FIELD OF THE INVENTION

The invention relates generally to the uses of biocatalytic processes to obtain branched alcohols. The invention also relates to fuel compositions comprising said alcohols.

BACKGROUND OF THE INVENTION

Demand for bio fuels as a substitute for petroleum is expected to increase because of economic and environmental concerns. The most common bio fuel, ethanol, is not ideal because it has a lower energy density than gasoline and must be mixed with gasoline at a limited concentration range in order to serve as a transportation fuel. Ethanol is hygroscopic and corrosive, which possesses a problem for storage and distribution systems. Thus, ethanol cannot completely replace existing-petroleum-based fuels. The use of higher alcohols can compensate for some of these issues.

It has been shown than n-butanol is a better bio-fuel than ethanol due to lower vapour pressure, high energy content and lesser susceptibility to phase separation in the presence of water. Furthermore, n-butanol can be blended at higher concentration than ethanol for use in standard vehicle engines and it does not require automobile manufacturers to compromise on performance to meet environmental regulations, n- Butanol can be obtained from an acetone/n-butanol/ethanol (ABE) mixture produced by the fermentation of carbohydrates by bacteria of the genus Clostridia. However, using fermentation processes, side products are always formed due to the presence of many different enzymes within the organisms; thus, n-butanol cannot be produced as a single fermentation product in Clostridium. In addition, conditions of the production process (temperature, pH, salts, etc.) are limited by the viability of the cells. Moreover, product purification is often the most costly process step in a fermentative process production. Thus, researchers focus on the identification of gene targets, mutations and pathways in several microorganisms for increased n-butanol production. Furthermore, the transference of the n-butanol pathway from Clostridium into other more tractable microorganisms, such as Escherichia coli or Saccharomyces cerevisiae is an area of active research. Enzymes often exhibit a great advantage compared to chemical catalysts because they can accept complex molecules as substrates, catalysing reactions with chiral (enantio-) and positional (regio-) selectivity. The best characterized mechanism for branched chain alcohol production is through the Ehrlich pathway which converts branched chain amino acids into alcohols. The carbon number (up to 5) of the alcohols derived from this type of pathway is limited by the carbon number in the branched chain amino acid pathways. Compared to linear alcohols, branched chain alcohols have high octane numbers. However, these alcohols cannot be synthetized economically using native microorganisms. This is a daunting task because a metabolic pathway usually involves the collective function of multiple enzymes which have to be engineered by rational design or directed evolution to perform non-natural activities.

Given the above considerations, there is a necessity for new synthetic pathways that go beyond known metabolic networks in order to produce branched chain alcohols for use as fuel components.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a process for obtaining a compound formula (I)

which comprises contacting a compound of formula (II)

with a polypeptide having aldehyde reductase activity,

under conditions suitable for the conversion of the compound of formula (II) into a compound of formula (I), wherein Ri is selected from the group consisting of linear or branched Ci-C ₆ alkyl, linear or branched C ₂ -C ₆ alkenyl and linear or branched C ₂ -C ₆ alkynyl, aryl, aryl-alkyl and benzyl and wherein R ₂ is selected from the group consisting of linear or branched C ₂ -C ₆ alkyl, linear or branched C ₂ -C ₆ alkenyl and linear, branched C2-C ₆ alkynyl, aryl, aryl-alkyl and benzyl; and wherein the sequence of said polypeptide has an identity of at least 45% to the sequences of SEQ ID NO: 1 or SEQ ID NO: 2.

In a second aspect, the invention relates to an in vivo process for the production of a compound of formula (I)

which comprises culturing a genetically modified microorganism expressing a polypeptide having aldehyde reductase activity and containing a compound of formula (II) under conditions suitable for the conversion of the compound of formula (II) into a compound of formula (I)

wherein Ri is selected from the group consisting of linear or branched Ci-C ₆ alkyl, linear or branched C ₂ -C ₆ alkenyl and linear or branched C ₂ -C ₆ alkynyl, aryl, aryl-alkyl and benzyl and wherein R ₂ is selected from the group consisting of linear or branched C ₂ -C ₆ alkyl, linear or branched C ₂ -C ₆ alkenyl and linear, branched C ₂ -C ₆ alkynyl, aryl, aryl-alkyl and benzyl; wherein the sequence of said polypeptide has an identity of at least 45% to the sequences of SEQ ID NO: 1 or SEQ ID NO: 2. In a third aspect, the invention relates to a fuel composition comprising the compound of formula (I). BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Total Ion Chromatogram (TIC) of 2-ethyl-butyraldehyde reaction with alcohol dehydrogenase from Z. lactis. Identity of compounds eluting at Peak 1 (2-ethyl- butyraldehyde) and Peak 2 (2-ethyl-l-butanol) was confirmed by the corresponding mass spectra.

Figure 2. (A) Average mass spectrum of peak 1 (7.88 to 7.98 min) from the reaction of 2-ethyl-butyraldehyde with alcohol dehydrogenase from L. lactis. (B) Mass spectrum of 2-ethyl-butyraldehyde in the NIST standard reference database.

Figure 3: (A) Average mass spectrum of peak 2 (10.05 to 10.10 min) from the reaction of 2-ethyl-butyraldehyde with alcohol dehydrogenase from Z. lactis. (B) Mass spectrum of 2-ethyl- 1 -butanol in the NIST standard reference database.

Figure 4: Total Ion Chromatogram of reaction with alcohol dehydrogenase from B. stearothermophilus . Identity of compounds eluting at Peak 1 (2-ethyl-butyraldehyde) and Peak 2 (2-ethyl-l-butanol) was confirmed by the corresponding mass spectra.

Figure 5. (A) Average mass spectrum of peak 1 (7.89 to 7.97 min) from the reaction of 2-ethyl-butyraldehyde with alcohol dehydrogenase from B. stearothermophilus. (B) Mass spectrum of 2-ethyl-butyraldehyde in the NIST standard reference database.

Figure 6. (A) Average mass spectrum of peak 2 (10.05 to 10.09 min) from the reaction of 2-ethyl-butyraldehyde with alcohol dehydrogenase from B. stearothermophilus. (B) Mass spectrum of 2-ethyl-l-butanol in the NIST standard reference database.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have designed a novel and efficient process for obtaining a long chain branched alcohols by using an enzyme which has a novel biological activity that uses branched long chain aldehydes. The process of the invention provides a non-naturally occurring pathway, which allows the production of 2-ethyl-l-butanol. This compound is useful as fuel additive and it is advantageously useful in fuel compositions due to its high proportion of carbons, low content in oxygen, and high energy density.

First Process of the invention

In a first aspect, the invention relates with a process, hereinafter

process of the invention", for obtaining a compound of formula (I)

which comprises contacting a compound of formula (II)

with a polypeptide having aldehyde reductase activity

under conditions suitable for the conversion of the compound of formula (II) into a compound of formula (I), wherein Ri is selected from the group consisting of linear or branched Ci-C ₆ alkyl, linear or branched C ₂ -C ₆ alkenyl and linear or branched C ₂ -C ₆ alkynyl, aryl, aryl-alkyl and benzyl and wherein R ₂ is selected from the group consisting of linear or branched C ₂ -C ₆ alkyl, linear or branched C ₂ -C ₆ alkenyl and linear, branched C ₂ -C ₆ alkynyl, aryl, aryl-alkyl and benzyl; and wherein the sequence of said polypeptide has an identity of at least 45% to the sequences of SEQ ID NO: 1 or SEQ ID NO: 2.

As used herein, the term "Ci_-C ₆ alkyl group" refers to a saturated straight chain or branched non-cyclic hydrocarbon having from 1 to 6 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n- hexyl; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-bvXy\, isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4- methylpentyl, 2,3-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl and the like. Alkyl groups included in compounds of this invention may be optionally substituted with one or more substituents.

As used herein, the term "C2-C6 alkyl group" refers to a saturated straight chain or branched non-cyclic hydrocarbon having from 2 to 6 carbon atoms. Representative saturated straight chain and branched alkyls have been cited above and include, without limitation, . ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl, 3- methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, 2,2- dimethylbutyl, 3,3-dimethylbutyl. In a preferred embodiment, R ₂ is an alkyl group.

As used herein, the term "C?-C ₆ alkenyl group", means a straight chain or branched non-cyclic hydrocarbon having from 2 to 6 carbon atoms and having at least one carbon-carbon double bond. Representative straight chain and branched (C2-C6) alkenyls include vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-l-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, and the like. Alkenyl groups may be optionally substituted with one or more substituents.

As used herein, the term "C?-C ₆ alkynyl group", means a linear chain or branched non-cyclic hydrocarbon having from 2 to 6 carbon atoms and having at least one carbon-carbon triple bond. Representative straight chain and branched (C2-C6) alkynyls include ethyne, propyne, butyne, pentyne, hexyne and the like.

"Aryl" refers to an aromatic hydrocarbon radical such as phenyl, naphthyl or anthracyl. The aryl radical may be optionally substituted by one or more substituents such as hydroxy, mercapto, halo, alkyl, phenyl, alkoxy, haloalkyl, nitro, cyano, carboxy, amino, dialkylamino, aminoalkyl, acyl and alkoxycarbonyl.

"Aryl- alkyl" as defined herein, refers to an alkyl radical, as defined above attached to an aryl radical, as defined above, such as phenyl.

"Benzyl", as defined herein, refers to the group -CH ₂ -phenyl.

The term "polypeptide having aldehyde reductase activity", as used herein, refers to a polypeptide which catalyzes the NAD(P)H-dependent conversion of branched aldehydes or ketones to their respective alcohols, wherein the term NAD(P)H refers to either NADH or NADPH as the cofactor. The polypeptide having aldehyde reductase activity for use in the present invention is a biological catalyst; thus the polypeptide having aldehyde reductase activity for use in the invention is an enzyme.

The mechanism of the catalytic reaction carried out by the polypeptide having aldehyde reductase activity begins with the binding of the NAD(P)H molecule to the polypeptide having aldehyde reductase activity. The aldehyde substrate, such as the compound of formula (II), binds to the complex formed between NAD(P)H and the polypeptide having aldehyde reductase activity. A hydride transfer from the nicotinamide ring to the carbonyl of the substrate and the addition of a proton from an active residue of said polypeptide forms the hydroxyl group of the alcohol product, such as the compound of formula (I). Release of the alcohol product is followed by an enzyme conformational change, involving in part a nucleotide-clamping loop with release of the oxidized NAD(P) ⁺ coenzyme and replacement with NAD(P)H to start a new catalytic cycle, where NAD(P) can mean NAD or NADP .

The aldehyde reductase activity of the polypeptide having aldehyde reductase activity can be determined by any method known in the art suitable for the determination of the activity of NAD(P)H-dependent aldehyde reductase activity. In a preferred embodiment, the enzymatic activity of the polypeptide having aldehyde reductase activity is determined by contacting the polypeptide the activity of which is to be determined with butyraldehyde or 2-ethyl-butyraldehyde as shown in example 1 of the present application. The aldehyde reductase activity of the polypeptide is then determined by monitoring the conversion of NAD(P)H into NAD(P) ⁺ , e.g. by determining the decrease in absorbance at 340 nm in a spectrophotometer, or by determining the appearance of the reaction product (e.g. 1-butanol or 2-ethyl-l-butanol when butyraldehyde or 2-ethyl-butyraldehyde are used as substrates, respectively) by using any method known in the art such as gas chromatography coupled to mass spectrometry.

Polypeptide having aldehyde reductase activity for use in the present invention include polypeptides having specific activities in the range of at least 0.001, at least 0.01, at least 0.1, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95 and/or at least 100 mU/mg when using 1-butyraldehyde as substrate or at least 0.001, at least 0.01, at least 0.1, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95 or at least 100 mU/mg when using 2-ethyl-butyraldehyde as substrate.

According to the present invention, the sequence of polypeptide having aldehyde reductase activity has an identity of at least 45% to the sequences of SEQ ID NO: 1 or SEQ ID NO: 2.

SEQ ID NO: l corresponds to NAD(+)-Dependent Alcohol Dehydrogenase from

Lactococcus lactis and which is encoded b the polynucleotide of SEQ ID NO.3. SEQ ID NO:2 corresponds to the aldehyde dehydrogenase from Bacillus stearothermophilus, and which is encoded b the polynucleotide of SEQ ID NO: 4.

In a preferred embodiment, the sequence of polypeptide having aldehyde reductase activity has an identity of at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more to any of the sequences of SEQ ID NO: 1. In a preferred embodiment, the above identity value is determined over the complete length of the polypeptides being compared.

In a preferred embodiment, the sequence of polypeptide having aldehyde reductase activity has an identity of at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more to any of the sequences of SEQ ID NO: 2. In a preferred embodiment, the above identity value is determined over the complete length of the polypeptides being compared

"Percentage of sequence identity" and "percentage homology" are used interchangeably herein to refer to comparisons among polynucleotides or polypeptides, which are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. MoT Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al, eds., Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (1995 Supplement)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, 1990, J. Mol. Biol. 215: 403-410 and Altschul et al, 1977, Nucleic Acids Res. 3389-3402, respectively as well as the BLASTP algorithm (BLAST Manual, Altschul, S. et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J., 1990, Mol. Biol. 215:403-410). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score, T, when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89: 10915). Exemplary determination of sequence alignment and percent sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided, or the ClustalW multiple alignment program (available from the European Bioinformatics Institute, Cambridge, UK), using, in some embodiments, the parameters below.

For purposes of the present disclosure, in some embodiments, the degree of percent amino acid sequence identity can be obtained by ClustalW analysis (version W 1.8), counting the number of identical matches in the alignment and dividing such number of identical matches by the length of the reference sequence, and using the following default ClustalW parameters to achieve slow/accurate pairwise optimal alignments - Gap Open Penalty: 10; Gap Extension Penalty: 0.10; Protein weight matrix: Gonnet series; DNA weight matrix: IUB; Toggle Slow/Fast pairwise alignments = SLOW or FULL Alignment. "Comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e. gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

The process to obtain the compound of formula (I) of the present invention involves starting from a compound of formula (II) and then contacting said compound of formula (II) with the polypeptide having aldehyde reductase activity under conditions suitable for the conversion of the compound of formula (II) into a compound of formula (I), that is to say under conditions which allow the reduction of the carbonyl group of the compound of formula (II) into an alcohol group. The process of the invention is shown in Scheme 1.

The expression "contacting the compound of formula (II) with the polypeptide having aldehyde reductase activity" as used herein, refers to incubation of said polypeptide and said compound of formula (II) in conditions suitable for the interaction between said compound and said polypeptide; thereby leading to the formation of the compound of formula (I).

The conditions required for the polypeptide having aldehyde reductase activity to reduce the aldehyde group of compound (II) to an alcohol group producing the compound of formula (I) include an appropriate ratio between the polypeptide and the substrate in terms of concentration and appropriate conditions of salt, pH, temperature, time of reaction, appropriate concentrations of a reducing agent as the cofactor NADH, appropriate concentrations of the substrate and appropriate concentrations of the polypeptide having aldehyde reductase activity. In a preferred embodiment, the first process of the invention is carried in vitro, i.e. by contacting the substrate and the enzyme having aldehyde reductase activity in a reactor, preferably in the absence of any cell.

Suitable salt conditions for carrying the process according to the invention include, without limitation, any buffer commonly known such as phosphate buffer, a bicarbonate buffer, a carbonate buffer, Tris buffer, a borate buffer, a succinate buffer, a histidine buffer or a citrate buffer at concentrations of at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mN, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 200 mM, at least 300 mM or more. In a preferred embodiment, the process according to the present invention is carried out in 50 mM phosphate buffer.

Suitable pH values for carrying the process according to the invention include, without limitation, values of 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5. In a preferred embodiment, the process is carried out at a pH of 7.5.

Suitable temperature conditions for carrying the process according to the invention include, without limitation, about 20°C, about 25°C, about 30°C, about 35°C, about 37°C. In a preferred embodiment, the process is carried out at a temperature between 25°C and 37°C.

Suitable reaction times for carrying the process according to the invention can be determined by carrying out small scale reactions and determining the progress of the reaction by measuring the decrease in absorbance at 340 nm. Suitable times include, without limitation, at least 1 ms, at least 1 s, at least 15 s, at least 30 s, at least 1 min, at least 2 min, at least 3 min, at least 4 min, at least 5 min, at least 6 min, at least 7 min, at least 8 min, at least 9 min, at least 10 min, at least 15 min, at least 20 min, at least 25 min, at least 30 min, at least 35 min, at least 40 min, at least 45 min, at least 50 min, at least 55 min, at least 1 h, at least 2h, at least 3 h, at least 4h, at least 5h, at least 6h, at least 7h, at least 8h, at least 9h, at least 10 h or more.

Suitable concentrations of the NADPH or NADH cofactor for carrying the process according to the invention include, without limitation, about 10 nM, about 50 nM, about 0.1 mM, about 0.5 mM, about 1 mM, about 5 mM, about 10 mM or more. The term "cofactor" as used herein refers to a non-protein chemical compound that is required for the protein's biological activity. In one embodiment, the molecule NADPH acts as cofactor in the process for obtaining a compound of formula (I) defined above. NADPH acts as a reducing agent that donates electrons which are necessary for the formation of the hydroxyl group (-OH) from the carbonyl group (=0). Consequently, NADPH is transformed to its oxidized form, NADP ⁺ . In another embodiment, the molecule NADH acts as cofactor in the process for obtaining compound of formula (I) defined above. NADH acts as a reducing agent that donates electrons which are necessary for the formation of the hydroxyl group (-OH) from the carbonyl group (=0). Consequently, NADH is transformed to its oxidized form, NAD ⁺ .

Suitable substrate concentrations for carrying the process according to the invention include, without limitation, about 1 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 100 nM, about 500 nM, about 1 μΜ, about 10 μΜ, about 20 μΜ, about 30 μΜ, about 40 μΜ, about 50 μΜ, about 100 μΜ, about 500 μΜ, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 100 mM or more. In a preferred embodiment, the substrate concentration is between 0.1 and 0.5 mM.

Suitable concentrations of the polypeptide having aldehyde reductase activity for carrying the process according to the invention include, without limitation, about 1 μg/mL, about 2 μg/mL, about 3 μg/mL, about 5 μg/mL, about 8 μg/mL, about 10 μg/mL, about 20 μg/mL, about 30 μg/mL, about 50 μg/mL, about 75 μg/mL, about 100 μg/mL, about 200 μg/mL, about 300 μg/mL, about 500 μg/mL, about 750 μg/mL, about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 5 mg/mL, about 7.5 mg/mL, about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 50 mg/mL, about 75 mg/mL, about 100 mg/mL. In a preferred embodiment, the polypeptide concentration is between 0.05 and 1 mg/mL.

Suitable conditions for producing the compound of formula (I) are shown in

Examples 1 and 2 of the present patent application. Illustratively, suitable conditions for obtaining the compound of formula (I) according to the process of the invention, comprises using 0.1-0.5 mM of compound (II) as substrate, such as 2-ethyl- butaraldehyde at 25°C-37°C, at pH 7.5, in the presence of 1 mM NADPH as redox cofactor of the reaction and 0.2 mg/mL of the polypeptide having aldehyde reductase activity.

The process of the present invention can be carried out by using a polypeptide having aldehyde reductase activity in pure form in order to obtain high specific activity (which gives a measurement of the enzyme purity in the mixture as the activity of an enzyme per milligram of total protein). The polypeptide having aldehyde reductase activity may be produced in any prokaryotic or any eukaryotic cell, or in an in vitro cell free protein expression system by using methods generally known in the art, based on techniques of recombinant protein production. Practically any cell type can be transformed with a polynucleotide encoding the polypeptide having aldehyde reductase activity (for example, cells can be transformed with a polynucleotide comprising the sequence SEQ ID NO: 3 or SEQ ID NO: 4) or any cell type can be transformed, transfected or infected by a recombinant vector containing the polynucleotide encoding the polypeptide having aldehyde reductase activity; for example animal cells, such as mammalian cells, bird cells, insect cells, etc.; plant cells; yeasts; bacteria; etc. Suitable vector systems are well-known in the art as evidenced by the vast amount of literature and materials available to the skilled person.

In general, of course, prokaryotes are preferred for the initial cloning of the nucleic acid which encodes polynucleotides which encode the polypeptide having aldehyde reductase activity (for example, polynucleotides comprising the sequence SEQ ID NO: 3 or SEQ ID NO: 4). Prokaryotes can be also utilized for expression, since efficient purification and protein refolding strategies are available. Strains from E. coli or bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may be used.

In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. The pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microorganism for expression.

Those promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase) and lactose promoter systems and a tryptophan (trp) promoter system. While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors. Certain genes from prokaryotes may be expressed efficiently in E. coli from their own promoter sequences, precluding the need for addition of another promoter by artificial means.

In addition to prokaryotes, eukaryotic microbes, such as yeast may also be used. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. Suitable promoter sequences in yeast vectors include the promoters for 3- phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde- 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mR A and termination. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase-2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.

In a preferred embodiment, the polypeptide having aldehyde reductase activity additionally comprises a peptide to facilitate its purification. The term "peptide to facilitate its purification" as used in the present invention, refers to a peptide useful for isolating or purifying the polypeptide having aldehyde reductase activity which binds to the N- or C-terminus end. Said peptide is thus capable of binding one or more ligands of an affinity matrix, such as an affinity chromatography. One example of said peptide is the histidine tag (His-tag) which can contain six histidine residues (His6 or H6), which can bind to a nickel or cobalt column with high affinity. Other examples of said peptides include, but are not limited to, Arg-tag, FLAG-tag, Strep-tag, an epitope capable of being recognized by an antibody, such as c-myc-tag (recognized by an anti- c-myc antibody), SBP-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, chitin-binding domain, glutathione S-transferase-tag, maltose-binding protein, NusA, TrxA, DsbA, Avi-tag, etc. After an appropriate expression period, cells are lysed and the tagged bait together with bound proteins. Then, the polypeptide is isolated using a specific chemical or biological ligand linked to a solid support. Eluted proteins can be separated by any suitable method known in the art such as chromatography or electrophoresis methods.

The starting compound for the process of the present invention is the compound of formula (II). In a preferred embodiment, Ri is a linear Ci-C ₆ alkyl group and R ₂ is a linear C ₂ -C ₆ alkyl group. In a more preferred embodiment, Ri is a methyl group and R ₂ is a linear C ₂ -C ₆ alkyl group. In yet another embodiment, Ri is a linear Ci-C ₆ alkyl group and R ₂ is an ethyl group. In another embodiment, R ₂ is an ethyl group and Ri is a linear Ci-C ₆ alkyl group, more preferably a methyl group. In another embodiment, Ri is a methyl group and R ₂ is an ethyl group. In yet another embodiment, the starting compound is defined by formula (II) wherein Ri is a methyl group. In another particular embodiment, the starting compound is defined by formula (II), wherein R ₂ is an ethyl group. In a still more particular and preferred embodiment of the invention, the starting compound is defined by formula (II), wherein Ri is a methyl group and R ₂ is an ethyl group, named 2-ethyl-butyraldehyde. In this particular and preferred embodiment of the invention, the process of the invention allows obtaining a compound of formula (I) wherein Ri is a methyl group and R ₂ is an ethyl group, named 2-ethyl-butanol. The process of this particular embodiment of the process of the invention is shown in scheme II.

H

2-ethyl-l-butyraldehyde 2-ethyl-l-butanol

Scheme II

The compounds of formula (II), can be obtained by any method known by the person skilled in the art by using standard synthetic methods known in the art or by acquiring said compound from any supplier of chemical compounds or from a chemical library such small organic molecule libraries.

In a particular embodiment, the polypeptide having aldehyde reductase activity comprises at least one GroES-like alcohol dehydrogenase domain and at least a Zinc binding dehydrogenase domain.

The term "GroES-like alcohol dehydrogenase domain" or "GroEs-like domain" as used herein, refers to a domain as shown under accession number PF08240 in the Pfam database which is a catalytic domain having a GroES-like structure. Proteins with a GroES fold structure have a highly conserved hydrophobic core and a glycil-aspartate dipeptide which is thought to maintain the fold.

The term "Zinc binding dehydrogenase domain" as used herein, refers to a domain having accession number PF00107 in the Pfam database and which forms the cofactor binding domain of the alcohol dehydrogenase enzyme. This domain is normally found towards the C-terminus and structural studies indicate that it forms a classical Rossmann fold that reversibly binds nicotinamide adenine dinucleotide (NAD+).

In a still more particular embodiment, the polypeptide having aldehyde reductase activity has the sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.

Second Process of the invention In another aspect, the invention relates to an in vivo process, hereinafter second process of the invention" for the production of a compound of formula (I)

which comprises culturing a genetically modified microorganism expressing a polypeptide having aldehyde reductase activity and containing a compound of formula (II) under conditions suitable for the conversion of the compound of formula (II) into a compound of formula (I)

wherein Ri is selected from the group consisting of linear or branched Ci-C ₆ alkyl, linear or branched C ₂ -C ₆ alkenyl and linear or branched C ₂ -C ₆ alkynyl, aryl, aryl-alkyl and benzyl and wherein R ₂ is selected from the group consisting of linear or branched C ₂ -C ₆ alkyl, linear or branched C ₂ -C ₆ alkenyl and linear, branched C ₂ -C ₆ alkynyl, aryl, aryl-alkyl and benzyl; and wherein the sequence of said polypeptide has an identity of at least 45% to the sequences of SEQ ID NO: 1 or SEQ ID NO: 2.

Thus, according to the second process of the invention, the contact between said compound of formula (II) and the polypeptide having aldehyde reductase activity is carried out in a genetically modified microorganism which expresses the polypeptide having an aldehyde reductase activity and which contains the compound of formula (II). The term "genetically modified microorganism" as used herein, refers to a microorganism whose genetic material has been altered using genetic engineering techniques.

Accordingly, the second process of the invention relates to obtaining compound of formula (I) by means of a genetically modified microorganism capable of producing said compound of formula (I) characterized in that said genetically modified microorganism expresses (i) a polypeptide having an aldehyde reductase activity as defined above and (ii) contains the compound of formula (II), compared to a corresponding non-modified control microorganism of the same strain. In the context of the present invention, the polypeptide having aldehyde reductase activity may be encoded by a gene found in the genome of the microorganism or may result from the introduction of a polynucleotide encoding said polypeptide into said microorganism. Generally, the polynucleotide encoding a polypeptide having an aldehyde reductase activity can be introduced into the microorganisms in any suitable form, e.g. comprised in a vector, a plasmid or as naked nucleic acid. The polynucleotide introduced into the microorganism may then be exogenous, on a vector/plasmid within the microorganisms [i.e. outside of the microbial chromosome(s)], or it may be incorporated into the microbial chromosome(s) by random (ectopic) or homologous recombination or any other suitable method as known in the art. If desired, the polypeptide encoding the polynucleotide having an aldehyde reductase can be codon-optimized, or modified to be optimized for expression in the recombinant microorganism. In this context of the invention, the genetically modified microorganism is cultured under appropriate conditions that allow the expression of the polypeptide having aldehyde reductase activity; and thereby producing the compound of formula (I). The person skilled in the art will understand that said conditions vary depending on the microorganism that is genetically transformed to be cultured. Suitable culture media for appropriate growth of different microorganisms are well known in the art; for example if the process of the invention is carried out in a Gram-negative genetically modified microorganism the culture media can comprise 1.5 g/L of KH ₂ PO ₄ , 4.54 g/L of K ₂ HP0 ₄ trihydrate, 4 g/L of (NH ₄ ) ₂ S0 ₄ , 0.15 g/L of MgS0 ₄ heptahydrate, 20 g/L of glucose, 200 mM of Bis-Tris buffer (pH 7.2), 1.25, and 1.25 mL/L of a vitamin solution. The vitamin solution can comprise, for example, 0.42 g/L of riboflavin, 5.4 g/L of pantothenic acid, 6 g/L of niacin, 1.4 g/L of pyridoxine, 0.06 g/L of biotin, and 0.04 g/L of folic acid.

In a preferred embodiment, polypeptide having aldehyde reductase activity contains a GroES-like alcohol dehydrogenase domain having accession number PF08240 in the Pfam database and a Zinc-binding dehydrogenase domain having accession number PF00107 in the Pfam database. In another embodiment, the polypeptide having aldehyde reductase activity has the sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.

The genetically modified organism contains the compound of formula (II). The expression "contain the compound of formula (II)" as used herein, means that said compound of formula (II) is endogenously produced by the microorganism. Said expression also encompasses that said compound of formula (II) is not endogenously produced by the microorganism and that said compound has exogenously introduced into the cell.

In one embodiment, wherein Ri is a methyl group. In another embodiment, R ₂ is an ethyl group. In yet another embodiment, the compound of formula (II) is 2-ethyl- butanaldehyde.

In a particular embodiment of the second process of the invention, the genetically modified microorganism has been previously contacted with the compound of formula (II). The term "contacting" the cell with the compound of formula (II) is understood, according to the present invention, as any possible way of introducing said compound into said microorganism. Suitable methods for introducing the compound of formula (II) are well known in the art. In the context of the invention, due to the low molecular weight of compound (II), it is sufficient to add said compound to the culture medium.

Any suitable microorganism can be genetically transformed in order to express the polypeptide having aldehyde reductase activity. In a particular embodiment, said microorganism is selected from the group comprising bacterium, yeast and fungus. The yeasts and filamentous fungi include Pichia sp. (for example, P. pastoris, P . finlandica, P. trehalophila, P. koclamae, P. membranaefaciens, P. minuta, P. opuntiae, P. thermotolerans, P. salictaria, P. guercuum, P. pijperi, P. stiptis, P. methanolica), Saccharomyces (S. cerevisiae), Schizosaccharomyces pombe, Kluyveromyces (for example, K. lactis, K. fragilis, K. bulgaricus, K. wickeramii, K. waltii, K. drosophilarum, K. thernotolerans, and K. marxianus,K.yarrowia), Trichoderma reesia, Neurospora crassa, Schwanniomyces, Schwanniomyces occidentalis, Penicillium, Totypocladium, Aspergillus (Tor example, A. nidulans, A. niger, A. oryzae), Hansenula polymorpha, Candida, Kloeckera, Torulopsis, and Rhodotorula, Hansenula, Kluyveromyces sp. (Tor example, Kluyveromyces lactis), Candida albicans, Aspergillus sp. (Tor example, Aspergillus nidulans, Aspergillum niger, Aspergillus oryzae), Trichoderma reesei,Chrysosporium luchiowense, Fusarium sp. (for example, Fusarium gramineum, Fusarium venenatum), Physcomitrella patens. Examples of bacteria are, without limitation, Escherichia coli, Shigella spp., Bacillus cereus, Yersinia pestis, Pseudomonas spp., Bordetella pertussis, Borrelia burgdorferi, Campylobacter pylori, Chlamydia trachomatis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Legionella pneumophila, Listeria monocytogenes, Mycobacterium diphtheriae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Salmonella spp., Shigella dysenteriae, Staphylococcus aureus, Streptococcus spp. and Vibrio cholerae.

In a preferred embodiment, said microorganism is selected from the group consisting of a bacterium of the genus Clostridium spp., Bacillus spp., Streptomyces spp., or Escherichia spp., yeast of the genus Saccharomyces spp., Pichia spp, Schhizosaccharomyces spp., Candida spp., or Hansenula spp., and a fungus of the genus Aspergillus spp. or Trichoderma spp. In a more preferred embodiment of the invention, said microorganism is Escherichia coli.

The gene encoding the aldehyde reductase can be placed under the control of a constitutive promoter or under the control of an inducible promoter. Suitable promoters for use in the microorganism for use in the second process of the invention have been described above in the context of the first process of the invention and include the β- lactamase (penicillinase) and lactose promoter systems and a tryptophan itrp) promoter system. In a preferred embodiment, the promoter is the lactose promoter and the induction is carried out using isopropyl-P-D-l-thiogalactopyranoside (IPTG). In a preferred embodiment, induction is carried out using 0.01 to 1 mM IPTG, more preferably 1 mM IPTG. Induction is carried out during the time and at the temperature needed to achieve sufficient aldehyde reductase activity in the cell. In a preferred embodiment, induction is carried out at 18-37°C, preferably at 25°C. In another embodiment, induction is carried out for 1 to 16 h, preferably during 5 h. In a preferred embodiment, induction is carried out using IPTG at 25°C for 5 h. The cell culture is then placed in contact with the substrate. The concentration of the substrate during the contacting step is not particularly limiting. Suitable substrate concentrations for carrying the process according to the invention include, without limitation, about 1 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 100 nM, about 500 nM, about 1 μΜ, about 10 μΜ, about 20 μΜ, about 30 μΜ, about 40 μΜ, about 50 μΜ, about 100 μΜ, about 500 μΜ, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 100 mM or more. In a preferred embodiment, the substrate concentration is 5 to 200 mM, preferably 5 mM. In a preferred embodiment, the substrate is butyraldehyde and is used at 5 mM.

In one embodiment, the cells are concentrated prior to the contacting step. Typically, the concentration of the cells is carried out by centrifugation and resuspension of the cell pellet in a volume which results in an optical density which is higher than the optical density of the starting culture. In a preferred embodiment, the cells are resuspended at a cell density of about 0.5 to 10 optical density units, preferably at a cell density of about 4. Resuspension of the cells is carried out typically in a buffer solution in the presence of a carbon source. In a preferred embodiment, the buffer solution is a phosphate buffer at a pH of about 5 to 8.5, preferably at a pH of 7.5. In another embodiment, the carbon source is glucose. In a more preferred embodiment, glucose is added at a concentration of 5 to 200 mM, more preferably 40 mM.

Suitable reaction times for carrying the process according to the invention can be determined by performing a time course experiments wherein samples of the culture are taken at regular intervals and the amount of product formed determined by analysing the supernatant of the culture medium. Said detection is carried out preferably by chromatography (preferably gas chromatography) coupled to mass spectrometry. Suitable times for the in vivo process include, without limitation, between 1 h to 480 h, preferably between 2 h and 240 h, more preferably between 5 and 120 h and even more preferably between 24-48 h. The culture is carried out under aerobic conditions under shaking and at a temperature between 18 to 45°C, more preferably at 25°C.

In a particular embodiment, the second process of the invention comprises an additional step comprising recovering the compound of formula (I) from the culture and/or from said genetically modified microorganism by any suitable method known by the person skilled in the art. By way of illustration, the compound of formula (I) can be recovered from the microorganism by a single lysis step so that all biological membranes are disrupted, including the cell membrane and the mitochondrial membranes, thereby obtaining a whole cell lysate which can then be further processed in order to purify the compound of formula (I). The step of lysing the cells can be achieved by any convenient means, including, but not limited to, heat-induced lysis, adding a base, adding an acid, using enzymes such as proteases and polysaccharide degradation enzymes such as amylases, using ultrasound, mechanical lysis (i.e., subjecting the biomass to pressure sufficient to lyse the cells, termed "pressing"), using osmotic shock, infection with a lytic virus, or expression of one or more lytic genes. Lysis is performed to release intracellular molecules which have been produced by the cell. Each of these methods for lysing cells can be used as a single method or in combination simultaneously or sequentially. The extent of cell disruption can be observed by microscopic analysis. The lysate or extract can be isolated or extracted from a cell by adding a base to a suspension containing the cells. The base should be strong enough to hydrolyze at least a portion of the protein compounds of the cells. Bases which are useful for solubilizing proteins are known in the state of the art. Exemplary bases which are useful in the present invention include, but are not limited to, hydroxides, carbonates and bicarbonates of lithium, sodium, potassium, calcium, and mixtures thereof. The lysate or extract can be isolated or extracted from a cell by using an enzyme. Suitable enzymes to be used according to the present invention include but are not limited to, proteases and polysaccharide-degrading enzymes such as lysozyme, hemicellulase, pectinase, cellulase, driselase, proteases, chymotrypsin, and proteinase K. Any combination of a protease and a polysaccharide-degrading enzyme can also be used. The lysate or extract can be isolated or extracted from a cell using an expeller press. In this process, cells are forced through a screw-type device at high pressure, lysing the cells and causing the intracellular product to be released and separated from the protein and fiber (and other components) in the cell. The lysate or extract can be isolated or extracted from a cell by using ultrasound, i.e., sonication. Thus, cells can also be lysed with high frequency sound. The sound can be produced electronically and transported through a metallic tip to an appropriately concentrated cellular suspension. This sonication (or ultrasonication) disrupts cellular integrity based on the creation of cavities in cell suspension. The lysate or extract can be isolated or extracted from a cell by mechanical lysis. Cells can be lysed mechanically and optionally homogenized to facilitate collection of the product. For example, a pressure disrupter can be used to pump a cell containing slurry through a restricted orifice valve. High pressure is applied, followed by an instant expansion through an exiting nozzle. Typically, cell disruption is accomplished by three different mechanisms: impingement on the valve, high liquid shear in the orifice, and sudden pressure drop upon discharge, causing an explosion of the cell. The method releases intracellular molecules. Alternatively, a ball mill can be used. In a ball mill, cells are agitated in suspension with small abrasive particles, such as beads. Cells break because of shear forces, grinding between beads, and collisions with beads. The beads disrupt the cells to release cellular contents. Cells can also be disrupted by shear forces, such as with the use of blending (such as with a high speed or Waring® blender), the french press, or even centrifugation in case of weak cell walls, to disrupt cells. The lysate or extract can be isolated or extracted from a cell by applying an osmotic shock. The lysate or extract can be isolated or extracted from a cell by infecting the cells with a lytic virus. A wide variety of viruses to lyse cells suitable for use in the present invention, are well known by the person skilled in the art. Alternatively, compound I can be extracted from the cell by contacting the cells with permeabilizmg agents. Said permeabilizmg agents are chemical compounds which greatly affect the cell envelope (in general) and even the outer membrane structure of cells, and low (or even high) molecular weight compounds may leak out of the cell (e.g. cofactors). These permeabilizmg agents are known by the person skilled in the art and include, but are not limited to ionic and non ionic detergents (Triton XI 00, Tween 20), organic solvents (acetone, ethanol, methanol, isopropyl alcohol, toluene, lactic acid), chelating agents (EDTA) and certain polycationic substances (such as polyethylenimine, polymyxin and its derivatives, polylysines and protamine). These compounds compromise cell viability, but also affect bioprocess performance. In bioprocesses in which growth and biotransformation are uncoupled, cell proliferation is not necessary to maintain productivity. Alternatively, compound I can be extracted from the cell by expressing in the cell membrane transport systems able to export organic compounds, namely said compound I. These membrane transport proteins are known by the person skilled in the art and include, but are not limited to periplasmic porins, multidrug efflux pumps and solvent efflux pumps.

The terms "Ci-C ₆ alkyl group", "C ₂ -C ₆ alkyl group", "C ₂ -C ₆ alkenyl group", "C ₂ - C ₆ alkynyl group", "aryl group", "aryl-alkyl group", "benzyl group", "polypeptide having aldehyde reductase activity" and the particulars thereof have been defined in the context of the first aspect of the invention and equally apply to the second aspect of the invention.

Fuel compositions

The inventors have surprisingly found that the compound of formula (I) is advantageously useful as fuel or as fuel additive due to its high octane rating and high energy content. Accordingly, in another aspect, the invention relates to a fuel composition, hereinafter "the fuel composition of the invention" comprising the compound of formula (I), wherein the compound of formula (I) is as defined above. In another aspect the invention relates to the use of the compound of formula (I) as fuel. In another aspect, the invention relates to a fuel composition comprising the compound of formula (I) obtained according to the method of the invention. All embodiments mentioned above regarding the processes of the invention are equally applicable to the fuel composition comprising the compound of formula (I) obtained according to the process defined in the first or in the second aspect of the invention.

In a preferred embodiment, Ri is a linear Ci-C ₆ alkyl group and R ₂ is a linear C ₂ - C ₆ alkyl group. In a more preferred embodiment, Ri is a methyl group and R ₂ is a linear C ₂ -C ₆ alkyl group. In yet another embodiment, Ri is a linear Ci-C ₆ alkyl group and R ₂ is ethyl group. In another embodiment, Ri is a methyl group and R ₂ is ethyl group. In yet another embodiment, Ri is a methyl group. In another particular embodiment, R ₂ is an ethyl group. In a still more particular and preferred embodiment of the invention, Ri is a methyl group and R ₂ is an ethyl group, named 2-ethyl-butyraldehyde.

The term "fuel" as used herein, refers to any material that store potential energy in forms that can be practicably released and used for work or as heat energy. The term fuel includes solid fuels, liquid fuels and gaseous fuels. Solid fuels refers to various types of solid material that are used as fuel to produce energy and provide heating, usually released through combustion, solid fuels include wood, charcoal, peat, coal, hexamine fuel tablets, and pellets made from wood, corn, wheat, rye and other grains. Liquid fuels are combustible or energy-generating molecules than can be harnessed to create mechanical energy, usually producing kinetic energy; they also must tale the shape of the container. Liquid fuels include but are not limited to diesel, gasoline, kerosene, liquefied petroleum gas (LPG), coal tar, naphtha and ethanol. Gaseous fuels include but are not limited to hydrogen, propane, coal gas, water gas, blast furnace gas, coke oven gas and compressed natural gas (CNG). In a preferred embodiment, by fuel is meant liquid fuels.

The compound of formula (I), particularly 2-ethyl-l-butanol, is preferably obtained by the process defined in the first aspect of the present invention.

In another aspect, the invention relates to the use of the compound of formula (I) as a fuel additive. The term "fuel additive" as used herein refers to a compound which is added to fuels to improve performance (such as increasing octane rating) or meet governmental or established regulations. In general terms, fuel additives are added in minor amounts. Illustrative examples of fuel additives include but are not limited to oxygenates, detergents, dispersants, lubricant agents, cetane improvers, cold flow improvers, metal deactivators, demulsifiers, defoamants, dyes, corrosion inhibitors and the like. Thus, the compound of formula (I), particularly 2-ethyl-butanol, may be added to a fuel composition such as gasoline or diesel fuel or mixtures thereof. In preferred embodiments of the invention, the compound of formula (I) is compatible with ordinary fuel systems and does not require any modification to an engine before use.

The person skilled in the art will understand that the percentage in which the compound of formula (I) is added as fuel additive to a fuel composition will be adjusted according to obtain optimal gasoline specification.

Thus, the compound of formula (I) used as fuel or as fuel additive and may be used in internal combustion engines; thereby, the compound of formula (I) may be used for road motor transport vehicles, private or commercial passenger vehicles, marine engines, train engines, marine application (such as jet skis, outboard motors and inboard motors), recreational application (such as snowmobiles, mopeds and motorcycles), residential application (such as lawnmowers, string trimmers and chain saws), powered constructions equipment or potable water or stationary powered electric generators.

In still more particular embodiment, the fuel composition comprises from 0.1% to 40% (v/v) of said compound of formula (I). The term "v/v" as used herein, refers to volume percent concentration defined as [(volume of solute)/(volume of solution)] x 100%.

Typically, the fuel composition of the invention comprises from 3% to 15% (v/v) of said compound of formula (I). In a preferred embodiment, said fuel composition comprises from 5% to 10% (v/v) of said compound of formula (I). In another preferred embodiment the fuel composition comprises 0.2%>, 0.3%>, 0.4%>, 0.5%>, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40% of said compound of formula (I).

In preferred embodiments of the invention, the components of the fuel composition are mixed (or blended) in appropriated proportions so that the fuel composition meets the quality specification established by the governments. Typical quality specifications of fuel compositions include but are not limited to density at 15°C, sulphur content, vapour pressure, distillation curve, olefins content, aromatic hydrocarbons content, benzene content, Research Octane Number and Motor Octane Number. Therefore, the fuel composition is made by means of using any suitable fuel blending process known in the art.

In a particular embodiment, the fuel composition of the invention further comprises at least one hydrocarbon. The term "hydrocarbon", as used herein, refers to an organic compound consisting of hydrogen and carbon atoms. Hydrocarbons from which one hydrogen atom has been removed are functional groups, called hydrocarbyls. Hydrocarbons are classified into alkanes, unsaturated hydrocarbons which include alkenes and alkynes, cycloalkanes and aromatic hydrocarbons. Hydrocarbons can be gases (such as methane and propane), liquid (such as hexane and benzene), waxes or low melting solid (such as paraffin wax and naphthalene) or polymers (such as polyethylene, polyethylene and polystyrene). Hydrocarbons of the fuel composition of the invention are typically derived from petroleum. The refining process of crude oil or petroleum leads to obtaining different complex mixtures of hydrocarbons called naphtha. "Naphtha" is defined as the fraction of hydrocarbons in petroleum boiling between 30°C and 200°C. In a still more particular embodiment of the invention, said at least one hydrocarbon is selected from the group consisting of an aromatic hydrocarbon, paraffin, an olefin, a naphthene and combinations thereof.

The term "aromatic hydrocarbon", as used herein, is meant as hydrocarbons that have at least one aromatic ring. Examples of aromatic hydrocarbons include but are not limited to benzene, toluene, ethylbenzene, /^-xylene, m-xylene, mesitylene, durene, 2- phenylhexane, biphenyl, phenol, aniline and nitrobenzene.

The term "paraffin", as used herein, refers to any of the saturated hydrocarbons having the general formula C _n H _2n+2 (C being a carbon atom and H being a hydrogen atom).

The term "olefin", as used herein, refers to any unsaturated hydrocarbon containing one or more pairs of carbon atoms linked by a double bond. The olefins are cyclic or acyclic (aliphatic), depending on if the double bond is located between carbon atoms forming part of a cyclic (closed ring) or of an open-chain grouping, respectively.

The term "naphthene" or "cycloalkane", as used herein refers to hydrocarbon compounds having one or more rings of carbon atoms in the chemical structure of the molecule. The general formula of naphthene is C _n H ₂ ( _n+ i_ _g ), wherein n is the number of carbon atoms, and g is the number of rings in the molecule.

In a particular embodiment, the fuel composition of the invention comprises from 40% to 99.9% (v/v) of said at least one hydrocarbon. In a preferred embodiment, the fuel composition of the invention comprises the compound of formula (I) as defined above and 40% (v/v) of said at least one hydrocarbon. In another preferred embodiment, the fuel composition of the invention comprises the compound of formula (I) as defined above and 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.9%) (v/v) of said at least one said hydrocarbon. Generally, the fuel composition of the invention comprises more than one hydrocarbon. An Illustrative example of a combination of said hydrocarbon composition may comprise benzene, at least one aromatic hydrocarbon different from benzene and an olefin. In this context, the person skilled in the art understands that the percentage of any compound can vary according to different specification. For example, the mixture of hydrocarbons may be blended forming a composition according to the present invention having 40% (v/v) of hydrocarbons content, wherein 18% (v/v) are olefins, 35% (v/v) are aromatic hydrocarbons including benzene and 1% (v/v) benzene.

Other illustrative examples of fuel compositions which are included in the context of the present invention include fuel compositions comprising 35% of aromatic hydrocarbons different from benzene, 18% olefins and 1% benzene; another example of fuel composition according to the present invention includes 20% of aromatic hydrocarbons including benzene 5%, benzene and 14% olefins.

The determination of the hydrocarbon content on the fuel composition of the invention, such as the determination of the aromatic hydrocarbons and olefins can be determined by any suitable method known in the art. In preferred embodiments of the invention, the hydrocarbon content on the fuel composition of the invention is measured by a multidimensional gas chromatography method. In a still more preferred embodiments of the invention, said content is determined by the internal standard procedure ISO 22854. Said procedure specifies the gas chromatographic method for the determination of saturated, olefmic and aromatic hydrocarbons in automotive motor gasoline and automotive ethanol fuel. Additionally, the benzene content, oxygenate compounds and the total oxygen content can be determined. This international standard defines two procedures, A and B.

Procedure A is applicable to compositions with a total volume fraction of aromatics up to 50%, a total volume fraction of olefins from about 1.5% up to 30%; a volume fraction of oxygenates from 0.8% up to 15%; a total mass fraction of oxygen from about 1.5% to about 3.7%; and a volume fraction of benzene up to 2%. The system may be used for a volume fraction of ethers with 5 or more carbons up to 22%. Although specifically developed for the analysis of automotive motor gasoline that contains oxygenates, this method may also be applied to other hydrocarbon streams having similar boiling ranges such as naphthas and reformates.

Procedure B describes the procedure for the analysis of oxygenated groups. The composition is diluted with an oxygenate- free component to lower the oxygenate content to a value below 20% (v/v) before the analysis by gas chromatographic. If the oxygenated component content is known, the dilution factor can be established accordingly. If, on the contrary, it is unknown, it is advised to use a dilution 4: 1 when analysing the sample.

In another particular embodiment, the fuel composition of the invention further comprises from 0.1% to 22% (v/v) of ethyl tert-butyl ether (ETBE), ethanol or methyl tert-butyl ether (MTBE).

The term "ethyl tert-butyl ether", 2-ethoxy-2-methyl-propane" or "ETBE", as used herein, refers to an oxygenate gasoline additive to increase the octane rating of the gasoline. ETBE is typically produced from the catalytic etherification of ethanol with C ₄ branched olefins such as isobutene.

The term "ethanol", as used herein, refers to a 2-carbon alcohol, having the formula C ₂ H ₆ 0. It is used as gasoline additive to increase the octane number. Ethanol can be chemically synthesized or, alternatively can be obtained from cellulose biomass materials, starch crops, paper or rice-straw by microbiological fermentation processes.

The term "methyl tert-butyl ether", 2-methoxy-2-methylpropane" or "MTBE" is an organic compound using as gasoline additive to raise the octane number. MTBE is typically produced via the chemical reaction of methanol and isobutylene.

In a still more preferred embodiment, the total oxygen content in the composition of the invention is not more than 3.7% (w/w). The term "total oxygen content" as used herein refers to the ratio of the mass of oxygen to the total mass amount of the fuel composition of the invention. Thus, if the fuel composition of the invention comprising the compound of formula (I) further comprises from 2% to 22% of an oxygenate component such ETBE, the total oxygen content of the fuel composition the percentage of ETBE in the composition will be considered when determining the total oxygen content in the fuel composition of the invention.

The determination of the oxygen content on the fuel composition of the invention, can be carried out by method known in the art. In preferred embodiments of the invention, the oxygen content is determined using the methods described by the European Normatives EN1601, EN13132 and EN ISO 22854.

In another embodiment, the fuel composition of the invention further comprises up tol500 mg/kg sulphur. Sulphur is a naturally occurring compound in crude oil. Typically, the content in sulphur is from 150 to 300 mg/kg. In a preferred embodiment of the invention, the composition of the invention comprises from 10 to 12 mg/kg sulphur. In another preferred embodiment, the fuel composition of the invention comprises 10 mg/kg sulphur, 50 mg/kg sulphur, 80 mg/kg sulphur, 150 mg/kg sulphur, 300 mg/kg sulphur, 500 mg/kg sulphur or 1000 mg/kg sulphur.

The determination of sulphur contents in the fuel composition of the invention can be carried out by any suitable method such as ASTM (Society for testing Materials standard) D5453, which uses ultraviolet fluorescence to measure sulphur concentrations in liquid hydrocarbons. Other specified methods for testing sulphur contents may also be used such X-ray spectrometry.

In another embodiment, the fuel composition of the invention has a Reid vapor pressure up to 103 kPa. The term "Reid vapor pressure" as used herein refers to the absolute vapor pressure exerted by a liquid at 100°F (37.8°C). The matter of vapor pressure is important relating to the function and operation of gasoline powered, especially carbureted, vehicles. High levels of vaporization are desirable for winter starting and operation and lower levels are desirable in avoiding vapor lock during summer heat. Fuel cannot be pumped when there is vapor in the fuel line (summer) and winter starting will be more difficult when liquid gasoline in the combustion chambers has not vaporized. Typically, the Reid vapor pressure of a fuel composition is from. 54 to 103 kPa, or from 35 to 55 kPa, or from. 70-90 kPa depending on the specification. In a preferred embodiment, the fuel composition, of the invention has a Reid vapor pressure from 55 to 58 kPa. In another preferred embodiments of the invention, the fuel composition of the invention has a Reid vapor pressure of 45 kPa, 50 Kpa, 55 kPa, 60 kPa, 65 kPa, 70 kPa, 75 kPa, 80kPA, 85 kPa, 90 kPa, 95 kPa or 100 kPa.

The Reid vapor pressure can be determined by any suitable test known in the state of the art such as the ASTDM D6378, ASTM D5188, ASTM D6377, ASTM D6897 and ASTM-D5 1 1 test. In preferred embodiments of the invention, the Reid vapor pressure is determined by the ASTM D5191 method. Said method covers the use of automated vapor pressure instruments to determine the total vapor pressure exerted in vacuum by air-containing volatile, liquid petroleum products, including automotive spark ignition fuels with or without oxygenates. The calculation of the vapor pressure according to said test is determined by the equation (0.965 X)-A, wherein the X parameter corresponds with the measurement of total vapor pressure, in units consistent with A; and A has the 3.78 kPa value.

In another particular embodiment, the fuel composition of the invention has a density at 15°C up to 775 kg/nf. The term, "density" as used herein refers to a mass per unit volume i.e. mass of a substance divided by its volume as a specific temperature.

The density of the fuel composition of the invention can be measured by any suitable method known in the state of the art. In a preferred embodiment, the density of the fuel composition of the invention is determined by the ASTM D4052 test. This test method covers the determination of the density or relative density of petroleum distillates (for example gasoline, gasoline-oxygenate blends, diesei, jet fuel, base stocks) and viscous oils that can be handled in a normal fashion as liquid at test temperatures between 15°C and 35°C by means of a density meter.

In another particular embodiment, the fuel composition of the invention has a Research Octane Number from. 83 to 98 and/or a Motor Octane Number from 75 to 85.

The term "octane rating" or "octane number", as used herein, refers to a standard measure of the performance of motor or aviation fuel. The higher the octane number, the more compression the fuel can withstand. High compressibility of the fuel matters in proportion to the compression the engine is designed to generate. The most common type of octane rating is the Research Octane Number (RON), which is determined by running the fuel in a test engine with a variable compression ratio under controlled conditions, and comparing the results with those mixtures of isooctane and n- heptane. The motor octane number (MON) is another type of octane rating which provides a measure of how the fuel behaves when under load, as it is determined at 900 rpm engine speed, instead of the 600 rpm for RON. MON testing uses a similar test engine to that used in RON testing, but with a preheated fuel mixture, higher engine speed, and variable ignition timing to further stress the fuel ^' s knock resistance.

In a preferred embodiment, the fuel composition of the invention has a RON of

83. In another preferred embodiment, the composition of the invention has a RON of

84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97 or 98.

In a preferred embodiment, the fuel composition of the invention has a MON of

75. In another preferred embodiment, the composition of the invention has a MON of

76, 77, 78, 79, 80, 81 , 82, 83, 84 or 85. Any method can be used to determine the RON and MON of the fuel composition of the invention. For example, the RON and MON of a spark-ignition engine fuel is determined using a standard test engine and operating conditions to compare its knock characteristics with those of primary reference fuel blends of known octane number. Compression-ratio and fuel-air ratio are adjusted to produce standard knock intensity for the sample fuel, as measured by a specific electronic denotation meter instrument system. The specific procedure for the RON can be found in ASTM D2699 and the MON can be found in ASTM D2700.

A fuel composition according to the present invention is exemplified in Example 4 of the present application. The fuel composition of the invention comprises suitable components for gasoline blends. Conventional gasoline components include but are not limited to reformed naphtha, debenzeneized reformed naphtha, fluid catalytic cracking naphtha, isopentane, ETBE, isomerate, alkylate, butanes, MTBE, hydrocracker naphtha and straight run naphtha. Other components that can be included in the gasoline are the ones derived from Fischer-Tropsch processes, using biomass, natural gas, coal or other raw materials, from pyrolysis of biomass or other raw materials, and other oxygenates (alcohols, ethers, etc.) among others. The person skilled in the art will understand that depending on the blending process at the refinery, the components and the percentage of each component may vary. Typically a refinery may have from butane, the most volatile component to heavy naphtha and include several gasoline naphthas from crude distillation, catalytic cracking and thermal processing units in addition to alkylate, isomerate and reformate. Gasoline may blended to meet different quality standards as has been detailed above. These standards include limits to some properties, such as vapour pressure;% volume evaporated at 70°C, 100°C and 150°C, final boiling point, sulfur content; color; stability; aromatic content; olefin content; octane measurements; and other local government or market restrictions. Fuel standards differ by geographical region and markets and are subject to frequent review to reflect changes in legislation and vehicle technology development. Since each of the individual components contribute uniquely in each of these quality areas and each bears a different cost of manufacture, the proper allocation of each component into its optimal disposition can be set using linear programming tools. The invention is detailed below by means of the following examples which are merely illustrative and by no means limiting for the scope of the invention. EXAMPLES

EXAMPLE 1: Synthesis of 2-ethyl-l-butanol from 2-ethyl-l-butyraldehyde with aldehyde reductase from Lactococcus lactis.

The gene encoding aldehyde reductase from Lactococcus lactis (SEQ ID NO: 3) was cloned into a multicopy plasmid with a N-terminal polyhistidine tag. This plasmid was transformed into E. coli BL21(DE3) for protein production.

Protein expression was induced with 1 mM IPTG at an initial optical density at 600 nm (OD) of 0.6. After overnight incubation at 21°C with vigorous agitation (200- 250 rpm), cells were collected by centrifugation for 10 minutes at 4500 rpm and resuspended in 50 mM phosphate buffer (pH 7.5). Protein was extracted by sonication and clarified by centrifugation for 30 min at 8000 rpm. The soluble protein fraction was affinity purified using resins with immobilized nickel or cobalt metal ions and eluted using 300 mM imidazole (in 50 mM phosphate buffer pH 7.5). Purified protein was used for subsequent transformation assays.

Enzyme activity was assessed using butyraldehyde or 2-ethyl-butyraldehyde as substrates. Reactions were carried out at 25-37°C in 50 mM phosphate buffer (pH 7.5). Substrate concentration was 0.1-0.5 mM. 1 mM NADH was used as redox cofactor. The reaction was monitored by measuring the decrease in absorbance at 340 nm in a multiplate reader with temperature control. One unit of enzyme activity was defined as the amount of enzyme capable to catalyze the transformation of 1 μιηοΐ of substrate per minute.

The alcohol dehydrogenase of Lactococcus lactis (SEQ ID NO: 1) thus prepared showed a specific activity of 3.9 ± 0.1 mU/mg using 0.1 mM butyraldehyde as substrate and 1 mM NADH as cofactor at 37°C.

The alcohol dehydrogenase of Lactococcus lactis thus prepared showed a specific activity of 22 ± 9 mU/mg using 0.1 mM 2-ethyl-butyraldehyde as substrate and 1 mM NADH as cofactor at 37°C. Production of 2-ethyl-butanol from 2-ethyl-butyraldehyde by alcohol dehydrogenase from Z. lactis was confirmed by GC-MS. 0.5 mM 2-ethyl-butyraldehyde and 1 mM NADH in 50 mM phosphate buffer (pH 7.5) were incubated overnight with alcohol dehydrogenase from L. lactis at room temperature. Reaction mixture was filtered and analyzed using a GC-MS with Twister-Thermal Desorption Unit. Two peaks were detected in the Total Ion Chromatogram (TIC), corresponding to 2-ethyl- butyraldehyde (peak 1) and 2-ethyl-l-butanol (peak 2), respectively (Figure 1).

The mass spectrum of the first peak was searched against the NIST standard reference database, which confirmed that it corresponded to 2-ethyl-l-butyraldehyde (Figure 2).

The mass spectrum of the second peak was searched against the NIST standard reference database, which confirmed that it corresponded to 2-ethyl-l-butyraldehyde (Figure 3). EXAMPLE 2: Synthesis of 2-ethyl-l-butanol from 2-ethyl-l-butyraldehyde with aldehyde reductase from Bacillus stearothermophilus.

The gene encoding aldehyde reductase from Bacillus stearothermophilus (SEQ ID NO: 4) was cloned in a multicopy plasmid with a N-terminal polyhistidine tag. This plasmid was transformed into E. coli BL21(DE3) for protein production.

Protein expression was induced with 1 mM IPTG at an initial optical density at

600 nm (OD) of 0.6. After 3 hours of incubation at 37°C with vigorous agitation (200- 250 rpm), cells were collected by centrifugation for 10 minutes at 4500 rpm and resuspended in 50 mM phosphate buffer (pH 7.5). Protein was extracted by sonication and clarified by centrifugation for 30 min at 8000 rpm. The soluble protein fraction was affinity purified using resins with immobilized nickel or cobalt metal ions and eluted using 300 mM imidazole (in 50 mM phosphate buffer pH 7.5). Purified protein was used for subsequent transformation assays.

Enzyme activity was assessed using butyraldehyde or 2-ethyl-butyraldehyde as substrates. Reactions were carried out at 25-37°C in 50 mM phosphate buffer (pH 7.5). Substrate concentration was 0.1-0.5 mM. 1 mM NADH was used as redox cofactor. The reaction was monitored by measuring the decrease in absorbance at 340 nm in a multiplate reader with temperature control. One unit of enzyme activity was defined as the amount of enzyme capable to catalyze the transformation of 1 μιηοΐ of substrate per minute.

The alcohol dehydrogenase of B. stearothermophilus (SEQ ID NO: 2) thus prepared showed a specific activity of 11 1 ± 23 mU/mg using 1 mM NADH as cofactor at 37°C and 0.1 mM butyraldehyde as substrate.

The alcohol dehydrogenase of B. stearothermophilus thus prepared showed a very low specific activity using 0.1-0.5 mM 2-ethyl-butyraldehyde as substrate and 1 mM NADH as cofactor, which could not be quantified in short term kinetic assays (up to 30 minutes) using spectrophotometric detection of NADH oxidation at 340 nm.

Nonetheless, production of 2-ethyl-butanol from 2-ethyl-butyraldehyde by alcohol dehydrogenase from B. stearothermophilus was confirmed by GC-MS in long term activity assays (up to 16 hours). 0.5 mM 2-ethyl-butyraldehyde and 1 mM NADH in 50 mM phosphate buffer (pH 7.5) were incubated overnight (c.a. 16 hours) with alcohol dehydrogenase from B. stearothermophilus at room temperature. Reaction mixture was filtered and analyzed using a GC-MS with Twister-Thermal Desorption Unit. Two peaks were detected in the Total Ion Chromatogram (TIC), corresponding to 2-ethyl-butyraldehyde (peak 1) and 2-ethyl-l-butanol (peak 2), respectively (Figure 4). The identity of these two compounds was confirmed by searching the mass spectra of these two peaks against the NIST standard reference database (Figures 5-6).

Example 3: In vivo biotransformation of 2-ethyl-butyraldehyde into 2-ethyl- butanol with transformed E. coli resting cells

E. coli cells transformed with aldehyde reductase expressing plasmids such as those described in this patent are able to biotransform branched aldehydes into the corresponding alcohols.

The procedure is carried out with E. coli BL21(DE3)T1 cells transformed with the expression plasmids carrying the genes of the aldehyde reductases described in this patent. Cells are grown in an orbital shaker (operated at 250 rpm) under aerobic conditions in LB growth medium for 2-3 hours or until an absorbance at 600 nm of approximately 0.6 is reached. Cultures are then induced using 1 mM isopropyl-P-D-l- thiogalactopyranoside (IPTG) as inducer and setting temperature to 25°C. After inducing cells for 5 h, cells are harvested by centrifugation at 3000 rpm (5 min at room temperature to avoid damaging the cells). Cell pellets obtained were resuspended in a biotransformation medium consisting of phosphate buffer 50 mM, pH 7.5, supplemented with 40 mM glucose and 5 mM 2-ethyl-butyraldehyde. Cell concentration is adjusted to an optical density (absorbance at 600 nm) of around 4.

Cells are incubated in this biotransformation medium at 25°C for 24-48 h under aerobic conditions (orbital shaking at 250 rpm) or under anaerobic conditions.

After incubation, the biotransformation medium is separated from cells by centrifugation at 4500 rpm (15 min). Supernatant is analyzed by GC (preferably coupled to MS detection) in order to determine the 2-ethyl-butyraldehyde to 2-ethyl-butanol biotransformation yield.

EXAMPLE 4: In vivo biotransformation of 2-ethyl-butyraldehyde into 2-ethyl- butanol with transformed E. coli growing cells.

E. coli cells transformed with aldehyde reductase expressing plasmids such as those described in this patent are able to biotransform branched aldehydes into the corresponding alcohols.

The procedure is carried out with E. coli BL21(DE3)T1 cells transformed with the expression plasmids carrying the genes of the aldehyde reductases described in this patent. Cells are grown in an orbital shaker (operated at 250 rpm) under aerobic conditions in LB growth medium for 2-3 hours or until an absorbance at 600 nm of approximately 0.6 is reached. Cultures are then induced using 1 mM isopropyl-P-D-l- thiogalactopyranoside (IPTG) as inducer and setting temperature to 25°C.

After inducing cells for 5 h, cells, culture medium is supplemented with 40 mM glucose and 5 mM 2-ethyl-butyraldehyde. Cultures are then incubated at 25°C for another 24 to 48 hours under aerobic conditions (in an orbital shaker operated at 250 rpm) or under anaerobic conditions. Cell growth is monitored in all cultures by measuring absorbance of the culture at 600 nm every 1-4 hours.

After incubation, the growth medium is separated from cells by centrifugation (4500 rpm, for 15 minutes). Supernatant is analyzed by GC (preferably coupled to MS detection) in order to determine the 2-ethyl-butyraldehyde to 2-ethyl-butanol biotransformation yield. EXAMPLE 5: Comparison of 2-ethyl-l-butanol properties vs ethanol

The compound 2-ethyl-l-butanol presents beneficial properties for its use in gasoline formulation that are superior to ethanol, the most spread biocomponent fuel for gasoline.

In the following table, some of these properties are compared. Gasoline properties correspond to EN 228:2013, the European gasoline standard, except for energy density and solubility in water, which are typical values.

Table 1 : Properties of pure 2-ethyl-l-butanol, pure ethanol, pure iso-butanol and fossil gasoline. Data for ethanol, iso-butanol and fossil gasoline were obtained from the literature. The RON and MON values for 2-ethyl-l- butanol have been determined experimentally using standard procedures. * Vapour pressure in a mixture with hydrocarbons, up to 10% alcohol content.

While ethanol presents very high octane rating numbers, 2-ethyl-l-butanol RON and MON values are also above the gasoline standard. On the other hand, energy density of 2-ethyl-l-butanol is very high, slightly above typical values for gasoline and clearly above ethanol and iso-butanol. This higher energy content means that the fuel consumption in the engine would be lower and higher mileages could be achieved. The higher consumption compared to fossil gasoline is one of the most important drawbacks in the use of ethanol. Another drawback that ethanol presents is its high miscibility with water, which generates corrosion problems as well as logistics issues. Higher alcohols are more similar to hydrocarbons, and present low solubility in water.

Another advantage of 2-ethly-l-butanol over other alcohols is its relative lower oxygen content, as it only contains one atom of oxygen per 6 atoms of carbon, compared to other alcohol fuels such as ethanol (1 oxygen per 2 carbons) or butanol (1 oxygen per 4 carbons).

Density and vapour pressure of 2-ethlyl-l -butanol correspond to a heavy compound in the gasoline mix. This allows for the introduction of light compounds in the gasoline blend, such as light naphtha or butanes, which are often limited due to their high vapour pressure or low density.

EXAMPLE 6: Gasoline Blending Formulation with 2-ethyl-l-butanol

This example illustrates the suitability of the product 2-ethyl-l-butanol as component for gasoline blends.

Samples of conventional gasoline components were collected from a refinery and characterized in the laboratory, following the international standard test methods in Table 2. For this example, conventional blending components including reformed naphtha, de-benzenized reformed naphtha, FCC naphtha, isopentane and ETBE. Other streams may also be present in the blending, such as isomerate, alkylate, MTBE, Hydrocracker naphtha, straight run naphtha etc. Their availability at the refinery depends on its scheme and type of processing units. Depending on the availability of streams, the percentage of each component in the gasoline may differ greatly.

Property Test method

Density at 15°C ASTM

D4052

Sulphur ASTM

D5453

Vapour pressure ASTM

D5191

Distillation curve ASTM D86

Research Octane Number ASTM

(RON) D2699 Motor Octane Number (MON) ASTM

D2700

Olefins EN ISO

22854

Aromatics EN ISO

22854

Benzene EN ISO

22854

Table 2: Test methods followed for the characterization of fuel properties

A sample of 2-ethyl-l-butanol was purchased from Aldrich. The properties that are critical in the gasoline blending were analyzed in the laboratory, following the same test methods in Table 2. The results for this characterization are shown in Table 3.

Table 3 : Property values for alcohol molecules Streams representing the conventional blending component and the C6 alcohol was introduced as blending feeds in the "Refinery Blender" model from Process Simulation software Petro-SIM, by KBC. Streams were modelled using the analytical data obtained in the laboratory. The final product (gasoline) specifications were set according to European standard EN 228:2013. The two oxygen content qualities in the specification were simulated: maximum 2.7% m/m content and maximum 3.7% m/m content. Specifications for season classes A and C were simulated, for both oxygen quantity qualities. In total, four specification sets were used. Using the model, formulations for four different gasolines with 2-ethyl-l-butanol as an ingredient were calculated.

Gasoline blends were prepared in the laboratory using the refinery samples and 2-ethyl-l-butanol by following the compositions calculated in the model. The different components were refrigerated at 4°C prior to the blending, to avoid losses of the lighter compounds. The blending was performed on a weight basis. Experimental characterization of the gasolines corroborated the calculated properties of the blend, with the exception of two of the blends with highest alcohol content. In these cases, the formulation had to be readjusted for RON and MON specification in order to meet the minimal required specifications.

The corrected formulations comprised alcohol contents up to 9.7% in gasolines with 3.7% maximum oxygen content. Final formulations are collected in Table 4. As mentioned before, refinery conventional components in this example include ETBE, which is produced at a refinery unit but contains oxygen. Oxygen content in ETBE also accounts for the specification in maximum oxygen content in the final gasoline.

Table 4: Formulation of gasolines with 2-ethyl-l-butanol Please note that other formulations are possible, and 2-ethyl-l-butanol blending rates may vary according to the composition of the fossil streams. Naphthas with better gasoline properties, such as isomerate or alkylate, would allow for a higher introduction of 2-ethyl-l-butanol in the gasoline and a decrease in ETBE content.

The alcohol content was limited to the current maximum oxygen contents in the specification. However, simulations without oxygen limit were also run with other Naptha compositions. The results showed it was possible to obtain gasoline blends within specification for the rest of properties with alcohol contents up to 27%. EXAMPLE 7: Engine tests

Performance of the blends with the higher alcohol content was tested in engine. Samples for the equivalent gasoline specifications formulations, not containing alcohols, were also prepared and tested for comparison.

The engine used was a 1.2TSI Volkswagen, from a SEAT Leon 105CV - 77kW vehicle.

The power (kW) and the consumption (liters/h) performance were recorded during a test. For this test the engine was set at maximum speed (5000 rpm) and 100% throttle during 25 seconds. The test was repeated three times. Power in samples with alcohols was slightly higher than in conventional gasoline, while consumption was also slightly higher. The differences were negligible, and attributed to the different oxygen content in the samples.

Drivability test were also conducted in order to see if the presence of alcohol provoked problems during fast acceleration. Two tests were performed: at 55% throttle and speed between 1000 and 3000 rpm and at 100% throttle and speed between 1000 and 3000 rpm. The tests were performed three times on each fuel sample. No differences with conventional gasoline tests were observed.