TECHNICAL FIELD

The invention relates to process for the production of alkyl alkanoates from alcohol and Acyl coenzyme A using polypeptides having alcohol acetyl transferase activity (hereinafter referred to as AAT). The process is further directed to the use of a recombinant expression vector or plasmid or host cell comprising the nucleotide sequence encoding the polypeptide. Alcohol acetyl transferase catalyzes the transfer of the acetyl group of acetyl-coenzyme A to an alcohol (EC2.3.1 .84) and produces Coenzyme A and the corresponding ester.

BACKGROUND Ethyl acetate is an environmentally friendly solvent with many industrial applications. Also, the higher alkyl alkanoates are interesting compounds for the industry. The production of ethyl acetate currently proceeds by energy-intensive petrochemical processes which are based on natural gas and crude oil without exception. The following methods play a larger role in industrial-scale production (Lin et al. 1998; Colley et al.2004; Arpe 2007);

a) Fischer esterification: reaction of ethanol with acetic acid in the presence of strong acids as a catalyst, in part combined with removal of water for shifting the equilibrium toward the ester b) Tischtschenko reaction: addition of two equivalents acetaldehyde by disproportionation under the action of alkoxides, especially aluminum triethoxide, as a catalyst.

c) Dehydrogenation of ethanol: ethanol is dehydrogenated to acetaldehyde, the aldehyde is then added to ethanol to form a hemiacetal which in turn is dehydrogenated to generate ethyl acetate

(Inui et al. 2002)

d) Avada process developed by BP Chemicals: Avada abbreviates Advanced acetates by direct addition" and means synthesis of ethyl acetate by catalytic addition of ethylene and acetic acid in the gas phase

e) Partial oxidation of ethanol plus esterification: this process combines catalytic oxidation of ethanol and esterification of the formed acetic acid with excess ethanol (Lin et al. 1998)

The economy of a given process depends on regional specifics such as availability of raw materials, expenses for all resources, and on fiscal conditions too. All the chemical reactions l occur at elevated temperature and often at high pressure. Additional sub-processes are required for recovery of ethyl acetate and for recycling of residual precursors. The conversion process is frequently incomplete due to reverse reactions, and catalysts are commonly required.

Current production of ethyl acetate is exclusively based on fossil hydrocarbons. This becomes clearly visible for the ethylene-based process as ethylene is produced by steam cracking of natural-gas and crude-oil constituents, but this also applies to all other processes since their precursors originate from ethylene; ethanol is formed by its hydration, acetaldehyde is obtained by its oxidation, and acetic acid arises by oxidation of acetaldehyde or ethylene. Alternative acetic acid production by addition of carbon monoxide and methanol does not change the situation since both are synthesized from natural gas (steam-methane reforming). All these processes require catalysts and a high input of energy (heat and pressure). Fossil hydrocarbons are limited resources with steadily growing expenses for their recovery.

Ethanol as a precursor for chemical synthesis of ethyl acetate could be produced from sugar by fermentation (Silveira et al. 2005; Aziz et al. 2009; Guimaraes et al. 2010; Rodrussamee et al. 201 1 ), however the direct microbial production of alkyl esters such as ethyl acetate would provide more advantages. Microbial synthesis of alkyl alkanoate could become an interesting alternative. Although the ability of yeasts for producing larger amounts of this ester is known for a long time, these native microorganisms are not able to produce acetate esters in yields that are suitable for industrial bulk production.

Moreover, it was not known which enzymes and corresponding genes are actually involved. This research was mainly of scientific interest, and large-scale ester production from renewable raw materials has not yet been achieved. In Applied Microbiol Biotechnol (2014) 98, p. 5397-5415 an overview is given of the research that has been done on the perspectives for the biotechnical production of ethyl acetate by yeasts.

In US 2013/0295616 the productivity of acetic acid, acetyl coenzyme A and products derived thereof is improved by increasing the availability of acetyl Coenzyme A in yeast. The enzyme used here is an ATF1 .

The production of ethyl acetate has been associated with three enzymatic reactions:

Esterase reaction wherein alcohol and acetate forms alkyl acetate with formation of water.

Hemiacetal dehydrogenase reaction (HADH) wherein alcohol and acetaldehyde form hemiacetal, which is formed into alkyl acetate, and

- Alcohol acetyl transferase (AAT) reaction wherein alcohol and acetyl coenzyme A is converted into alkyl acetate and co-enzyme A. Since the delta Gibbs Free energy of this reaction is strongly negative, this reaction is most suitable for industrial alkyl acetate and also higher alkyl alkanoate production. See formulae below:

Ethanol + Acetaldehyde

spontaneous Ethanol + Acetyl-CoA

Etha.no! + A.cetate

Esterase

Ethyl acetate

Ethyl acetate Ethyl acetate

AG _r ' (kJ/mol) +28.8 -18.2 -18.3 It is however not really clear which enzymes are involved exactly. In Microb. Biotechnol. 3, 165— 177 (2010) it is indicated that in S. cerevisiae two AATs, Atf1 and Atf2 are involved in synthesis of ethyl acetate and isoamyl acetate during wine and beer fermentation. However, a Aatf1Aatf2 strain of S. cerevisiae still retained 50% of its ethyl acetate production, suggesting that other ester producing enzymes are also active in S. cerevisiae. In Knight, M. J., Bull, I. D. & Curnow, P. Yeast s , n/a-n/a (2014) and J. Biol. Chem. 281 , 4446-4456 (2006) it is mentioned that Eht1 and Eeb1 produce medium chain ethyl esters in S. cerevisiae. They do not resemble Atf1 and Atf2 on a protein level and contain an alpha/beta hydrolase fold. The reaction typically associated with alpha/beta hydrolases is hydrolysis, but Eht1 and Eeb1 show AAT, thioesterase and esterase activities. Eht1 and Eeb1 are not known to produce ethyl acetate, but since no combined gene deletions of Eht1 and Eeb1 and aft 1 and aft2 have been reported, their involvement in the remaining ethyl acetate synthesis is unconfirmed.

The nature of the enzymes that are involved in ethyl acetate formation in typical high ethyl acetate producing yeast species is even less clear. Reports on the enzymatic mechanism are inconsistent, as AATs, HADHs and esterases have all been suggested to contribute to ethyl acetate synthesis, at times even in the same organism.

SUMMARY OF THE INVENTION An object of this invention is to provide a process for the production of alkyl alkanoates using a polypeptide with alcohol acetyl transferase activity to convert:

- C3-C10 alkanol and acetyl coenzyme A or,

- C1 -C10 alkanol with C3-C10 acyl coenzyme A, or

- Methanol with acetyl coenzyme A, into an alkyl alkanoate.

The alkanoate may be an acetate, but also higher carboxyl acid residues up to decanoyi may be formed, thus, the term "C3-C10 acyl". As indicated by the term "C3-C10 alkanol" it is indicated that propanol up to decanol, i.e. C10 alcohol, may be converted. Both the alcohols and the acyls may concern linear as well as branched compounds. The genes encoding these polypeptides, when expressed in a suitable host, are able to produce alkyl alkanoate in a yield suitable for industrial scale.

The group of polypeptides that can be used in the process according to the invention was found to represent enzymes that have alcohol acetyl transferase activity and whose three-dimensional structure contains an alpha-beta hydrolase fold and an active site comprising a serine, histidine and optionally aspartic acid, forming a dyad of serine- histidine or a triad of serine-aspartic acid- histidine wherein the histidine of the serine-histidine dyad or serine-apartic acid -histidine triad in the polypeptide according to the invention, is present in a polypeptide fragment that has a glutamic acid-arginine- proline (ERP) fragment, a glutamic acid-asparagine-proline (ENP) fragment, or glutamic-acid- methionine- proline (EMP) fragment as the 5 ^th , 6 ^th and 7 ^th amino acid from the histidine on the C-terminal side of the polypeptide. The polypeptides that turned out to be a non- alkyl C3-C10 alkanoate producer did not have such a ERP, ENP or EMP fragment.

In an aspect of the invention the amino acid sequence of the polypeptide comprises an amino acid sequence that has:

- an amino acid sequence according to SEQ ID NO: 1 : or an amino acid sequence that has at least 70% identity with the sequence as shown in SEQ ID NO: 1 and/or,

- an amino acid sequence according to SEQ ID NO: 3: or an amino acid sequence that has at least a 70 % identity with SEQ ID NO: 3 and/or,

- an amino acid sequence according to SEQ ID NO: 5: or an amino acid sequence that has at least 70 % identity with SEQ ID NO: 5 and/or,

- an amino acid sequence according to SEQ ID NO: 7: or an amino acid sequence that has at least 70% identity with the sequence as shown in SEQ ID NO: 7 and/or, - an amino acid sequence according to SEQ ID NO: 9: or an amino acid sequence that has at least a 70 % identity with SEQ ID NO: 9 and/or,

- an amino acid sequence according to SEQ ID NO: 1 1 : or an amino acid sequence that has at least 70 % identity with SEQ ID NO: 1 1 and/or,

an amino acid sequence according to SEQ ID NO: 13: or an amino acid sequence that has at least 70% identity with the sequence as shown in SEQ ID NO: 13 and/or,

- an amino acid sequence according to SEQ ID NO: 15: or an amino acid sequence that has at least a 70 % identity with SEQ ID NO: 15 and/or,

- an amino acid sequence according to SEQ ID NO: 17: or an amino acid sequence that has at least 70 % identity with SEQ ID NO: 17 and/or,

- an amino acid sequence according to SEQ ID NO: 19: or an amino acid sequence that has at least 70% identity with the sequence as shown in SEQ ID NO: 19.

In an aspect of the invention the nucleotide sequence encoding for the polypeptide according to the invention comprises,

a nucleotide sequence according to SEQ ID NO:2, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 2 and/or,

a nucleotide sequence according to SEQ ID NO:4, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 4 and/or,

- a nucleotide sequence according to SEQ ID NO:6, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 6,

a nucleotide sequence according to SEQ ID NO:8, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 8 and/or,

a nucleotide sequence according to SEQ ID NO:10, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 10 and/or,

a nucleotide sequence according to SEQ ID NO:12, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 12 and/or,

a nucleotide sequence according to SEQ ID NO:14, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 14 and/or,

- a nucleotide sequence according to SEQ ID NO:16, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 16 and/or,

a nucleotide sequence according to SEQ ID NO:18, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 18 and/or, a nucleotide sequence according to SEQ ID NO:20, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 20.

The invention is further directed to the process using a recombinant vector, plasmid or a host cell comprising the nucleotide sequence encoding for the polypeptide used in the process invention. Said host cell may be selected from bacteria, yeasts and filamentous fungi.

Preferred bacteria are selected from Escherichia species and Bacillus species. A preferred Escherichia species is preferably E. coli and a preferred Bacillus species is Bacillus subtilis. Other suitable bacteria are the ones that are used in industrial production such as

Corynebacterium glutamicum.

Species that are especially preferred are the thermophilic species since they allow production at high temperatures. Examples thereof are Geobacillus thermoglucosidasius, Caldicellulosiruptor bescii, Clostridium thermocellum, Thermoanaerobacterium aotearoense,

Thermoanaerobacterium saccharolyticum, Thermoanaerobacter tengcongensis,

Thermoanaerobacter ethanolicus, Thermoanaerobacter mathrani, Thermococcus kodakarensis, Pyrococcus furiosus.

Preferred yeasts are the ones from which the polypeptides according to the invention originate such as Wickerhamomyces anomalus, Wickerhamomyces ciferrii, Kluyveromyces marxianus Kluyveromyces lactis, Cyberlindnera jadinii, Hanseniaspora u varum, Eremothecium cymbalarie, and Saccharomyces cerevisiae, but also yeasts that are often used in the industry as production yeasts such as Saccharomyces cerevisiae species, Pichia species such as P. pastoris and Schizosaccharomyces species. Preferred filamentous fungi are selected from Aspergillus species, Trichoderma species or Penicillium species; Preferred Aspergillus species are preferably A. niger, A. oryzae, and A. nidulans. Also Monascus ruber is suitable.

The process may be conducted at a temperature of between 10 and 130 °C. Preferably the polypetides used in the process are produced in a host cell. Also the alcohol and the acetyl coenzyme A, or Acyl coenzyme A may be produced in a host cell. The alkyl alkanoate formed may be isolated from the host cell and optionally purified. The invention is further directed to the use of the hosts cells according to the invention in the preparation of alkyl alkanoates. A sugar compound may be used as a growth substrate for the host cell.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 sets out the amino acid sequence of AAT1 obtained from Wickerhamomyces anomalus DSM 6766)

SEQ ID NO:2 sets out the nucleotide sequence encoding for AAT1. SEQ ID NO:3 sets out the amino acid sequence of AAT 2 obtained from Wickerhamomyces ciferrii CBS 111

SEQ ID NO:4 sets out the nucleotide sequence encoding for AAT2.

SEQ ID NO:5 sets out the amino acid sequence of AAT 3 obtained from Kluyveromyces marxianus DSM 5422.)

SEQ ID NO:6 sets out the nucleotide sequence encoding for AAT3.

SEQ ID NO:7 sets out the amino acid sequence of AAT4 obtained from Kluyveromyces lactis CBS 2359

SEQ ID NO:8 sets out the nucleotide sequence encoding for AAT4.

SEQ ID NO:9 sets out the amino acid sequence of AAT 5 obtained from Cyberlindnera jadinii DSM 2361

SEQ ID NO:10 sets out the nucleotide sequence encoding for AAT5.

SEQ ID NO:1 1 sets out the amino acid sequence of AAT 6 obtained from Cyberlindnera fabianii CBS 5640

SEQ ID NO:12 sets out the nucleotide sequence encoding for AAT6. SEQ ID NO:13 sets out the amino acid sequence of AAT7 obtained from Hanseniaspora uvarum CECT 11105

SEQ ID NO:14 sets out the nucleotide sequence encoding for AAT7. SEQ ID NO:15 sets out the amino acid sequence of AAT 8 obtained from Hanseniaspora uvarum CECT 11105

SEQ ID NO:16 sets out the nucleotide sequence encoding for AAT8.

SEQ ID NO:17 sets out the amino acid sequence of AAT 9 obtained from Eremothecum cymbalariae).

SEQ ID NO:18 sets out the nucleotide sequence encoding for AAT 9.

SEQ ID NO:19 sets out the amino acid sequence of AAT 10 obtained from Saccharomyces cerevisiae NCYC 2629

SEQ ID NO:20 sets out the nucleotide sequence encoding for AAT10.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present specification and the accompanying claims, the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows. The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

The inventors have found a novel group of polypeptides. The genes of this group when expressed in a suitable host, were able to produce alkyl alkanoates, and may also produce alkyl alkanoate esters, in a yield suitable for industrial scale. A preferred polypeptide to be used in the process according to the invention was encoded by a gene isolated from the genome of W. anomalus, hereinafter referred to as AAT 1 . The amino sequence of AAT 1 is given under SEQ ID No:1 , while the nucleotide sequence encoding for AAT 1 is given in SEQ ID NO: 2. When using BLASTP on the sequence of AAT 1 no hits were found with known AATs, indicating that AAT 1 shares no close identity with known AATs and is a newly found polypeptide for use in acetate ester production.

Upon investigating homologs of AAT 1 according to the invention it was found that these homologs contain a highly conserved nucleophilic elbow (GYSLG) at Ser 121 . Moreover, Asp 145, Asp 178, and His 295 were also highly conserved. It was found that homologs of AAT 1 had the same highly conserved serine, aspartic acid and histidine. Upon visualization with a three-dimensional model of the first AAT found (AAT 1 ), Ser 121 , Asp 145 and His 295 showed to be in the correct spatial proximity to form a catalytic Ser-Asp-His triad that occurs in alpha/beta hydrolases. Some homologs however did not have the aspartic acid at a

corresponding site. Since these homologs also showed the ability to produce ethyl acetate, it is thought that an active site of at least serine and histidine is present in the polypeptides according to the invention. Normally, an alpha/beta hydrolase fold is associated with esterases, proteases, lipases, peroxidases, epoxide hydrolases and dehalogenases, but the newly found polypeptides proved to have alcohol acetyl transferase activity. Thus, a novel group of polypeptides was found to be enzymes that have alcohol acetyl transferase activity and have in their three-dimensional structure an alpha-beta hydrolase fold and an active site comprising a serine, histidine dyad or a serine, aspartic acid, histidine triad. This triad and alpha/beta hydrolase fold is not present in the known aftl and aft2 enzymes of S. cerevisiae.

Upon further investigating the sequences of the polypeptides that proved to have ethyl acetate production activity (such as in AAT 1 to AAT 10, hereinafter referred to as Producers) and sequences of polypeptides that did not prove to have ethyl acetate production activity

(hereinafter referred to as Non-producers), it was found that in the producers, the histidine of the serine-aspartic acid-histidine triad or serine-glycine-histidine triad was present in a polypeptide fragment that has a glutamic acid-arginine- proline (ERP) fragment a glutamic acid-asparagine - proline (ENP) fragment or glutamic-acid- methionine- proline (EMP) fragment as the 5 ^th , 6 ^th and 7 ^th amino acid from the histidine on the C-terminal side of the polypeptide. The polypeptides that turned out to be a non-producer did not have such a ERP, ENP or EMP fragment.

It was found that polypeptides with AAT activity and the structural features as described above, had a good yield in alkyl alkanoate production when expressed in a proper host cell. Other preferred polypeptides to be used according to the invention were genes isolated from Kluyveromyces marxianus, Kluyveromyces lactis, Wickerhamomyces ciferrii, Cyberlindnera jadinii, Hanseniaspora uvarum, Eremothecium cymbalarie and Saccharomyces cerevisiae. These genes showed roughly 50% identity to AAT 1 , but some of them showed even less identity with AAT 1 . Upon expression in a host, these genes also have alkyl C3-10 alkanoate producing AAT activity, similar to AAT 1. These new enzymes are referred to as AAT 2 to AAT 10. The amino sequence of these AAT 2-10 are given under SEQ ID No:3, SEQ ID No:5, SEQ ID No:7, SEQ ID No:9, SEQ ID No:1 1 , SEQ ID No:13, SEQ ID No:15, SEQ ID No:17 and SEQ ID No: 19, respectively. The nucleotide sequences encoding for AAT 2-10, are given in SEQ ID NO: 4, SEQ ID No: 6, SEQ ID NO: 8, SEQ ID No: 10, SEQ ID NO: 12, SEQ ID No: 14, SEQ ID NO: 16, SEQ ID No: 18, and SEQ ID NO: 20, respectively.

Upon further investigating the sequences of the polypeptides that proved to have AAT activity (such as in AAT 1 to AAT 10, hereinafter referred to as Producers) and sequences of polypeptides that did not prove to have AAT activity (hereinafter referred to as Non-producers), it was found that in the producers, the histidine of the serine-aspartic acid-histidine triad or serine- histidine dyad was present in a polypeptide fragment that has a glutamic acid-arginine- proline (ERP) fragment or glutamic-acid- methionine- proline (EMP) fragment as the 5 ^th , 6 ^th and 7 ^th amino acid from the histidine on the C-terminal side of the polypeptide. The polypeptides that turned out to be a non-producer did not have such a ERP or EMP fragment.

The invention is further directed to the use of polypeptides in the process according to the invention which have:

- an amino acid sequence according to SEQ ID NO: 1 : or an amino acid sequence that has at least 70% identity with the sequence as shown in SEQ ID NO: 1 and/or,

- an amino acid sequence according to SEQ ID NO: 3: or an amino acid sequence that has at least a 70 % identity with SEQ ID NO: 3 and/or,

- an amino acid sequence according to SEQ ID NO: 5: or an amino acid sequence that has at least 70 % identity with SEQ ID NO: 5 and/or,

- an amino acid sequence according to SEQ ID NO: 7: or an amino acid sequence that has at least 70% identity with the sequence as shown in SEQ ID NO: 7 and/or,

- an amino acid sequence according to SEQ ID NO: 9: or an amino acid sequence that has at least a 70 % identity with SEQ ID NO: 9 and/or,

- an amino acid sequence according to SEQ ID NO: 1 1 : or an amino acid sequence that has at least 70 % identity with SEQ ID NO: 1 1 and/or,

- an amino acid sequence according to SEQ ID NO: 13: or an amino acid sequence that has at least 70% identity with the sequence as shown in SEQ ID NO: 13 and/or,

- an amino acid sequence according to SEQ ID NO: 15: or an amino acid sequence that has at least a 70 % identity with SEQ ID NO: 15 and/or,

- an amino acid sequence according to SEQ ID NO: 17: or an amino acid sequence that has at least 70 % identity with SEQ ID NO: 17,and/or,

- an amino acid sequence according to SEQ ID NO: 19: or an amino acid sequence that has at least 70% identity with the sequence as shown in SEQ ID NO: 19.

Optionally the polypeptides have the structural features as described above.

The terms "homology", "sequence identity" and the like are used interchangeably herein. For the purpose of this invention, it is defined herein that in order to determine the degree of sequence identity shared by two amino acid sequences or by two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). Such alignment may be carried out over the full lengths of the sequences being compared. Alternatively, the alignment may be carried out over a shorter comparison length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids.

The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The degree of identity shared between sequences is typically expressed in term of percentage identity between the two sequences and is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions (i.e. overlapping positions) x 100). Preferably, the two sequences being compared are of the same or substantially the same length.

The skilled person will be aware of the fact that several different computer programs are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percentage identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/qcq/), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

In one embodiment the polypeptide comprises a substantially homologous amino acid sequence that has of at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with any of the amino acid sequences shown in SEQ ID NO: 1 and/or SEQ ID NO:3, and/or SEQ ID NO 5, and/or SEQ ID NO: 7 and/or SEQ ID NO:9, and/or SEQ ID NO 1 1 , and/or SEQ ID NO: 13 and/or SEQ ID NO:15, and/or SEQ ID NO 17, and or SEQ ID NO 19.

The enzyme may be a polypeptide derived from amino acid SEQ ID NO: 1 to 19 by substitution, deletion or addition of one or several amino acid residues in any of the amino acid sequences of SEQ ID NO: 1 and/or SEQ ID NO:3, and/or SEQ ID NO 5, and/or SEQ ID NO: 7 and/or SEQ ID NO:9, and/or SEQ ID NO 1 1 , and/or SEQ ID NO: 13 and/or SEQ ID NO:15, and/or SEQ ID NO 17, and or SEQ ID NO 19 and having the same activity as any of the amino acid residue sequences of SEQ ID NO: 1 -19.

In one of the embodiments of the invention the nucleotide sequence encoding for the

polypeptide according to the invention comprises:

-a nucleotide sequence according to SEQ ID NO:2, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 2 and/or,

a nucleotide sequence according to SEQ ID NO:4, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 4 and/or,

a nucleotide sequence according to SEQ ID NO:6, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 6,

a nucleotide sequence according to SEQ ID NO:8, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 8 and/or,

a nucleotide sequence according to SEQ ID NO:10, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 10 and/or,

a nucleotide sequence according to SEQ ID NO:12, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 12 and/or,

a nucleotide sequence according to SEQ ID NO:14, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 14 and/or,

- a nucleotide sequence according to SEQ ID NO:16, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 16 and/or,

a nucleotide sequence according to SEQ ID NO:18, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 18, and/or,

a nucleotide sequence according to SEQ ID NO:20, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 20. In one embodiment the nucleic acid molecule comprises a nucleotide sequence encoding a protein, comprising a substantially homologous nucleotide sequence of at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the nucleotide sequences shown in nucleotide SEQ ID NO: 2 and/or SEQ ID NO: 4 and/or SEQ ID NO: 6, SEQ ID NO: 8 and/or SEQ ID NO: 10 and/or SEQ ID NO: 12, SEQ ID NO: 14 and/or SEQ ID NO: 16 and/or SEQ ID NO: 18, SEQ ID NO: 20.

The present invention provides a gene, which hybridizes selectively under stringent conditions to all or part of the DNA as shown in any of nucleotide SEQ ID NO: 2-20 to all or part of a DNA complementary to the sequence as shown in any of nucleotide SEQ ID NO: 2-20 and which encodes a protein having the activity of alcohol acetyl transferase.

As used herein, the term "selectively hybridizing", "hybridizes selectively" and similar terms are intended to describe conditions for hybridization and washing under which nucleotide sequences at least 66%, at least 70%, at least 75%, at least 80%, more preferably at least 85%, even more preferably at least 90%, preferably at least 95%, more preferably at least 98% or more preferably at least 99% homologous to each other typically remain hybridized to each other. That is to say, such hybridizing sequences may share at least 45%, at least 50%, at least 55%, at least 60%, at least 65, at least 70%, at least 75%, at least 80%, more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, more preferably at least 98% or more preferably at least 99% sequence identity.

A preferred, non-limiting example of such stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 ^q C, followed by one or more washes in 1 X SSC, 0.1 % SDS at about 50 ^< €, preferably at about 55 ^< €, preferably at about 60 °C and even more preferably at about 65 °C. Highly stringent conditions include, for example, hybridization at about 68 °C in 5x SSC/5x Denhardt's solution / 1 .0% SDS and washing in 0.2x SSC/0.1 % SDS at room temperature. Alternatively, washing may be performed at 42 °C.

The skilled artisan will know which conditions to apply for stringent and highly stringent hybridization conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).

Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double- stranded cDNA clone).

Homologous gene sequences can be isolated, for example, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of nucleotide sequences as taught herein.

The template for the reaction can be total chromosomal DNA from the strain known or suspected to express a polynucleotide according to the invention. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new oxidoreductase nucleic acid sequence, or a functional equivalent thereof. Alternatively, the template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from strains known or suspected to express a polynucleotide according to the invention. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new oxidoreductase nucleic acid sequence, or a functional equivalent thereof.

The PCR fragment can then be used to isolate a full-length cDNA clone by a variety of known methods. For example, the amplified fragment can be labelled and used to screen a

bacteriophage or cosmic cDNA library. Alternatively, the labelled fragment can be used to screen a genomic library.

PCR technology also can be used to isolate full-length cDNA sequences from other organisms. For example, RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source. A reverse transcription reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.

The resulting RNA/DNA hybrid can then be "tailed" (e.g., with guanines) using a standard terminal transferase reaction, the hybrid can be digested with RNase H, and second strand synthesis can then be primed (e.g., with a poly-C primer). Thus, cDNA sequences upstream of the amplified fragment can easily be isolated. For a review of useful cloning strategies, see e.g.,Sambrook et al., supra; and Ausubel et al., supra.

Another aspect of the invention pertains to vectors, including cloning and expression vectors, comprising a polynucleotide of the invention encoding alcohol acetyl transferase protein or a functional equivalent thereof and methods of growing, transforming or transfecting such vectors in a suitable host cell, for example under conditions in which expression of a polypeptide of the invention occurs. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

The term "recombinant" as used herein with reference to a nucleotide sequence present in a host organism, microorganism or cell refers to a nucleotide sequence which is not naturally present in said organisms, microorganism or cell. This includes nucleotide sequences which are foreign to said organisms, microorganism or cell and nucleotide sequences which are introduced at a position other than their natural position in the genome and endogenous gene sequences which have been modified.

The term "heterologous" is used herein with reference to a nucleotide sequence present in a host organism, microorganism or cell refers to a nucleotide sequence that has been derived from a different organism.

The term "homologous" is used herein with reference to a nucleotide sequence present in a host organism, microorganism or cell refers to a nucleotide sequence that has been derived from the same organism as the host organism.

Polynucleotides of the invention can be incorporated into a recombinant replicable vector, for example a cloning or expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below. The vector into which the expression cassette or polynucleotide of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of the vector will often depend on the host cell into which it is to be introduced.

A vector to be used in the process according to the invention may be an autonomously replicating vector, i.e. a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e. g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" can be used interchangeably herein as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as cosmid, viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) and phage vectors which serve equivalent functions.

Vectors to be used in the process according to the invention may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.

A vector to be used in the process of the invention may comprise two or more, for example three, four or five, polynucleotides of the invention, for example for overexpression.

The recombinant expression vectors used in the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a vector, such as an expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell), i.e. the term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence such as a promoter, enhancer or other expression regulation signal "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences or the sequences are arranged so that they function in concert for their intended purpose, for example transcription initiates at a promoter and proceeds through the DNA sequence encoding the polypeptide according to the invention.

The present invention also provides a vector or a plasmid containing the nucleotide sequence encoding for the polypeptide for use in the process according to the invention. Said plasmid can be introduced into proper host cells. Said host cell may contain a homologous, or a heterologous gene, which is capable of generating an amino acid sequence that has at least 70% identity with the sequence shown in any of the amino acid SEQ ID NO: 1 -19. In the case of a homologous gene, this gene may be incorporated into the host cell in such a way so as to increase the alkyl alkanoate yield compared to the alkyl alkanoate yield that can be obtained via the gene in its natural position in the genome and with endogenous gene sequences. These recombination techniques are known in the art and need no further elucidation here.

The present invention further provides proper host cells for use on the process according to the invention, which comprises a nucleotide sequence exhibiting at least 70% identity with that shown in any of the nucleotide SEQ ID NO: 2-20.

Suitable host organisms are selected from bacteria, yeasts, or filamentous fungi. Preferred bacteria are selected from Escherichia species and Bacillus species. A preferred Escherichia species is preferably E. coli and a preferred Bacillus species is Bacillus subtilis. Other suitable bacteria are the ones that are used in industrial production such as

Corynebacterium glutamicum.

Species that are especially preferred are the thermophilic species since they allow production at high temperatures. Examples thereof are Geobacillus thermoglucosidasius, Caldicellulosiruptor bescii, Clostridium thermocellum, Thermoanaerobacterium aotearoense,

Thermoanaerobacterium saccharolyticum, Thermoanaerobacter tengcongensis,

Thermoanaerobacter ethanolicus, Thermoanaerobacter mathrani, Thermococcus kodakarensis, Pyrococcus furiosus.

Preferred yeasts are the ones from which the polypeptides according to the invention originate such as Wickerhamomyces anomalus, Wickerhamomyces ciferrii, Kluyveromyces marxianus, Kluyveromyces lactis, Cyberlindnera jadinii, Hanseniaspora u varum, Eremothecium

cymbalariae, but also yeasts that are often used in the industry as production yeasts such as Saccharomyces cerevisiae species, Pichia species such as P. pastoris and

Schizosaccharomyces species. Preferred filamentous fungi are selected from Aspergillus species, Trichoderma species or Penicillium species; Preferred Aspergillus species are preferably A. niger, A. oryzae, and A. nidulans. Also Monascus ruber is suitable.

Appropriate culture media and conditions for the above-described host cells are known in the art.

The process of the present invention is directed to the use of host cells comprising the nucleotide sequence encoding for the polypeptide with of alcohol acetyl transferase activity according to this invention. In said process alcohol and acetyl coenzyme A or its higher carboxyl acid counterpart is converted in the presence of the polypeptide with AAT activity to form alkyl alkanoate. The acetyl coenzyme A or its higher carboxyl acid counterpart may be produced by the host cell. The alcohol can either be added to the reaction or the conditions are arranged such that the alcohol is also produced in the host cell. In particular, said use is described as follows: suitable host cells were transformed with said DNA by conventional methods in the field, and the recombinant enzyme produced by the recombinant cells after transformation convert alcohol and acetyl coenzyme A or its higher carboxyl acid counterpart to alkyl alkanoate in the host cell.

The invention is further directed to a process for the production of alkyl alkanoate wherein the polypeptide with AAT activity according to the invention converts alcohol and acyl coenzyme A into alkyl alkanoate. The polypeptide according to the invention may be produced in a host cell. The formed polypeptide may be used after isolation from the host cell, or the process may be a microbial process, taking place in the host cell. The process may also be arranged so that the alcohol needed to form alkyl alkanoate is formed by fermentation of sugar compounds either separately from the conversion to alkyl alkanoate or simultaneously. Preferably, the process is conducted as a full microbial process wherein both alcohol and acyl coenzyme A are formed within the host cell and converted into alkyl alkanoate. The process according to the invention may be conducted at a temperature of between 10 and 130 °C. This has clear advantages over any known chemical process for the production of ethyl acetate and higher alkyl alkanoates, where much higher temperatures, higher amounts of energy and increased pressures are needed. Thermophilic hosts allow the process to be conducted at relatively high temperature such as between 45 and 130 °C, preferably between 50 and 100 °C. It goes without saying that the polypeptide to be incorporated in a thermophilic host should be able to undergo the growth- and production temperatures used.

Preferred sugar compounds to be used as the growth substrates are selected from compounds comprising both hexose and pentose sugars. The sugar compound may be a hexose sugar such as glucose, fructose, galactose, glycan or other polymers of glucose, hexose oligomers such as sucrose, lactose, maltose, maltotriose and isomaltotriose, panose, and fructose oligomers. In particular embodiments, the micro-organism is modified to have the ability to ferment pentose sugars, and the medium includes a pentose sugar such as xylose, xylan or other oligomer of xylose. In particular embodiments, the organisms are cultivated on combinations of hexose and pentose sugars.

The sugars can be hydrolysates of a hemicellulose, an amylose or cellulose-containing biomass. In particular embodiments, the micro-organism is modified to ensure degradation of the biomass to monomers (e.g. expression of cellulase genes). Accordingly, in particular embodiments, the substrate comprises a sugar oligomer or polymer such as cellulose, hemicellulose or pectin. Additionally or alternatively, enzymes can be added to the fermentation medium to ensure degradation of the substrate into fermentable monomers.

With the AATs used in the process according to the invention yields were obtained of up to 35% on mole to mole glucose basis in shake flask tests. In these tests no process optimalization has taken place yet. With the AATs used in the process according to the invention yields may be reached of up to 60 % alkyl alkanoate on mole to mole glucose basis. In general, the

polypeptides according to the invention are able to have a yield ranging from 10 to 55% mole alkyl alkanoate per mole glucose. In most cases, a yield of 45 to 55 % mole of alkyl alkanoate per mole of glucose may be reached.

In further embodiments, the invention provides methods for producing a alkyl alkanoate which, in addition to the steps detailed above further comprise the step of recovering the C3-C10 alkyl alkanoate of interest. This comprises recovery of the C3-C10 alkyl alkanoate from the host cell mixture and optionally additional purification.

Suitable purification can be carried out by methods known to the person skilled in the art such as by using distillation, extraction, ion exchange resins, electrodialysis, nanofiltration, etc

EXAMPLES

Strains and plasmids construction

A table of strains is available in Table I. pYES2 derived plasmids were constructed by inserting genes into the multiple cloning site, either by using appropriate restriction enzymes, or by in vivo yeast assembly (Saccharomyces cerevisiae. Bio-protocol 5, (2015) pCUP1 derived plasmids were constructed by replacing the GAL1 promoter of pYES2 with the S. cerevisiae NCYC 2629 CUP1 promoter and inserting a gene of interest with in vivo yeast recombination, using S.

cerevisiae W '303 or CEN.PK2-1 D. S. cerevisiae transformations were performed according to the protocol of Gietz and Woods {Methods Enzymol. 350, 87-96 (2002). The pYES2 and pCUP1 derived plasmids were characterized in S. cerevisiae INVSd and CEN.PK2-1 D, respectively. pET26b:harm AAT 1 -His was constructed by cloning the E. coli codon harmonized AAT 1 gene with Ndel and Xhol in accordance with the method described in PLoS One 3, e2189 (2008). All plasmids were propagated in E. coli NEB® 5-alpha and are listed in Table II. Site directed mutagenesis in pCUP1 :AAT 1 was performed with Quickchange ® (Agilent).

TABLE I Strains used in the Experiments

Host Escherichia, coli BL21 (DE3) fhuA2 [Ion] ompT gal (λ DE3) [dcm]

AhsdS

λ DE3 = λ sBamHIo AEcoRI-B int::(lacl::PlacUV5::T7gene1 ) i21 Δηίηδ

Cloning strain for Escherichia coli NEB® 5-alpha fhuA2 A(argF-lacZ)U169 phoA glnV44 E.coli plasm id Φ80 A(lacZ)M15 gyrA96 recA 1 relA 1 endA 1 thi-1 hsdRU

TABLE II PLASMIDS AND HOSTS CONTAINING PLASMIDS

pYES2:AAT 1 S. cerevisiae, GAL1 W. anomalus DSM 6766

(WANOMALA_5543)

pCUP1:AAT 1 S. cerevisiae, CUP1 W. anomalus DSM 6766

WANOMALA_5543

(WANOMALA_5543)

pCUP1:AAT 1 Ser121Ala S. cerevisiae, CUP1 W. anomalus DSM 6766

(WANOMALA_5543), serine 121 substituted for alanine

pCUP1:AAT 1 Asp145Ala S. cerevisiae, CUP1 W. anomalus DSM 6766

(WANOMALA_5543), aspartate 145 substituted for alanine

pCUP1:AAT 1 His295Ala S. cerevisiae, CUP1 W. anomalus DSM 6766

(WANOMALA_5543), histidine 295 substituted for alanine

pCUP1:AAT 11 ( Non- S. cerevisiae, CUP1 W. anomalus DSM 6766

producer) (WANOMALA_5545)

pCUP1:AAT2 S. cerevisiae, CUP1 W. cil 'err ii CBS 111

(XP_011273049.1)

pCUP1:AAT 12 ( Non- S. cerevisiae, CUP1 K. marxianus DSM 5422

producer)

pCUP1:AAT3 S. cerevisiae, CUP1 K. marxianus DSM 5422 () pCUP1:AAT4 S. cerevisiae, CUP1 K. lactis CBS 2359 of (XP_455051.1) pCUP1:AAT 13 ( Non- S. cerevisiae, CUP1 C. jadinii DSM 2361 (CEP25159.1) producer)

pCUP1:AAT5 S. cerevisiae, CUP1 C. jadinii DSM 2361 (CEP25158.1) pCUP1:AAT 14 ( Non- S. cerevisiae, CUP1 C. fabianii CBS 5640 (CDR40570.1) producer)

pCUP1:AAT6 S. cerevisiae, CUP1 C. fabianii CBS 5640 (CDR40574.1) pCUP1:AAT7 S. cerevisiae, CUP1 H. uvarum CECT 11105

(D499_0A01740)

pCUP1:AAT8 S. cerevisiae, CUP1 H. uvarum CECT 11105

(D499_0F00170) pCUP1 :AAT 9 S. cerevisiae, CUP1 E. cymbalarie CBS 270.75 homolog of

WANOMALA_5543 (Ecym_7076) pCUP1 :AAT 10 S. cerevisiae, CUP1 S. cerevisiae NCYC 2629 YGR015C

(YGR015C)

pCUP1 :AAT 15 ( Non- S. cerevisiae, CUP1 S. cerevisiae NCYC 2629 IM032 producer) (YGR015C)

pET26b ( ex Novagen™) E. coli, T7 /

pET26b:harm AAT 1 -His E. coli, T7 Codon harmonised

WANOMALA_5543

Cultivation conditions

Wild type yeast strains were routinely cultured and propagated in YPD medium (20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract). Uracil auxotrophic yeast strains were routinely cultured and propagated in YS (yeast synthetic) medium (6.7 g/L Yeast nitrogen base without amino acids, 1.92 g/L Medium Supplements without uracil) with 20 g/L glucose E. coli strains were routinely cultured and propagated in LB or M9 medium supplemented with 50 μg/mL ampicillin or kanamycin. Yeast and E. coli strains were grown at 30 °C, the plasmids were cloned at 37 °C, respectively, unless stated otherwise. S. cerevisiae INVSd strains carrying pYES2 derived plasmids were characterized in 250 mL Erlenmeyers containing 50 mL YS medium with 20 g/L galactose and 10 g/L raffinose. Erlenmeyers were inoculated from an overnight culture and cultivated 48-72 hours at 250 rpm. K. lactis CBS 2359 Aku80, K. lactis CBS 2359

Aku80Akla5543, C. fabianii CBS 5640 and W. ciferrii CBS 1 1 1 were tested in 1 L Erlenmeyers containing 250 mL YM (yeast minimal) medium (Can. J. Microbiol. 24, 440-447 (1978). Iron was omitted from the medium. 1 mL 1000X vitamins mix (0.1 g/L thiamine, riboflavin, panthothenic acid, D-biotin, folic acid, p-aminobenzoic acid, cobalamin and 0.5 g/L nicotinic acid,

pyridoxamine-HCI, pyridoxal-HCI) was added to the medium. Flasks were inoculated with 1 mL overnight YPD cultures and shaken at 150 rpm.

Batch bioreactors

Batch S. cerevisiae and E. coli fermentations were performed in 1.4 L DasGip ® bioreactors (ex Eppendorf) in 0.5 L defined medium ( Yeast 8, 501-517 (1992). 1 mM CuSC was added to the medium in S. cerevisiae fermentations and 10 g/L NaCI and 15 g/L ethanol in E. coli fermentations. pH was kept at 7 by automatic addition of 3M KOH. S. cerevisiae fermentations were not sparged and were stirred at 600 rpm. E. coli fermentations were sparged with 2 L/h air and were kept aerobic (DO>25%) by gradually increasing the stirring speed from 200 to 800 rpm and increasing the fraction of 0 ₂ in the sparging gas. All batch fermentations were operated at 30 °C. The S. cerevisiae and £. co// ^' pre-cultures were made by inoculating 50 mL YS medium with 20 g/L glucose or 50 mL M9 medium, respectively in a 250 mL Erlenmeyer flask to an initial OD of 0.5. The cultures were grown at 250 rpm and 30°C until an OD of 3. The cells were centrifuged at 4700 rpm for 5 minutes, resuspended in 50 mL sterile water and transferred to the fermenter. E. coli cultures were induced with 0.2 mM IPTG after 4 hours of growth.

Continuous bioreactors

Continuous culturing of W. anomalus was performed in a 3 L Infors ® fermenter with a working volume of 2 L. pH was controlled at 5 by addition of 8M KOH. The fermenter was stirred at 800 rpm and the temperature controlled at 30 °C. The culture was kept glucose limited by adding medium that contained 10 g/L glucose, 5 g/L (NH^SO^ 2.5 g/L KH2PO4, trace elements without iron ²¹ , 50 μΜ FeCI ₃ , 0.4 mM CaCI ₂ , 2 mM MgS0 ₄ , 15 mg/L ethylenediaminetetraacetic acid and 0.15 mL/L Antifoam 204 (ex Sigma). The pH of the medium vessel was lowered to 2.0 by adding 37% HCI. The dilution rate was 0.1 h ^_1 . Ethyl acetate production was controlled by aeration. Producing (aerobic) and non-producing (oxygen limited) conditions were achieved by sparging the fermenter with 3 L/h air or a 0.3 L/h air, 2.7 L/h N ₂ mixture, respectively. Steady state was achieved after 50 h. 50 mL medium was withdrawn, centrifuged and the pellet frozen at -80 °C.

RNA isolation and RNAseq analysis

The pellets from the continous cultures were kept on ice as much as possible during the protocol. The pellets were resuspended in 0.5 mL cold TE buffer (10 mM Tris-HCI, pH 8.0, 1 mM EDTA) and divided into two 2 mL screw capped tubes containing 0.5 g zirconium beads, 30 μί 10 %SDS, 30 μί 3 M sodium acetate (pH 5.2) and 500 μί Roti-® Phenol (pH 4.5-5.0, Roth). The cells were disrupted with a FastPrep apparatus (MP biomedials) at speed 6 for 40 seconds and centrifuged at 4 °C and 14.000 rpm for 5 minutes. 400 μί of the aqueous phase from each tube was combined. 400 μί of chloroform-isoamyl alcohol (ex Roth) was added and centrifuged at 4 °C at 14000 rpm for 6 minutes. 300 μί of the aqueous phase was mixed with 300 μί of the lysis buffer from the High Pure RNA Isolation kit ® ( ex Roche). All subsequent steps were performed according to the kit instructions, except the DNAse treatment which was performed for 45 minutes. The RNAseq analysis was performed by BaseClear and the reads mapped to the annotated genome of 1/1/. anomalus DSM 6766, See FEMS Yeast Res. 12, 382-386 (2012).

Bioinformatics

Homologs of AAT 1 were identified by performing standard protein BLAST searches against the non-redundant protein sequences database. Phylogenetic analysis was performed by making a structural alignment of wan5543 and close homologues using the Tcoffee (Espresso) server (http://tcoffee.crg. cat/apps/tcoffee/do:expresso) [1]. A three dimensional structure of Wan5543 was modelled using the PHYRE2 Protein Fold Recognition Server

(http://www.sbg.bio.ic.ac.uk/phyre2) on intensive mode [2]. Six templates (pdb: 2y6v, 4d9j, 1 cr6, 3i1 i, 3i28, & 2vav) were selected based on heuristics and 52 residues were modelled by ab initio. In the final model 87% of wan5543 was modelled at >90% confidence. The structure was analysed and visualized using PyMol [3].

1. Expresso: automatic incorporation of structural information in multiple sequence alignments using 3D-Coffee. Armougom F, Moretti S, Poirot O, Audic S, Dumas P, Schaeli B, Keduas V,

Notredame C. Nucleic Acids Res. 2006

2. Kelley LA et al. Nature Protocols 10, 845-858 (2015).

3. The PyMOL Molecular Graphics System, Version 1.8 Schrodinger, LLC.

AAT 1 purification

AAT 1 was purified from E. coli BL21 (DE3) pET26b:harmAAT 1 -His. Three 1 L Erienmeyers with 250 mL M9 medium were inoculated with 2 mL overnight preculture and cultivated at 37 °C and 200 rpm. After 4 hours the cultures were chilled on ice for 15 minutes and induced with 0.2 mM IPTG. The cultures were then incubated at 20 °C and 200 rpm. After 18 hours, the cells were combined, harvested by centrifugation at 5000 rpm for 5 minutes, washed with 50 mL 50 mM phosphate buffer (KPi, pH 7.5) and stored at -20 °C. To extract the protein, the cells were resuspended in 20 mL buffer HA (50 mM KPi, 300 mM NaCI, pH 8) and passed twice through a chilled French Press Cell (Thermo Scientific) at 20000 psi. 25 mg DNAsel was added to the lysate, which was centrifuged at 4 °C and 18000 rpm for 20 minutes. The supernatant was filtered through 0.45 μιτι filter and was used for protein purification. An AKTA Purifier system was used to purify AAT 1 . The cell free extract was loaded on a 1 ml_ HisTrap HP column (GE Healthcare Life Sciences) that was equilibrated with buffer HisA. The protein was eluted with a gradient of buffer HB (50 mM KPi, 300 mM NaCI, 500 mM imidazole, pH 8). The fractions containing the protein were desalted over three connected 5 ml_ HiTrap Desalting columns (GE Healthcare Life Sciences), equilibrated with buffer CA (50 mM KPi, pH 7). The desalted protein fractions were loaded on a 1 mL HiTrap HP SP column (GE Healthcare Life Sciences). AAT 1 was eluted with a gradient of buffer CB (50 mM KPi, pH 7, 1 M NaCI). The fractions with the highest content of protein were combined and used for further analyses. Analytical

Glucose and organic acids were analysed by HPLC using an ICS5000 HPLC system (Thermo Scientific) equipped with a Dionex DP pump, Dionex AS-AP autosampler, Dionex VWD UV detector operated at 210 nm and Shodex Rl detector operated at 35 °C. An Aminex HPX-87H cation-exchange column was used with a mobile phase of 0.16 N H ₂ S0 ₄ and was operated at 0.8 mL/min and 60 °C. 10 mM dimethylsulfoxide in 0.04 N H ₂ S0 ₄ was used as internal standard. Volatile compounds were analysed on a Shimadzu 2010 gas chromatograph equipped with a 20i-s autosampler. 0.5 μί of liquid sample was injected on a Stabilwax column (30 m x 0.25 mm, 0.5 μηη coating, Restek). The column temperature was held the temperature at 60 °C for 1 minute and increased to 120^ at a rate of 20 ^q C/minute. The split ratio was 20. 10 mM 1 -butanol was used as internal standard.

Enzyme assays

All assays were performed in 50 mM KPi, pH 7.5 with 150 mM NaCI and 30 °C, using 0.164 mg purified protein/mL reaction. AAT activity was assayed at 1 mM acetyl-coenzymeA and varying concentrations of ethanol (1 -80 mM). The assay was measured in liquid reactions by directly measuring ethyl acetate on a Shimadzu GC 2010 equipped with a temperature controlled 20i+s autosampler. 0.5 μί of liquid sample was injected on a Stabilwax column (30 m x 0.25 mm, 0.5 μηη coating, Restek). The column temperature was held the temperature at 60 °C for 1 minute and increased to 85 at a rate of 20 ^q C/minute. The split ratio was 5.

Example 1 Identification of AATs in W. Anomalus

The sequenced and annotated genome of ethyl acetate producing Wickerhamomyces anomalus DSM 6766 contains five putative Atf1 or Atf2 homologs and one Eht1 homolog. To see if they are involved in ethyl acetate production they were expressed in S. cerevisiae INVSc (See Table I). The transformants 0.005 g/L ethyl acetate at most. However, they did produce 3 - 15 fold more isoamyl acetate from the endogenously produced isoamyl alcohol. Overexpression of the S. cerevisiae atfl gave similar results, but S. cerevisiae INVSd expressing atf2 and ehtl showed poor growth and did not produce esters.

The poor ethyl acetate production of the known AAT homologs suggests that other enzymes are responsible for most of ethyl acetate synthesis in W. anomalus. To search for such enzymes, we compared the transcriptome of W. anomalus DSM 6766 under ethyl acetate producing and non- producing conditions in glucose-limited continuous cultures. Ethyl acetate was not produced under fully aerobic conditions, but oxygen limitation induced formation of fermentation products, including 0.64 ± 0.10 g/L ethyl acetate. 168 genes were more than 4-fold upregulated under ethyl acetate producing conditions. Many of the changes are due to the metabolic shift from an aerobic to fermentative metabolism. These genes were not further considered. The known homologs of Atf1 , Atf2 and Eht1 did not show a significant change in expression levels. The overexpressed genes that were not upregulated due to the metabolic shift from an aerobic to fermentative metabolism during ethyl acetate production (AAT 1 and WANOMALA 7754 from the same strain, respectively) code for two hypothetical proteins with an alpha/beta hydrolase fold. This fold has been observed in esterases, as well as some AATs, see J. Biol. Chem. 281 , 4446-4456 (2006). Both types of enzymes are involved in ester metabolism in yeast, making AAT 1 and WANOMALA 7754 potential candidates for explaining ethyl acetate formation in W. anomalus. Their protein products are 99% identical and only AAT 1 was studied further.

The enzyme was expressed in S. cerevisiae INVSd pYES2-AAT 1 , which produced 0.13 ± 0.01 g/L ethyl acetate. This is 26- fold higher than the best W. anomalus Atf1 , Atf2 or Eht1 homolog. S. cerevisiae INVSd was grown on galactose, which also served as the inducer for gene expression.

Example 2 Expression of AAT 1 in E.coli Codon harmonized AAT 1 , according to PLoS One 3, e2189 (2008), was expressed in E. coli BL21 (DE3) pET26b:harmAAT 1 -His. This led to the peak production of 3.5 ± 0.12 g/L ethyl acetate from 20 g/L glucose and 15 g/L ethanol. £. coli produced 29-fold more ethyl acetate than S. cerevisiae (26 % of the theoretical pathway maximum).

Example 3 Biochemical characterization of AAT 1

Automatic annotation predicted an α/β hydrolase fold in AAT1. Based on this we assumed that AAT 1 makes ethyl acetate either as an AAT or as a reversed esterase, and excluded HADH. AAT 1 was purified from £. coli BL21 (DE3) pET26b:harm AAT 1 -His by Ni/NTA affinity chromatography, followed by cation exchange. The Purified AAT1 protein fraction was subjected to a GC assay for AAT activity. To this end both ethanol and acetyl coenzyme A were contacted with AAT1 . When omitting either ethanol and/or acetyl coenzyme A no ethyl acetate was formed. This confirms that AAT 1 produces ethyl acetate as an AAT, and not as a reversed esterase.

Example 4A: Activity measurements with ethanol

Several activity measurements were done using AAT 1 using the AAT activity assays described above. Increasing ethanol concentrations in the AAT assay led to a higher final concentration of ethyl acetate. Increasing the acetyl-coenzyme A concentration up to 4 mM resulted in faster AAT reaction rates. The results of the assay show that both ethanol and acetyl-coenzyme A concentrations contribute significantly to the efficiency of the AAT reaction.

Example 4B: Activity measurement with other alcohols

Codon harmonized AAT 1 , according to PLoS One 3, e2189 (2008), expressed in £. coli BL21 (DE3) pET26b:harmAAT 1 -His. To this end Aerobic shake flasks were inoculated with 500 μ\- of an overnight pre-culture of £. coli BL21 (DE3) (pET26b:harmAAt1 -His) in 50 mL M9 medium containing 100mM methanol, ethanol, 1 -propanol or 1 -butanol. Strains were cultivated in 250 mL non-baffled Erlenmeyer flasks. Flasks were incubated at 30 ^S C at 180 rpm. The expression of AAT1 was induced with 0.2 mM IPTG after 4 hours of growth. Ester production was determined after 30 hours of incubation. Figure 1 gives the resulting yields in methyl acetate, ethyl acetate, propyl acetaat and buthyl actetate. Example 5. Homologues of AAT 1 and their structural features

To identify the catalytic residues, we used BIASTP to find homologs of AAT 1 and used them to compile a multiple sequence alignment of homologs from other yeasts. A highly conserved nucleophilic elbow (GYSLG) was present at Ser 121 . Moreover, Asp 145, Asp 178, and His 295 were also highly conserved. The catalytic site was then visualized with a 3D model of AAT 1 . Ser 121 , Asp 145 and His 295 are in the correct spatial proximity to form a catalytic Ser-Asp-His triad that occurs in alpha/beta hydrolases. To confirm the function, we substituted the three residues individually by alanine and expressed the proteins in S. cerevisiae CEN.PK2-1 D. None of the AAT 1 variants formed ethyl acetate, confirming the Ser-Asp-His catalytic triad.

Example 6 Activity of homologues of AAT 1

The AAT 1 homologs used to compile the multiple sequence alignment originate from other ethyl acetate producing yeast species. When the homologs were expressed in S. cerevisiae

CEN.PK2-1 D several of the transformants produced ethyl acetate upon expression in a host. The growth and product production profiles of the transformants resembled that of CEN.PK2-1 D pCUP1 :AAT 1 , but the final titer of ethyl acetate varied per homolog. The amino acid sequences and the nucleotide sequences of the homologs that proved to have ethyl actetate production activity are compiled in sequence listings. At least one ethyl acetate producing homolog of AAT 1 was present in each ethyl acetate producing yeast. The homologs found which proved to have ethyl acetate producing activity were genes isolated from Kluyveromyces marxianus,

Kluyveromyces lactis, Wickerhamomyces ciferrii, Cyberlindnera jadinii Hanseniaspora uvarum Eremothecium cymbalarie and S. cerevisiae. These genes showed roughly 50% identity to AAT 1 . These new enzymes are referred to as AAT 2 to AAT 10. The amino sequence of these AAT 2-10 are given under SEQ ID No:3, SEQ ID No:5, SEQ ID No:7, SEQ ID No:9, SEQ ID No:1 1 , SEQ ID No:13, SEQ ID No:15, SEQ ID No:17 and SEQ ID No: 19, respectively. The nucleotide sequences encoding for AAT 2-10, are given in SEQ ID NO: 4, SEQ ID No: 6, SEQ ID NO: 8, SEQ ID No: 10, SEQ ID NO: 12, SEQ ID No: 14, SEQ ID NO: 16, SEQ ID No: 18, and SEQ ID NO: 20, respectively. Upon further investigating the sequences of the polypeptides that proved to have ethyl acetate production activity (such as in AAT 1 to AAT 10, hereinafter referred to as Producers) and sequences of polypeptides that did not prove to have ethyl acetate production activity

(hereinafter referred to as Non-producers), it was found that in the producers, the histidine of the serine-aspartic acid-histidine triad or serine-glycine-histidine triad was present in a polypeptide fragment that has a glutamic acid-arginine- proline (ERP) fragment a glutamic acid-asparagine - proline (ENP) fragment or glutamic-acid- methionine- proline (EMP) fragment as the 5 ^th , 6 ^th and 7 ^th amino acid from the histidine on the C-terminal side of the polypeptide. The polypeptides that turned out to be a non-producer did not have such a ERP, ENP or EMP fragment. See TABLE III

TABLE III Fragment containing histidine of catalytic site

Example 7 shake flask tests of AAT 2 and AAT 6

AAT 1 homologs were also present in Cyberlindnera fabianii CBS 5640 and Wickerhamomyces ciferrii CBS 1 1 1 (AAT 6 and AAT 2, respectively). These strains have, to our knowledge, not been studied for ethyl acetate production before. Therefore, their potential for ethyl acetate production was tested in shake flasks in YM medium. C. fabianii and W. ciferrii produced 4.1 and 5.7 g/L ethyl acetate from 50 g/L glucose, respectively.

Example 8 Importance of AAT 4 and AAT 10 for ethyl acetate production

AAT 4 of K. lactis CBS2359 Aku80 prepared according to Yeast 21 , 781-792 (2004) was knocked-out to investigate its importance to ethyl actetate production. It resulted in a 91 % decrease in ethyl acetate production.

Similar, upon disruption of AAT 10 from S. cerevisiae IMX585, the ethyl acetate production was reduced approximately 50% compared to the parental strain IMX585