comprising an amino acid sequence which is at least 17% identical to

SEQ ID NO : 2 (for example, selected from the sequences specified in Table 5 herein, such as SEQ ID NOs : 2 and 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 and 37) . The recombinant host cell may comprise a polypeptide

comprising both of SEQ ID NOs : 1 and 2 and/or an amino acid sequence at least 21% identical to SEQ ID NO : 1 (e.g., any of the sequences in Table 4, such as SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26) and an amino acid sequence at least 17% identical to SEQ ID NO : 2 (e.g., any of the sequences in Table 5, such as SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37). Such a polypeptide may be, for example, a fusion protein. In exemplary embodiments, the recombinant host cell may comprise one or more of the polynucleotide sequences SEQ ID NOs: 3, 4, 5, 6 and/or 7. In other embodiments, the recombinant host cell may comprise one or more of the polynucleotide sequences SEQ ID NOs: 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,

57, 58 and/or 59.

In a third aspect of the invention, a suitable polynucleotide may preferably be introduced into the cell by homologous recombination and/or may form part of an expression vector comprising at least one of the

polynucleotide sequences SEQ ID NOs: 3, 4, 5, 6 and/or 7 (or SEQ ID NOs:38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 and/or 59) or a complement thereof. Suitable vectors for construction of such an expression vector are well known in the art

(examples are mentioned above) and may be arranged to comprise the polynucleotide operably linked to one or more expression control sequences, so as to be useful to express the required enzymes in a host cell, for example a micro-organism as described above.

In some embodiments, the recombinant or genetically modified host cell, as mentioned throughout this

specification, may be any micro-organism or part of a micro-organism selected from the group consisting of fungi (such as members of the genus Saccharomyces) , protists, algae, bacteria (including cyanobacteria) and archaea. The bacterium may comprise a gram-positive bacterium or a gram-negative bacterium and/or may be selected from the genera Escherichia , Bacillus,

Lactobacillus, Rhodococcus, Pseudomonas or Streptomyces . The cyanobacterium may be selected from the group of Synechococcus elongatus, Synechocystis, Prochlorococcus marinus, Anabaena variabilis, Nostoc punctiforme,

Gloeobacter violaceus, Cyanothece sp . and Synechococcus sp . The selection of a suitable micro-organism (or other expression system) is within the routine capabilities of the skilled person. Particularly suitable micro-organisms include Escherichia coli and Saccharomyces cerevisiae, for example.

In a related embodiment of the invention, a Fdcl and/or Padl polypeptide or functional variant or fragment of either of these may be expressed in a non-micro- organism cell such as a cultured mammalian cell or a plant cell or an insect cell. Mammalian cells may include CHO cells, COS cells, VERO cells, BHK cells, HeLa cells, Cvl cells, MDCK cells, 293 cells, 3T3 cells, and/or PC12 cells .

The recombinant host cell or micro-organism may be used to express the enzymes mentioned above and a cell- free extract then obtained by standard methods, for use in the method according to the first aspect of the invention .

The present invention also encompasses variants of the polypeptides as defined herein. As used herein, a "variant" means a polypeptide in which the amino acid sequence differs from the base sequence from which it is derived in that one or more amino acids within the sequence are substituted for other amino acids. For example, a variant of SEQ ID NO : 1 has similar or

identical ferulic acid decarboxylase characteristics as SEQ ID NO:l, being classified in enzyme class EC 4.1.1.- by the Enzyme Nomenclature of NC-IUBMB as mentioned above. It may have an amino acid sequence at least about 21% identical to SEQ ID NO:l, for example, at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,

99% or about 100 % identical. Suitable variants may include the proteins listed in Table 4, with particular examples being any of SEQ ID NOs: 16-26. The variants are functional variants in that the variant sequence has similar or, preferably, identical functional enzyme activity characteristics to the enzyme having the non- variant amino acid sequence specified herein (and this is the meaning of the term "functional variant" as used throughout this specification) . The similar or identical ferulic acid decarboxylase characteristics as SEQ ID

NO:l, mentioned above, may be assessed, for example, by comparing the rate of conversion of cinnamic acid to styrene by a variant (in the presence of SEQ ID NO : 2 ) to the rate achieved by SEQ ID NO : 1 in the presence of SEQ ID NO : 2. For a functional variant, this rate may be the same or similar, for example at least about 60%, 70%, 80%, 90% or 95% the rate achieved by SEQ ID NO : 1 (S.

cerevisiae Fdcl protein) .

Likewise, a variant of SEQ ID NO : 2 has similar or identical phenacrylate decarboxylase characteristics as

SEQ ID NO : 2 and is classified in enzyme class EC 4.1.1.-. It may have an amino acid sequence at least about 17% identical to SEQ ID NO: 2, for example, at least about 20%, 25'o 30%, 35%, 37%, 40%, 45%, 50%, 55%, 60%, 65%,

70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,

97%, 98%, 99% or about 100 % identical . Suitable variants may include the proteins listed in Table 5, with

particular examples being any of SEQ ID NOs: 27-37. Again, the activity of a variant of SEQ ID NO : 2 may be

determined by measuring the rate of conversion of

cinnamic acid to styrene by a variant (in the presence of SEQ ID NO:l) to the rate achieved by SEQ ID NO : 2 in the presence of SEQ ID NO : 1.

Amino acid substitutions may be regarded as

"conservative" where an amino acid is replaced with a different amino acid with broadly similar properties. Non-conservative substitutions are where amino acids are replaced with amino acids of a different type.

By "conservative substitution" is meant the

substitution of an amino acid by another amino acid of the same class, in which the classes are defined as follows :

Class Amino acid examples

Nonpolar: A, V, L, I, P, M, F, W

Uncharged polar: G, S, T, C, Y, N, Q

Acidic: D, E

Basic: K, R, H.

As is well known to those skilled in the art,

altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that polypeptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the polypeptide's conformation. In the present invention, non-conservative

substitutions are possible provided that these do not interrupt the enzyme activities of the polypeptides, as defined elsewhere herein.

Broadly speaking, fewer non-conservative

substitutions than conservative substitutions will be possible without altering the biological activity of the polypeptides. Determination of the effect of any

substitution (and, indeed, of any amino acid deletion or insertion) is wholly within the routine capabilities of the skilled person, who can readily determine whether a variant polypeptide retains the enzyme activity according to the invention, as discussed above. For example, when determining whether a variant of the polypeptide falls within the scope of the invention (i.e., is a "functional variant or fragment" as defined above) , the skilled person will determine whether the variant or fragment retains the substrate converting enzyme activity which is at least about 60%, preferably at least about 70%, more preferably at least about 80%, yet more preferably about

90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% the activity of the non-variant

polypeptide. In some cases, the variant may have enzyme activity which is greater than 100% the activity of the non-variant polypeptide, i.e., the variant may have improved enzyme activity compared to the non-variant and increase the rate of conversion of the substrate relevant to the particular enzyme compared to the rate achieved by the non-variant under the same conditions (e.g.,

substrate concentration, temperature) . All such variants are within the scope of the invention.

Using the standard genetic code, further nucleic acid sequences encoding the polypeptides may readily be conceived and manufactured by the skilled person, in addition to those disclosed herein. The nucleic acid sequence may be DNA or RNA and, where it is a DNA

molecule, it may for example comprise a cDNA or genomic DNA. The nucleic acid may be contained within an

expression vector, as described elsewhere herein.

The invention, therefore, encompasses variant nucleic acid sequences encoding the polypeptides of the

invention. The term "variant" in relation to a nucleic acid sequence means any substitution of, variation of, modification of, replacement of, deletion of, or addition of one or more nucleic acid(s) from or to a

polynucleotide sequence, providing the resultant

polypeptide sequence encoded by the polynucleotide exhibits at least the same or similar enzymatic

properties as the polypeptide encoded by the basic sequence. The term therefore includes allelic variants and also includes a polynucleotide (a "probe sequence") which substantially hybridises to the polynucleotide sequence of the present invention. Such hybridisation may occur at or between low and high stringency conditions. In general terms, low stringency conditions can be defined as hybridisation in which the washing step takes place in a 0.330-0.825 M NaCl buffer solution at a temperature of about 40-48°C below the calculated or actual melting temperature (T _m ) of the probe sequence (for example, about ambient laboratory temperature to about 55°C) , while high stringency conditions involve a wash in a 0.0165-0.0330 M NaCl buffer solution at a temperature of about 5-10°C below the calculated or actual T _m of the probe sequence (for example, about

65°C) . The buffer solution may, for example, be SSC buffer (0.15M NaCl and 0.015M tri-sodium citrate), with the low stringency wash taking place in 3 x SSC buffer and the high stringency wash taking place in 0.1 x SSC buffer. Steps involved in hybridisation of nucleic acid sequences have been described for example in Sambrook et al . (10) .

Suitably, nucleic acid sequence variants have about 55% or more of the nucleotides in common with the nucleic acid sequence of the present invention, more suitably 60%, 65%, 70%, 80%, 85%, or even 90%, 95%, 98% or 99% or greater sequence identity.

Variant nucleic acids of the invention may be codon- optimised for expression in a particular host cell.

Sequence identity between amino acid sequences can be determined by comparing an alignment of the sequences using the Needleman-Wunsch Global Sequence Alignment Tool available from the National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA, for example via http://blast.ncbi.nlm.nih.gov/Blast.cgi, using default parameter settings (for protein alignment, Gap costs Existence:ll Extension : 1 ) . Sequence comparisons and percentage identities mentioned in this specification have been determined using this software. When comparing the level of sequence identity to, for example, SEQ ID NO:l, this suitably can be done relative to the whole length of SEQ ID NO : 1 (i.e., a global alignment method is used) , to avoid short regions of high identity overlap resulting in a high overall assessment of identity. For example, a short polypeptide fragment having, for

example, five amino acids might have a 100% identical sequence to a five amino acid region within the whole of

SEQ ID NO:l, but this does not provide a 100% amino acid identity according to the present definitions, unless the fragment forms part of a longer sequence which also has identical amino acids at other positions equivalent to positions in SEQ ID NO : 1. When an equivalent position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids at positions shared by the compared sequences. When

comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences, to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence

alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps.

Calculation of maximum percent identity involves the production of an optimal alignment, taking into

consideration gap penalties. As mentioned above, the percentage sequence identity may be determined using the

Needleman-Wunsch Global Sequence Alignment tool, using default parameter settings. The Needleman-Wunsch

algorithm was published in J. Mol. Biol. (1970) vol.

48 : 443-53.

Polypeptide and polynucleotide sequences for use in the methods, vectors and host cells according to the invention are shown in the Sequence Listing and in Tables 4 and 5.

According to a fourth aspect of the invention, there is provided a method of producing an alkane, comprising hydrogenation of an isolated alkene produced in a method according to the first aspect of the invention. In a preferred embodiment the present invention therefore provides a method for producing an alkane comprising or consisting of:

a) contacting one or more aliphatic unsaturated carboxylic acid(s), preferably comprising at least one a, β-mono-unsaturated alkenoic acid, with an Fdcl

polypeptide comprising an amino acid sequence with at least 21% sequence identity to SEQ ID NO : 1 and a Padl polypeptide comprising an amino acid sequence with at least 17% sequence identity to SEQ ID NO : 2 to produce one or more alkene(s), preferably comprising at least one terminal mono-unsaturated alkene;

b) isolating the one or more alkene (s), preferably comprising at least one terminal mono-unsaturated alkene to produce one or more isolated alkene (s), preferably comprising at least one isolated terminal mono- unsaturated alkene;

c) hydrogenating the one or more isolated alkene (s), preferably comprising at least one isolated terminal mono-unsaturated alkene, to produce one or more

alkane ( s ) .

Preferences for steps a) and b) are as described above for the first, second and third aspect of the invention .

The unsaturated bonds in the isolated alkene can be hydrogenated to produce the alkane. The hydrogenation may be carried out in any manner known by the person skilled in the art to be suitable for hydrogenation of

unsaturated compounds. The hydrogenation catalyst can be any type of hydrogenation catalyst known by the person skilled in the art to be suitable for this purpose. The hydrogenation catalyst may comprise one or more

hydrogenation metal (s), for example, supported on a catalyst support. The one or more hydrogenation metal (s) may be chosen from Group VIII and/or Group VIB of the

Periodic Table of Elements. The hydrogenation metal may be present in many forms; for example, it may be present as a mixture, alloy or organometallic compound. The one or more hydrogenation metal (s) may be chosen from the group consisting of Nickel (Ni), Molybdenum (Mo), Tungsten (W) , Cobalt (Co) and mixtures thereof. The catalyst support may comprise a refractory oxide or mixtures thereof, for example, alumina, amorphous silica-alumina, titania, silica, ceria, zirconia; or it may comprise an inert component such as carbon or silicon carbide.

The temperature for hydrogenation may range from, for example, 300°C to 450°C, for example, from 300°C to

350°C. The pressure may range from, for example, 50 bar absolute to 100 bar absolute, for example, 60 bar

absolute to 80 bar absolute.

A fifth aspect of the invention provides a method of producing a branched alkane, comprising

hydroisomerization of an isolated alkene produced in a method according to the first aspect of the invention, or an alkane produced in a method according to the fourth aspect of the invention. Hydroisomerization may be carried out in any manner known by the person skilled in the art to be suitable for hydroisomerization of alkanes.

The hydroisomerization catalyst can be any type of hydroisomerization catalyst known by the person skilled in the art to be suitable for this purpose. The one or more hydrogenation metal (s) may be chosen from Group VIII and/or Group VIB of the Periodic Table of Elements. The hydrogenation metal may be present in many forms, for example it may be present as a mixture, alloy or

organometallic compound. The one or more hydrogenation metal (s) may be chosen from the group consisting of

Nickel (Ni), Molybdenum (Mo), Tungsten (W) , Cobalt (Co) and mixtures thereof. The catalyst support may comprise a refractory oxide, a zeolite, or mixtures thereof.

Examples of catalyst supports include alumina, amorphous silica-alumina, titania, silica, ceria, zirconia; and zeolite Y, zeolite beta, ZSM-5, ZSM-12, ZSM-22, ZSM-23, ZSM-48, SAPO-11, SAPO-41, and ferrierite.

Hydroisomerization may be carried out at a

temperature in the range of, for example, from 280 to

450°C and a total pressure in the range of, for example, from 20 to 160 bar (absolute) .

In one embodiment, hydrogenation and

hydroisomerization are carried out simultaneously.

A sixth aspect of the invention provides a method for the production of a biofuel and/or a biochemical

comprising combining an alkene produced in a method according to the first aspect of the invention with one or more additional components to produce a biofuel and/or biochemical.

According to a seventh aspect of the invention, there is provided a method for the production of a biofuel and/or a biochemical comprising combining an alkane produced according to the fourth or fifth aspects of the invention with one or more additional components to produce a biofuel and/or biochemical.

In the sixth and seventh aspects, the alkane and/or alkene can be blended as a biofuel component and/or a biochemical component with one or more other components to produce a biofuel and/or a biochemical. By a biofuel or a biochemical, respectively, is herein understood a fuel or a chemical that is at least partly derived from a renewable energy source. Examples of one or more other components with which alkane and/or alkene may be blended include anti-oxidants , corrosion inhibitors, ashless detergents, dehazers, dyes, lubricity improvers and/or mineral fuel components, but also conventional petroleum derived gasoline, diesel and/or kerosene fractions.

A further aspect of the invention provides the use of a host cell according to the second aspect of the invention as a biofuel/biochemical hydrocarbon precursor source. A "biofuel/biochemical hydrocarbon precursor" is a hydrocarbon, suitably an alkene or mixture of alkenes, which may be used in the preparation of a biofuel and/or a biochemical, for example in a method according to the sixth or seventh aspects of the invention. The use of a host cell as the source of such a precursor indicates that the host cell according to the second aspect of the invention produces hydrocarbons suitable for use in the biofuel/biochemical production methods, the hydrocarbons being isolatable from the recombinant host cell as described elsewhere herein.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to" and do not exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In

particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Preferred features of each aspect of the invention may be as described in connection with any of the other aspects .

Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this

specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be shown, by way of example only, with reference to Figures 1-6 in which :

Figure 1 shows a series of SDS-PAGE gels of protein purification expression trials: for samples containing Fdcl, a band can be readily observed corresponding to the 58kDa his-tagged Fdcl; in contrast, Padl expression levels are very low;

Figure 2 shows heterologous expression of a Padl-TF fusion protein: the fusion protein is soluble and can be purified using affinity chromatography; the fusion protein binds flavin, indicative of Padl being folded;

Figure 3 shows GC-MS analysis of headspace samples from cells expressing Fdcl/Padl (third trace from top) and from pure protein extracts (top trace), confirming styrene production following incubation with cinnamic acid; the second trace from the top is a buffer only sample and the final trace is an empty pCOLAduet plasmid sample ;

Figure 4 shows the results of a high-throughput pH- based screening of enzymatic activity of purified

Fdcl /Padl;

Figure 5 shows direct monitoring of enzyme reactivity using UV-VIS spectroscopy: panel A shows the enzyme catalysed conversion of octotrienoic acid monitored over a 230-400nm range and B shows the linear decrease in absorbance at a single wavelength observed following enzyme addition; and

Figure 6 shows 1-pentene identification by GC-MS, indicating enzyme-catalysed production of 1-pentene from

2-hexenoic acid (A) compared to an enzyme-free control (B) .

EXAMPLES

Unless indicated otherwise any solutions and/or concentrations are in distilled water.

Heterologous expression

fdcl gene and padl gene were codon optimised for expression in E. coll and synthesised as detailed within this text. The codon optimized fdcl and padl genes were both cloned into a pCOLAduet-1 vector (commercially available from Merck Millipore) using polymerase chain reaction (PCR) , to ensure high expression of both subunits of the enzyme. The pCOLADuet-1 vector carries the COLA replicon, lacl gene and kanamycin resistance gene. Primers were used to complement the codon optimised gene sequences. The fdcl and padl genes were cloned using In-fusion Advantage PCR Cloning Kit (commercially obtained from Clontech, In-fusion is a trademark) . The primers used were as shown in Table 1 below.

Table 1 : Primers

Versions of yeast fdcl gene (genbank accession number NM_001180847.1 ) and yeast padl gene (genbank accession number NM_001180846 ) that were codon optimized for expression in E. coli (SEQ ID NOs : 3 & 4, respectively) were commercially obtained from GenScript and provided in pUC57 constructs.

For expression of each enzyme individually the fdcl gene and padl gene respectively were amplified from the pUC57 constructs by polymerase chain reaction (PCR) using a Pfu Ultra II fusion HS DNA polymerase (commercially obtainable from Agilent) . Template was removed by Dpnl (Diplococcus pneumonia nuclease-I) digestion

(commercially obtainable from New England Biolabs) and the gene was cloned into the Sacl /Ail I I sites of Multiple Cloning Site 1 (MCS1) in a pCOLAduet-1 vector

(commercially available from Merck Millipore) using an In-fusion Advantage PCR Cloning Kit as described below (commercially obtained from Clontech, In-fusion is a trademark) .

For expression of each enzyme individually the fdcl gene and padl gene respectively were individually cloned into the Sacl /Aflll sites of Multiple Cloning Site 1

(MCS1) in the pCOLAduet-1 vector (commercially available from Merck Millipore) . For co-expression of both genes, fdcl gene was cloned into the Sacl /Ail I I sites of MCS1 first and then padl gene was cloned into the Ndel/Xhol sites of Multiple Cloning Site 2 (MCS2) of the pCOLAduet-

1 vector.

To clone into the pCOLAduet-1 vector (also sometimes referred to as expression vector) , the pCOLAduet-1 vector was linearized by digestion with the two relevant

restriction enzymes (i.e. Sacl/AfIII respectively

Ndel/Xhol), and both the PCR product and the linearized pCOLAduet-1 vector were purified using a Qiaquick PCR purification kit (commercially obtainable from Qiagen) . The purified PCR product and purified linearized

pCOLAduet-1 vector were added in a 2:1 ratio (of PCR product to vector) to 2 ml of the 5XIn-fusion buffer and 1 ml In-fusion enzyme in a 10 ml reaction volume

(obtained from the In-fusion Advantage PCR Cloning Kit, Clontech) . The reaction was incubated at 37°C for 15 minutes, followed by 15 minutes at 50°C. Hereafter the reaction mixture was diluted to 50mL with a 10 mM Tris (tris (hydroxymethyl ) methylamino ) propane ) /CI pH 8.5 buffer and 2.5 ml transformed into 50 ml chemically competent E. coli NEB5a (commercially obtainable from New England Biolabs) . Plasmid from a number of transformants was purified using a Qiaprep miniprep kit (commercially obtainable from Qiagen) . The insertion of the gene into the pCOLAduet-1 vector was confirmed by restriction digest (as described above) and sequencing.

Fdcl and/or Padl polypeptides were expressed in E. coli B121(DE3) grown at 37°C in Luria-Bertani (LB) supplemented with 40 pg/ml (microgram/millilitre )

kanamycin. At mid-log phase cells were induced with 0.25 mM (millimolair , in distilled water) Isopropyl-β-Ο-Ι- thiogalactopyranoside (IPTG) and grown at 25°C overnight.

To purify Fdcl and/or Padl polypeptides, cell pellets were resuspended in a purification buffer (200 mM NaCl,

50 mM Sodium Phosphate (NaPi) in distilled water, pH 7.5) supplemented with about 1 mg/ml (milligrams/millilitre) hen egg white lysozyme (commercially obtainable from Sigma) , 10 g/ml (micrograms/millilitre)

Deoxyribonuclease I (commercially obtainable from Sigma) ,

10 g/ml Ribonuclease A (commercially obtainable from Sigma) and complete EDTA free protease inhibitor cocktail (commercially obtainable from Roche) . Cells were lysed by French press and about 48384 grams of lysate was

centrifuged to produce a supernatant and a pellet

residue. Imidazole was added to the clarified supernatant until a 10 mM (millimolair) imidazole final concentration was obtained. Subsequently the imidazole containing supernatant was applied to a 5 ml nickel-nitrilotriacetic (Ni-NTA) agarose (also referred to as Ni ²⁺ -agarose, commercially obtainable from Qiagen) . The column was washed with 3 column volumes of purification buffer supplemented with 10 mM imidazole and consequently another 3 column volumes of purification buffer with 50 mM imidazole, and the polypeptide eluted with 4x1 ml purification buffer containing 250 mM imidazole.

Fractions were analysed by SDS-PAGE (Sodium dodecyl- sulphate-polyacrylamide gel electrophoresis) to detect the presence of Fdcl/Padl polypeptides. Fractions

containing the desired polypeptides were pooled and buffer exchanged into 200 mM NaCl, 50 mM NaPi pH 7.5 using an Econo-Pac 10DG (i.e. comprising 10 ml Bio-GelP- 6DG gel) desalting column (BioRad) to remove imidazole.

Analysis of the Fdcl and/or Padl polypeptide yield under a variety of expression conditions/^", coli strains revealed that significant amounts of Fdcl polypeptide can easily be achieved (Figure 1) . In contrast, the Padl protein is not visible as a distinct band using SDS-PAGE, indicating low levels of expression. Polypeptide

purification of Fdcl polypeptide (from fdcl gene

containing cells grown with or without the padl gene present) is straightforward, giving rise to large amounts of pure Fdcl polypeptide. The presence of Padl

polypeptide can be detected, either through western blotting with anti-histidine (His ) -tag antibodies, or via direct detection of Padl polypeptides following an in-gel tryptic digest (not shown) . Padl levels were found to be <5% of the corresponding Fdcl levels.

The inventors have found that Padl levels can be increased through fusion of the protein to a trigger factor type chaperone. Cloning and expression of Padl trigger factor was achieved using amplification primers: PadlTFF (SEQ ID NO:14):

5 ' -TCGAAGGTAGGCATATGCTGCTGTTCCCGCGTCGCACC-3 '

PadlTFR (SEQ ID NO:15):

5 ' -ATTCGGATCCCTCGAGCTATTTGCTTTTGATACCTTCCCAGCGCGG-3 '

The Padl gene was amplified from the codon optimized gene in pUC57 vector by PCR using the Phusion DNA

polymerase (NEB) reaction. Template was removed by Dpnl digestion and the gene inserted into the Ndel/Xhol sites of linearized pCold TF vector (commercially obtainable from Takara Bio Inc.) using infusion HD cloning kit

(commercially obtainable from Clontech) . Takara 's pCold TF DNA Vector is a fusion cold shock expression vector that expresses Trigger Factor (TF) chaperone as a soluble fusion tag. Trigger Factor is an E.Coli originated prokaryotic ribosome-associated chaperone protein (48 kDa) . The pCold TF DNA Vector consists of the cspA promoter plus additional downstream sequences including a 5' untranslated region (5' UTR) , a translation enhancing element (TEE) , a His-Tag sequence, and a multicloning site (MCS) . A lac operator is inserted downstream of the cspA promoter to ensure strict regulation of expression. Additionally, recognition sites for HRV 3C Protease, Thrombin and Factor Xa are located between TF-Tag and the MCS and function to facilitate tag removal from the expressed fusion protein.

padl pCold TF in E. coli B121(DE3) was grown in LB broth supplemented with 50 pg/ml ampicillin at 37°C to OD ₆ o o of 0.6-0.8. Expression was induced by addition of 0.25mM ITPG and the cultures grown overnight at 15°C.

Cell pellets were resuspended in purification buffer (400 mM NaCl, 50 mM NaPi pH 7.5) supplemented with lysozyme, DNase, RNase and EDTA free complete protease inhibitor cocktail (Roche) . Cells were lysed by French press and the lysate centrifuged. The clarified

supernatant was supplemented with 10 mM imidazole and applied to a 5 ml Ni ²⁺ -agarose (Qiagen) . The column was washed with 3 column volumes of purification buffer supplemented with 10 mM imidazole and consequently another 3 column volumes of purification buffer with 50 mM imidazole, and the protein eluted with 4x1 ml

purification buffer supplemented with 250 mM imidazole. Fractions were analysed by SDS-PAGE to confirm the presence of trigger factor-Padl fusion protein (Figure 2A) .

Enzyme activity studies

In vivo enzyme activity assays were carried out using exogenous supplied substrates in the media (i.e. sorbic acid) . Significant enzyme activity leaded to a measurable pH increase (alkalinification through CO ₂ release and H ⁺ take-up) .

For in vivo pH assays, LB agar plates were

supplemented with 40 mg/ml kanamycin, 6 mM sorbic acid and 0.004% w/v phenol red in distilled water and buffered to pH 6.2. E. coli B121(DE3) cells containing either the co-expression plasmid or empty pCOLAduet-1 were

inoculated onto the plate and grown overnight at 37°C. Using phenol red as a pH indicator, the inventors could establish that substrate dependent alkalinification in vivo (i.e., yellow to red colour change observed) is clearly linked to the presence of the fdcl/padl genes. This allows rapid visual indication of enzyme activity levels.

For in vitro pH assays, the activity of purified His- Fdcl/Padl was assayed in 96 well plates containing 200 μΐ 50 mM KC1, 0.06-1 mM Hepes ( 4- ( 2-hydroxyethyl ) -1- piperazineethanesulfonic acid), 0.002% Phenol red and 0- 50 mM substrate pH 6.8. Rates of alkalinification were followed by the increase in Absorbance at 560nm

wavelength (A ₅ 6o) using a synergy High throughput

(HT)plate reader (commercially obtainable from BioTek) .

Following positive indication of in vitro enzyme activity, direct product analysis of volatile alkenes was performed using head space Gas Chromatography-Mass

Spectrometry (GC-MS) analysis. For in vivo analysis, fdcl/padl in pCOLAduet and empty pCOLAduet in E. coli B121(DE3) were grown in LB supplemented with 40 pg/ml kanamycin at 37 °C to an Οϋβοο of about 0.7 and induced with 0.25 mM IPTG. 11.5 ml induced culture and a solution of 3.5 ml 20 mM substrate in distilled water with a pH of about 7.5 were placed in 20 ml head space vials with crimp top seals. Cultures were incubated in shaking incubator at 25°C for about 12 - 48 hours.

For in vitro analysis, purified His-Fdcl /Padl polypeptide was buffer exchanged into a solution of 150 mM NaCl and 50 mM NaPi in water with pH 7.5. Subsequently solutions of 11.5 ml 150 mM NaCl and 50 mM NaPi with pH 7.5, of 3.5 ml 20 mM substrate with pH 7.5 and of 150 μΐ 30 mg/ml enzyme in water or buffer were added to 20 ml head space vials with crimp top seals. Reactions were incubated with shaking at 25 °C for about 12 - 48 hours.

Samples were analysed by GC-MS using a Thermofisher DSQ II. Vials were incubated for 3 minutes at 80°C with agitation. 0.5 ml headspace was injected with a 105°C syringe and a split ratio of 1/10. Analytes were

separated on a 30m 0.25mm 0.25μ ZB5-MS (commercially obtainable from Zebron) column run (alternatively a 30 m x 0.25 mm i.d. x 0.1 μ Varian VF-5HT columnmay be used) with a flow of 1.5 ml/min He. Oven temperature was held at 35 °C for 2 minutes before being ramped up to 250 °C at 10 °C /min. Products were identified by searching for the corresponding mass peak.

These GC-MS studies verified the presence of the anticipated decarboxylation products of sorbic acid, cinnamic acid and ferulic acid (i.e. 1 , 3-pentadiene, styrene and 4-vinylguaicol ) using in vivo enzymatic conversions (Figure 3) .

The inventors then turned to purification and in vitro assaying of enzyme activity. Using standard Ni- affinity chromatography purification (where needed combined with gel-filtration), Fdcl(/Padl) can easily be purified. Unfortunately, Padl levels are <5% of Fdcl level (Figure 1) . The (indirect) enzyme activity pH-test used for in vivo studies is easily extended to in vitro- studies on purified protein (Figure 4) . In this case, purified Fdcl (/Padl) was shown to lead to substrate dependent alkalinification of a range of substrates in absence of any externally added cofactors.

A direct observation of enzyme activity is possible by recording UV-VIS spectra of the solution. Aromatic substrates and aliphatic substrates with two or more double bonds conjugated to the acidic group absorb in the UV region. The activity of purified His-Fdcl /Padl was assayed against varying concentrations of these

substrates in 400 μΐ 50 mM KC1, 50 mM NaPi pH 6 in a 1 mm pathlength cuvette at 20°C. Substrate consumption was monitored using a Cary 50 Bio spectrophotometer (varian) either in scanning mode, or at a specific wavelength for kinetic experiments. The rate of cinnamic acid

consumption was monitored at 270 nm, ferulic acid at 312 nm, 2 , 4-pentadienoic acid at 242 nm, sorbic acid at 260 nm, 2 , 4 , 6-octatrienoic acid at 290 nm and 2 , 4-nonadienoic acid at 260 nm.

Most substrates displayed a spectrum distinct from the corresponding products. Enzyme dependent spectral changes can easily be monitored over time, and have confirmed the activity of the enzyme with a wide range of enolate-type substrates (Figure 5). This is, at present, the most rigorous and efficient method for enzyme

activity studies.

Finally, GC-MS product analysis (see above) has confirmed product formation using in vitro methods for standard substrates sorbic acid, cinnamic acid and ferulic acid (i.e. 1 , 3-pentadiene, styrene and 4- vinylguaiacol ; Figure 3) . The UV-vis method was used to assess Michaelis Menten parameters for a variety of substrates (sorbic acid, cinnamic acid, ferulic acid, octatrienoic acid) , most of which have a K _M in the sub-mM range (see Table 2 below) .

Mechanistic studies of Fdcl/Padl

The data herein points to Fdcl being responsible for catalysis, with the role of Padl remaining unclear.

Nevertheless, specific activity of Fdcl is significantly higher when Padl is present in the solution/cell, albeit at sub-stoichiometric levels.

Fdcl/Padl substrate range

To assess what limit the enzyme imposes on the extent of unsaturat ion, the inventors tested a wide range of saturated and unsaturated substrates, both in vivo and in vitro. Under no conditions was decarboxylation with saturated fatty acids observed and the enzyme clearly does not support alkane production. However, mono- unsaturated acids (mono unsaturated alkenoic acids) were found to give rise to corresponding CC-olefine (terminal alkene) compounds. The activity observed with the latter compounds could only be detected following prolonged incubation with high enzyme concentrations and using product detection, as follows.

GC-MS detection of terminal alkenes

As described above, for in vivo studies, Fdcl/Padl in pCOLAduet-1 and empty pCOLAduet-1 vector were transformed into E. coli B121(DE3) . Cultures were grown in LB

supplemented with 40 pg/ml kanamycin at 37°C to OD ₆₀ o ~0.7 and induced with 1 mM IPTG. 11.5 ml of induced culture was added to 3.5 ml 20 mM substrate pH -7.5 per tube in a sealed headspace vial.

For in vitro reactions, Fdcl/Padl partially purified by Ni ²⁺ affinity and buffer exchanged into 150 mM NaCl, 50 mM NaPi pH 7.5. 11.5 ml 300 mM NaCl, 100 mM NaPi pH

7.5, 3.5 ml 20 mM substrate pH 7.5 and 150 μΐ 30 mg/ml enzyme or enzyme free buffer were added to a sealed headspace vial.

All Fdcl/Padl reactions were incubated in a shaking incubator at 25°C for -36 hours.

Samples were analysed by GC-MS using a Thermofisher DSQ II. Vials were incubated for 3 minutes at 80°C with agitation. 0.5 ml headspace was injected with a 105°C syringe and a split ratio of 1/10. Analytes were

separated on a 30m 0.25mm 0.25μ ZB5-MS (or 30 m x 0.25 mm i.d. x 0.1 μ Varian VF-5HT ) column run with a flow of 1.5 ml/min He. Oven temperature was held at 35°C for 2 minutes before being raised to 250°C at 10°C/min.

Products were identified by searching for the

corresponding mass peak.

A split ratio of 1/100 was used with aromatic and di/tri-unsaturated aliphatic substrates as the quantities of product produced from these were far higher. Figure 6 shows that 1-pentene product has been detected with 2- hexenoic acid in vitro; it has also been detected in vivo. Activity with 2-heptenoic and 2-octenoic has so far only been detected in vivo.

In summary, high in vitro and in vivo activity was observed with 2 , 4-pentadienoic-, sorbic (2,4- Hexadienoic) -, 2, 4, 6-octatrienoic-, 2 , 4-nonadienoic-, cinnamic-, and ferulic acids. Lower level activity was observed with 2-hexenoic acid. In vivo activity has also been detected with 2-heptenoic or 2-octenoic acids. No activity was observed for saturated carboxylic acids. Direct identification of 1 , 3-butadiene, 1 , 3-pentadiene, 1, 3, 5-heptatriene, 1 , 3-octadiene, styrene, 4- vinylguaiacol ( 3-methoxy-4-vinylphenol ) , 1-pentene, 1- hexene and 1-heptene products have been observed by GC-

MS .

Table 2A: Aromatic substrates

Activity

Kinetics

Detected

Expected

Substrate uv

product GC

pH Km Kcat assay Vi (rtiM) is ^"1 )

MS

s

0.203 30.774

Ferulic 4-vinyl

✓ ✓ ✓ (±0.0 (±1.996 acid guaiacol

33) )

2,6-

Sinapinic dimethoxy-

✓

acid 4- vinylphenol

34.39

0.021

4

Cinnamic acid styrene ✓ ✓ ✓ (±0.003

(±1.2 ) 06) -Methyl 2-methyl

✓

cinnamic acid styrene

-Fluoro 2-fluoro

✓

cinnamic acid styrene

β-Methyl

1-methyl

cinnamic ✓

styrene

acid

1,2,3,4,5-

1,2,3,4,5-

Pentafluoro

Pentafluoro ✓

cinnamic

styrene

acid

4-Nitro 4-

✓

cinnamic acid nitrostyrene

trans-3- Indole 3-vinylindole ✓

acrylic acid Table 2B: Aliphatic substrates

* pH assays were non-positive as the method was not sensitive enough to measure the lower levels present.

Table 3: Identity of sequences included in

application

SEQ ID NO Description of sequence

1 Saccharomyces cerevisiae Fdcl protein

2 Saccharomyces cerevisiae Padl protein

3 Codon-opt imised cDNA encoding Fdcl (SEQ ID

NO: 1)

4 Codon-opt imised cDNA encoding Padl (SEQ ID

NO: 2)

5 Saccharomyces cerevisiae DNA encoding Fdcl and Padl

6 Saccharomyces cerevisiae cDNA encoding Fdcl

7 Saccharomyces cerevisiae DNA encoding Padl

8 PadlF Sacl amplification primer

9 PadlR Aflll amplification primer

10 PadlF Xhol amplification primer

11 PadlR Sacl amplification primer

12 FdclF Sacl amplification primer

13 FdclR Aflll amplification primer

14 PadlTFF amplification primer

15 PadlTFR amplification primer

16 GenBank accession no. BAG32392.1 S.

cerevisiae Fdcl homologue

17 GenBank accession no. EHN07793.1 S.

cerevisiae x S. kudriavzevii VIN7 Fdcl homologue

18 GenBank accession no. EDN60854.1 S.

cerevisiae Fdcl homologue

19 GenBank accession no. EGA83279.1 S.

cerevisiae Fdcl homologue

20 GenBank accession no. EHN02917.1 S.

cerevisiae x S. kudriavzevii VIN7 Fdcl homologue

21 GenBank accession no. GAA22747.1 S.

cerevisiae Kyokai No. 7 Fdcl homologue

22 GenBank accession no. XP_461563.1

Debaryomyces hansenii CBS 767 Fdcl homologue

23 GenBank accession no. XP_002499372.1

Zygosaccharomyces rouxii Fdcl homologue

24 GenBank accession no. XP_002421128.1 Candida dubliniensis CD36 Fdcl homologue

25 GenBank accession no. EEQ46648.1 Candida albicans WO-1 Fdcl homologue

26 GenBank accession no. XP_001390534.1

Aspergillus niger CBS 513.88 Fdcl homologue

27 GenBank accession no. AAA20484.1 S.

cerevisiae Padl homologue SEQ ID NO Description of sequence

28 GenBank accession no. EDN60853.1 S.

cerevisiae YJM789 Padl homologue

29 GenBank accession no. BAG32352.1 S.

cerevisiae Padl homologue

30 GenBank accession no. BAG32360.1 S.

cerevisiae Padl homologue

31 GenBank accession no. BAG32371.1 S.

cerevisiae Padl homologue

32 GenBank accession no. EHN02916.1 S.

cerevisiae x S. kudriavzevii VIN7 Padl homologue

33 GenBank accession no. EGA79325.1 S.

cerevisiae Vinl3 Padl homologue

34 GenBank accession no. XP_461564.2

Debaryomyces hansenii CBS 767 Padl homologue

35 GenBank accession no. XP_002499371.1

Zygosaccharomyces rouxii Padl homologue

36 GenBank accession no. XP_002421129.1 Candida dubliniensis CD36 Padl homologue

37 GenBank accession no. EEQ46647.1 Candida

albicans WO-1 Padl homologue

38 GenBank accession no. AB368818.1 S.

cerevisiae DNA encoding SEQ ID NO: 16

39 GenBank accession no. AGVY01000015.1 S.

cerevisiae x S. kudriavzevii VIN7 DNA

encoding SEQ ID NO: 17

40 GenBank accession no. AAFW02000145.1 S.

cerevisiae YJM789 DNA encoding SEQ ID NO: 18

41 GenBank accession no. ADVV01000025.1 S.

cerevisiae DNA encoding SEQ ID NO: 19

42 GenBank accession no. AGVY01000185.1 S.

cerevisiae x S. kudriavzevii VIN7 DNA

encoding SEQ ID NO: 20

43 GenBank gi 134956838 S. cerevisiae Kyokai No.

7 DNA encoding SEQ ID NO: 21

44 GenBank accession no. XM_461563.1

Debaryomyces hansenii CBS 767 mRNA encoding SEQ ID NO:22

45 GenBank accession no. XM_002499327.1

Zygosaccharomyces rouxii mRNA encoding SEQ ID NO: 23

46 GenBank accession no. XM_002421083.1 Candida dubliniensis CD36 mRNA encoding SEQ ID NO: 24

47 GenBank accession no. CM000312.1 Candida

albicans DNA encoding SEQ ID NO: 25

48 GenBank accession no. XM_001390497.1

Aspergillus niger CBS 513.88 mRNA encoding SEQ ID NO:26 SEQ ID NO Description of sequence

49 GenBank accession no. L09263.1 S. cerevisiae

DNA encoding SEQ ID NO : 27

50 GenBank accession no. AAFW02000145.1 S.

cerevisiae YJM789 DNA encoding SEQ ID NO: 28

51 GenBank accession no. AB368778.1 S.

cerevisiae DNA encoding SEQ ID NO: 29

52 GenBank accession no. AB368786.1 S.

cerevisiae DNA encoding SEQ ID NO: 30

53 GenBank accession no. AB368797.1 S.

cerevisiae DNA encoding SEQ ID NO: 31

54 GenBank accession no. AGVY01000185.1 S.

cerevisiae x S. kudriavzevii VIN7 DNA

encoding SEQ ID NO:32

55 GenBank accession no. ADXC01000024.1 S.

cerevisiae Vinl3 DNA encoding SEQ ID NO: 33

56 GenBank accession no. XM_461564.2

Debaryomyces hansenii CBS 767 mRNA encoding SEQ ID NO:34

57 GenBank accession no. XM_002499326.1

Zygosaccharomyces rouxii mRNA encoding SEQ ID NO: 35

58 GenBank accession no. XM_002421084.1 Candida dubliniensis CD36 mRNA encoding SEQ ID NO: 36

59 GenBank accession no. CM000312.1 Candida

albicans WO-1 DNA encoding SEQ ID NO:37

- -

Table 4 - Fdcl homologue polypeptides

GenBank Accession No. Overall % sequence identity with SEQ no. 1

NP_010828.1 100%

BAG32392.1 99%

BAG32381.1 99%

EHN07793.1 99%

EDN60854.1 99%

EGA83279.1 99%

EHN02917.1 89%

GAA22747.1 68%

XP_461563.1 60%

XP_002499372.1 59%

XP_002421128.1 59%

EEQ46648.1 59%

XP_718068.1 58%

XP_003195517.1 47%

XP_002564382.1 47%

XP_001261424.1 47%

EFX01410.1 47%

XP_001818650.1 47%

EGU80878.1 45%

EHK48809.1 46%

XP_001218695.1 46%

EFZ02049.1 46%

XP_003195387.1 45%

GAA90861.1 47%

CCD33957.1 46%

EDZ72732.1 46%

XP_001390534.1 46%

XP_001545682.1 46%

EHK20699.1 44%

XP_003039530.1 44%

EGU86506.1 43%

EHK46069.1 42%

EFX01795.1 43%

XP_384845.1 41%

EFY97969.1 41%

EDZ72739.1 37%

XP_003041887.1 40%

XP_003044170.1 40%

EGZ23463.1 41%

XP_386610.1 37%

AC072979.1 37%

EGU82090.1 42% - -

GenBank Accession No. Overall % sequence identity with SEQ no. 1

EHK21882.1 40%

EHK42408.1 38%

EFY85845.1 39%

CAK45837.1 40%

EHA28102.1 41%

XP_001395986.1 36%

GAA87759.1 35%

XP_002374905.1 38%

XP_001819606.1 38%

EHA25577.1 35%

ZP_07797950.1 37%

AE072739.1 37%

YP_001345734.1 38%

YP_788405.1 37%

ZP_06876238.1 37%

NP_248945.1 37%

YP_002437859.1 37%

EGM13823.1 37%

ZP_01363161.1 37%

ZP_04936675.1 37%

ZP_09387347.1 36%

XP_001393328.2 39%

GAA19696.1 37%

XP_572217.1 35%

YP_003114400.1 37%

ZP_06711456.1 35%

BAG32388.1 30%

XP_001217436.1 37%

YP_004902842.1 35%

ZP_09000373.1 35%

YP_003111840.1 36%

ZP_03780130.1 35%

ZP_09425197.1 34%

BAG32384.1 28%

XP_664768.1 27%

AAO72071.1 29%

YP_004828050.1 32%

YP_001535961.1 31%

ZP_09054416.1 28%

ABI94382.1 32%

ZP_05967166.1 32%

ZP_07293769.1 31%

YP_001007209.1 30%

CBY26238.1 30% - -

Table 5 - Padl homologue polypeptides

GenBank Accession No. Overall% sequence identity with SEQ no. 2

NP_010827.1 100%

AAA20484.1 99%

EDN60853.1 99%

BAG32352.1 98%

BAG32360.1 98%

BAG32371.1 98%

EHN02916.1 80%

EGA79325.1 85%

XP_461564.2 56%

XP_002499371.1 58%

XP_002421129.1 56%

XP_718069.1 56%

EEQ46647.1 56%

EFY97970.1 50%

XP_002374906.1 51%

XP_001819605.2 49%

XP_386611.1 48%

EHK46071.1 48%

EFZ02050.1 48%

XP_002380060.1 48%

XP_001818651.1 48%

EHK20701.1 47%

XP_002564375.1 47%

XP_001261423.1 45% - -

GenBank Accession No. Overall% sequence identity with SEQ no. 2

GAA90863.1 47%

XP_001390532.1 47%

XP_001545681.1 44%

EGU80879.1 45%

XP_001218694.1 17%

XP_001393326.1 46%

YP_004828051.1 42%

EHK21881.1 47%

XP_003195392.1 44%

EFX01028.1 38%

ZP_07293770.1 42%

EGU86507.1 41%

EFY85844.1 42%

NP_388245.1 44%

ZP_05227335.1 39%

YP_077665.1 42%

YP_004206319.1 43%

AEP89434.1 43%

EHA29008.1 43%

BAI83837.1 43%

YP_003944634.1 41%

YP_003868777.1 41%

AET58676.1 41%

YP_004517954.1 41%

YP_001211446.1 42%

ZP_08624297.1 42%

YP_004875917.1 43%

ZP_06875184.1 42%

CAD19476.1 40%

YP_174133.1 40%

ABV91288.1 40%

AEB22516.1 40%

ABI94381.1 39%

YP_003192438.1 42%

YP_003918919.1 41%

EHM06418.1 40%

CCF03850.1 41%

YP_001703499.1 40%

YP_003975944.1 41%

YP_004497687.1 40%

ZP_09000374.1 42%

ZP_08499357.1 40%

YP_001420010.1 41%

ZP_08113998.1 40%

XP_003306895.1 37% - -

GenBank Accession No. Overall% sequence identity with SEQ no. 2

ZP_06355238.1 40%

AEG59431.1 41%

EGA75335.1 42%

ZP_08273725.1 39%

YP_003211671.1 40%

YP_002330491.1 40%

YP_428976.1 40%

XP_001395985.1 40%

YP_217838.1 40%

NP_461842.1 40%

YP_002227663.1 40%

YP_003614566.1 40%

CBK86407.1 40%

YP_001354955.1 41%

ZP_09039132.1 40%

YP_001464062.1 39%

ZP_05970041.1 40%

EGA83277.1 41%

ZP_02667593.1 39%

YP_002413752.1 40%

NP_457310.1 39%

ZP_06541550.2 40%

EHP66767.1 40%

YP_002216887.1 39%

YP_004953406.1 40%

EFW76961.1 40%

ZP_02884445.1 41%

YP_001101250.1 40%

YP_002638497.1 39%

YP_145845.1 39%