PROCESS FOR PRODUCING ITACONIC ACID AND ITACONIC ACID ESTERS

UNDER ANAEROBIC CONDITIONS

Field of the invention

The present invention relates to a method for the production of itaconic acid and/or itaconate methylester by fermentation. The invention further relates to a fermentation broth comprising itaconic acid and/or itaconate methylester obtainable by such a process.

Background to the invention

Itaconic acid, an essential precursor to various products (e.g., acrylic fibers, rubbers, artificial diamonds, and lens), is in high demand in the chemical industry. Conventionally, itaconic acid is isolated from the filamentous fungus Aspergillus terreus. In addition, itaconic acid esters may be key intermediates for both commodity and specialty chemicals. The itaconic acid mono-methyl esters, i.e. 4-methyl itaconate and 1 -methyl itaconate are particularly interesting in this respect.

Recently, Aspergillus niger has been genetically modified to produce itaconic acid (WO2009014437, WO2009104958) by overexpressing c/ ^' s-aconitate decarboxylase (CAD) and/or a putative itaconic acid transporter. However, Aspergilli are less suitable for industrial production of itaconic acid due to its filamentous morphology, leading to oxygen transfer problems in large scale bioreactors.

E. coli has also been genetically modified to produce itacionic acid (US2010285546) by overexpressing CAD in combination with reduced isocitrate dehydrogenase (ICD) activity. This approach is problematic, however, since E. coli, and prokaryotes in general, are not tolerant to low pH. In a high pH fermentation (e.g. about pH7 which is optimal for E. coli), titration is needed to keep pH constant and this leads to the formation of itaconic salts instead of the acid. This in turn leads to increased DSP costs since recovery of the acid from the salt is more complex, as compared with a low pH fermentation process, where the acid can be directly recovered from the fermentation broth by crystallization.

More recently, a non-filamentous yeast, Yarrowia lipolytica, has been genetically modified to produce itaconic acid on glycerol (US201 10053232). However, the modified Y. lipolytica does not produce significant amounts of itaconic acid on sugar, one of the most commonly available renewable feedstocks.

Accordingly, there is a need to further improve itaconic acid production processes based on fermentation from sugar at low pH so that economically viable, large scale production may be achieved in industrial bioreactors.

Summary of the invention

The present invention is based on the unexpected identification of recombinant cells, i.e. a genetically modified cells, that may produce itaconic acid and/or an ester of itaconic acid under anaerobic conditions. These cells may be yeast cells. The advantage of using yeast cells is that it they are tolerant to low pH and are not filamentous, which allows for the optimal process conditions to produce itaconic acid and/or itaconic acid methyl ester. Advantages of anaerobic processes are in general, higher productivities and yields, while the energy requirements for mixing and cooling are lower than for aerobic processes.

According to the invention, there is thus provided a method for the production of itaconic acid or an ester of itaconic acid, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced.

In particular, the invention provides:

a method for the production of itaconic acid or an ester of itaconic acid, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced and wherein the recombinant cell comprises, for example overexpresses polypeptides catalysing the following reactions:

transportation of cytosolic itaconate to extracellular itaconic acid;

conversion of cytosolic cis-aconitate to itaconate; conversion of cytosolic citrate to cis-aconitate;

conversion of cytosolic oxaloacetate and acetyl-coenzyme-A to citrate; conversion of cytosolic acetaldehyde, NAD, and coenzyme-A to acetyl- coenzyme-A and NADH; and

conversion of cytosolic pyruvate and bicarbonate to oxaloacetate;

a method for the production of itaconic acid or an ester of itaconic acid, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced and wherein the recombinant cell comprises, for example overexpresses polypeptides catalysing the following reactions:

transportation of cytosolic itaconate to extracellular itaconic acid;

conversion of cytosolic cis-aconitate to itaconate;

conversion of cytosolic citrate to cis-aconitate;

transportation of mitochondrial citrate to the cytosol;

transportation of cytosolic oxaloacetate to the mitochondria; and conversion of cytosolic pyruvate and bicarbonate to oxaloacetate.

a method for the production of itaconic acid or an ester of itaconic acid, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced and wherein the recombinant cell comprises, for example overexpresses polypeptides catalysing the following reactions:

transportation of cytosolic itaconate to extracellular itaconic acid;

conversion of cytosolic cis-aconitate to itaconate;

conversion of cytosolic citrate to cis-aconitate;

conversion of cytosolic oxaloacetate and acetyl-coenzyme-A to citrate; conversion of cytosolic acetyl-phosphate to acetyl-coenzyme-A;

conversion of xylulose-5-phosphate and phosphate to acetyl-phosphate and glyceraldehyde 3-phosphate; and

conversion of cytosolic pyruvate and bicarbonate to oxaloacetate; and a method for the production of itaconic acid or an ester of itaconic acid, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced and wherein the recombinant cell comprises, for example overexpresses polypeptides catalysing the following reactions:

transportation of cytosolic itaconate to extracellular itaconic acid;

conversion of cytosolic cis-aconitate to itaconate;

conversion of cytosolic citrate to cis-aconitate;

conversion of cytosolic oxaloacetate and acetyl-coenzyme-A to citrate; conversion of cytosolic acetyl-phosphate to acetyl-coenzyme-A;

conversion of cytosolic acetate and ATP to acetyl-phosphate, ADP, and phosphate; and

conversion of cytosolic pyruvate and bicarbonate to oxaloacetate.

The invention further provides a fermentation broth comprising a itaconic acid and/or an ester of itaconate obtainable by a process of the invention.

Brief description of the drawings

Figure 1 a-d sets out metabolic pathways allowing the production of itaconic acid. Numbered reactions shows enzymes which may be overexpressed as follows. Reaction (1 ): pyruvate carboxylase. Conversion of cytosolic pyruvate and bicarbonate to oxaloacetate. Reaction (2): mitochondrial oxaloacetate transporter. Transportation of cytosolic oxaloacetate to mitochondrial oxaloacetate. Reaction (3): mitochondrial membrane citrate transporter. Transportation of mitochondrial citrate to cytosolic citrate and vice versa. Reaction (4): Aconitase. Conversion of citrate to aconitate. Reaction (5): cis-aconitate decarboxylase. Conversion of cis-aconitate to itaconate. Reaction (6): Itaconic acid transporter, transportation of cytosolic itaconate to extracellular itaconic acid. Reaction (7): citrate synthase, conversion of cytosolic oxaloacetate and acetyl coenzyme-A to citrate. Reaction (8): acetylating acetaldehyde dehydrogenase, conversion of cytosolic acetaldehyde, NAD, and coenzyme-A to acetyl-coenzyme-A and NADH. Reaction (9): Phosphoketolase. Conversion of xylulose 5-phosphate to acetyl phosphate, glyceraldehyde 3-phosphate, and water; or conversion of fructose 6- phosphate to acetyl phosphate, erythrose 4-phosphate, and water. Reaction (10): phosphate acetyltransferase. Conversion of coenzyme-A and acetyl phosphate to acetyl coenzyme-A and phosphate. Reaction (1 1 ): ATP:acetate phosphotransferase. Conversion of acetate and ATP to acetyl phosphate and ADP. The reactions highlighted by thicker arrow are the reactions expected to be relevant for conversion from glucose to itaonic acid and/or itaconate.

Figure 2 sets out metabolic pathways allowing the production of esters of itaconic acid.

Description of the sequence listing

A description of the sequences is set out in Table 4, 5 and 6. Sequences described herein may be defined with reference to the sequence listing or with reference to the database accession numbers also set out in Table 4, 5 and 6.

Detailed description of the invention

Throughout the present specification and the accompanying claims, the words "comprise", "include" and "having" 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.

In Aspergillus terreus, itaconic acid is synthesized from cis-aconitate, which is an intermediate of the tricarboxylic acid cycle. The enzyme responsible for converting cis- aconitate to itaconic acid is cis-aconitate decarboxylase. We have shown that this enzyme may be overexpressed in recombinant cells so that cells which do not typically produce itaconic acid may do so. Overexpression of one or more enzymes catalysing reactions to acetyl-CoA can further improve the amount of itaconic acid product. Also, such recombinant cells may produce an ester of itaconic acid by overexpressing one or more enzymes leading to the production of such an ester. We have further shown that such cells have a higher specific productivity of itaconic acid and/or an ester thereof when cultivated under anaerobic conditions.

According to the invention, there is thus provided a method for the production of itaconic acid or an ester of itaconic acid, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced.

Overexpression in the context of this invention indicates that a given nucleic acid sequence and/or amino acid sequence is expressed to a greater degree in a recombinant cell of the invention than a reference cell, which may typically be a corresponding wild type cell (i.e. a wild type cell of the same species). A nucleic acid and/or polypeptide may be overexpressed in the sense that a nucleic acid and/or polypeptide expressed in the reference cell is expressed to a greater degree in a recombinant cell of the invention (the reference cell may not express the nucleic acid and/or polypeptide at all). Overexpression may occur, for example, via overexpression of a nucleic acid and/or polypeptide which is endogenous (or homologous) to the reference cell. Overexpression may occurs, for example, via overexpression of a nucleic acid and/or polypeptide which is exogenous (or heterologous) to the reference cell. That is to say, overexpression may occur, for example, via overexpression of a nucleic acid and/or polypeptide which is natively occurs in the reference cell. Overexpression may occur, for example, via overexpression of a nucleic acid and/or polypeptide which is not present or not expressed at all in the reference cell.

A recombinant cell for use in a method of the invention may overexpress at least one an exogenous nucleic acid and/or polypeptide and overexpress at least one endogenous nucleic acid and/or polypeptide.

References herein to carboxylic acids or carboxylates, e.g. itaconic acid/itaconate, should be understood to include the protonated carboxylic acid (free acid), the corresponding carboxylate (its conjugated base) as well as a salt thereof, unless specified otherwise.

According to this invention, there is thus provided a method for the production of itaconic acid or an ester of itaconic acid, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced.

The recombinant cell may be a recombinant yeast comprising one or more nucleotide sequence(s) encoding (or, optionally, overexpressing):

a polypeptide having cis-aconitate decarboxylase activity; and

a genetic modification leading to an increase in flux towards acetyl-CoA. According to this invention, elevated levels of itaconic acid and itaconate methyl ester production are achieved by increasing combinations of various metabolic reactions rates for the production of one or more of the precursors, including, cis- aconitate, citrate, oxaloacetate, acetyl-Coenzyme-A, and acetyl-phosphate and fermenting the resulting cells under anaerobic conditions. That is to say, nucleic acid sequences encoding polypeptides carrying out such reactions may be overexpressed.

Accordingly, combinations of two or more of the following reactions may be organized into one or more metabolic pathways (the following numbering follows that set out in Fig 1 a-d):

Reaction (1 ): pyruvate carboxylase. Conversion of cytosolic pyruvate and bicarbonate to oxaloacetate.

Reaction (2): mitochondrial oxaloacetate transporter. Transportation of cytosolic oxaloacetate to mitochondrial oxaloacetate.

Reaction (3): mitochondrial membrane citrate transporter. Transportation of mitochondrial citrate to cytosolic citrate and vice versa.

Reaction (4): Aconitase. Conversion of citrate to aconitate.

Reaction (5): cis-aconitate decarboxylase. Conversion of cis-aconitate to itaconate.

Reaction (6): Itaconic acid transporter, transportation of cytosolic itaconate to extracellular itaconic acid.

Reaction (7): citrate synthase, conversion of cytosolic oxaloacetate and acetyl coenzyme-A to citrate.

Reaction (8): acetylating acetaldehyde dehydrogenase, conversion of cytosolic acetaldehyde, NAD, and coenzyme-A to acetyl-coenzyme-A and NADH.

Reaction (9): Phosphoketolase. Conversion of xylulose 5-phosphate to acetyl phosphate, glceraldehyde 3-phosphate, and water; or conversion of fructose 6- phosphate to acetyl phosphate, erythrose 4-phosphate, and water.

Reaction (10): phosphate acetyltransferase. Conversion of coenzyme-A and acetyl phosphate to acetyl coenzyme-A and phosphate. This enzyme may be referred to as acetyl-CoA:Pi acetyltransferase or acetyl-CoA: phosphate acetyltransferase.

Reaction (1 1 ): ATP:acetate phosphotransferase. Conversion of acetate and ATP to acetyl phosphate and ADP.

Preferred combinations are:

A. Reaction (1 ), (2), (3), (4), (5) and (6) - see Figl a. B. Reaction (1 ), (8), (7), (4), (5) and (6) - see Fig1 b.

C. Reaction (1 ), (9), (10), (7), (4), (5) and (6) - see Fig1 c.

D. Reaction (1 ), (1 1 ), (10), (7), (4), (5) and (6) - see Fig 1 d.

Any suitable sequence nucleic acid sequence encoding a polypeptide carrying out the stated reaction may be used in the invention. Examples include:

Reaction (1 ): SEQ ID NO: 25 or a sequence having at least 50% sequence identity thereto(or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (2): SEQ ID NO: 23 or a sequence having at least 50% sequence identity thereto(or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (3): SEQ ID NO: 21 or 47 or a sequence having at least 50% sequence identity to either of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (4): SEQ ID NO: 15, 17 or 19 or a sequence having at least 50% sequence identity to any of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (5): SEQ ID NO: 7, 9, 1 1 or 13 or a sequence having at least 50% sequence identity to any of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (6): SEQ ID NO: 1 , 3 or 5 or a sequence having at least 50% sequence identity to any of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (7): SEQ ID NO: 27, 29 or 31 or a sequence having at least 50% sequence identity to any of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (8): SEQ ID NO: 33 or a sequence having at least 50% sequence identity thereto (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto). Reaction (9): SEQ ID NO: 35 or 37 or a sequence having at least 50% sequence identity to either of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (10): SEQ ID NO: 41 , 43 or 45 or a sequence having at least 50% sequence identity to any of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (1 1 ): SEQ ID NO: 39 or a sequence having at least 50% sequence identity thereto (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Accordingly, a cell according to the invention may express and/or overexpress a polypeptide carrying out the stated reaction. Any polypeptide carrying out the stated reaction may be suitable. Examples include:

Reaction (1 ): SEQ ID NO: 26 or a sequence having at least 50% sequence identity thereto (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (2): SEQ ID NO: 24 or a sequence having at least 50% sequence identity thereto (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (3): SEQ ID NO: 22 or 48 or a sequence having at least 50% sequence identity to either of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (4): SEQ ID NO: 16, 18 or 20 or a sequence having at least 50% sequence identity to any of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (5): SEQ ID NO: 8, 10, 12 or 14 or a sequence having at least 50% sequence identity to any of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (6): SEQ ID NO: 2, 4 or 6 or a sequence having at least 50% sequence identity to any of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (7): SEQ ID NO: 28, 30 or 32 or a sequence having at least 50% sequence identity to any of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (8): SEQ ID NO: 34 or a sequence having at least 50% sequence identity thereto (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (9): SEQ ID NO: 36 or 38 or a sequence having at least 50% sequence identity to either of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (10): SEQ ID NO: 42, 44 or 46 or a sequence having at least 50% sequence identity to any of said sequences (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Reaction (1 1 ): SEQ ID NO: 40 or a sequence having at least 50% sequence identity thereto (or at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

As set out above, combinations of two or more of these reactions may be organized into one or more of the following metabolic pathways including:

PATHWAY 1 comprises at least one or more of the following reaction(s), typically one or more of which are overexpressed:

transportation of cytosolic itaconate to extracellular itaconic acid (eg. SEQ ID NO: 1 , 3 or 5 or a sequence having at least 50% sequence identity to any one of said sequences);

conversion of cytosolic cis-aconitate to itaconate (eg. SEQ ID NO: 7, 9, 1 1 or 13 or a sequence having at least 50% sequence identity to any one of said sequences); conversion of cytosolic citrate to cis-aconitate (eg. SEQ ID NO: 15, 17 or 19 or a sequence having at least 50% sequence identity to any one of said sequences);

transportation of mitochondrial citrate to the cytosol (eg. SEQ ID NO: 21 or 47 or a sequence having at least 50% sequence identity to any one of said sequences); conversion of mitochondrial oxaloacetate and acetyl-coenzyme-A into mitochondrial citrate;

transportation of cytosolic oxaloacetate to the mitochondria (eg. SEQ ID NO: 23 or a sequence having at least 50% sequence identity thereto); and

conversion of cytosolic pyruvate and bicarbonate to oxaloacetate (eg. SEQ ID NO: 25 or a sequence having at least 50% sequence identity thereto).

Preferably, in pathway 1 , nucleic acids encoding polypeptides having the following activities are overexpressed in a recombinant cell of the invention:

transportation of cytosolic itaconate to extracellular itaconic acid (eg. SEQ I D NO: 1 , 3 or 5 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences);

conversion of cytosolic cis-aconitate to itaconate (eg. SEQ ID NO: 7, 9, 1 1 or 13 or a sequence having at least 50, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences);

conversion of cytosolic citrate to cis-aconitate (eg. SEQ ID NO: 15, 17 or 19 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences);

transportation of mitochondrial citrate to the cytosol (eg. SEQ ID NO: 21 or 47 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to either of said sequences);

transportation of cytosolic oxaloacetate to the mitochondria (eg. SEQ ID NO: 23 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto); and

conversion of cytosolic pyruvate and bicarbonate to oxaloacetate (eg. SEQ ID NO: 25 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

PATHWAY 2 comprises at least one or more of the following reaction(s), typically one or more of which are overexpressed: transportation of cytosolic itaconate to extracellular itaconic acid (eg. SEQ ID NO: 1 , 3 or 5 or a sequence having at least 50% sequence identity to any one of said sequences);

conversion of cytosolic cis-aconitate to itaconate (eg. SEQ ID NO: 7, 9, 1 1 or 13 or a sequence having at least 50% sequence identity to any one of said sequences); conversion of cytosolic citrate to cis-aconitate (eg. SEQ ID NO: 15, 17 or 19 or a sequence having at least 50% sequence identity to any one of said sequences;

conversion of cytosolic oxaloacetate and acetyl-coenzyme-A to citrate (eg. SEQ ID NO: 27, 29 or 31 or a sequence having at least 50% sequence identity to any one of said sequences);

conversion of cytosolic acetaldehyde, NAD, and coenzyme-A to acetyl- coenzyme-A and NADH (eg. SEQ ID NO: 33 or a sequence having at least 50% sequence identity thereto);

conversion of cytosolic pyruvate to acetaldehyde and carbon dioxide; and conversion of cytosolic pyruvate and bicarbonate to oxaloacetate (SEQ ID NO: 25 or a sequence having at least 50% sequence identity thereto).

Preferably, in pathway 2, nucleic acids encoding polypeptides having the following activities are overexpressed in a recombinant cell of the invention:

transportation of cytosolic itaconate to extracellular itaconic acid (eg. SEQ ID NO: 1 , 3 or 5 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences);

conversion of cytosolic cis-aconitate to itaconate (eg. SEQ ID NO: 7, 9, 1 1 or 13 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences);

conversion of cytosolic citrate to cis-aconitate (eg. SEQ ID NO: 15, 17 or 19 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences;

conversion of cytosolic oxaloacetate and acetyl-coenzyme-A to citrate (eg. SEQ ID NO: 27, 29 or 31 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences); conversion of cytosolic acetaldehyde, NAD, and coenzyme-A to acetyl- coenzyme-A and NADH (eg. SEQ ID NO: 33 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto); and

conversion of cytosolic pyruvate and bicarbonate to oxaloacetate (eg. SEQ ID NO: 25 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

PATHWAY 3 comprises at least one or more of the following reaction(s), typically one or more of which are overexpressed:

transportation of cytosolic itaconate to extracellular itaconic acid (eg. SEQ ID NO: 1 , 3 or 5 or a sequence having at least 50% sequence identity to any one of said sequences);

conversion of cytosolic cis-aconitate to itaconate (eg. SEQ ID NO: 7, 9, 1 1 or 13 or a sequence having at least 50% sequence identity to any one of said sequences); conversion of cytosolic citrate to cis-aconitate (eg. SEQ ID NO: 15, 17 or 19 or a sequence having at least 50% sequence identity to any one of said sequences);

conversion of cytosolic oxaloacetate and acetyl-coenzyme-A to citrate (eg. SEQ ID NO: 27, 29 or 31 or a sequence having at least 50% sequence identity to any one of said sequences);

conversion of cytosolic acetyl-phosphate to acetyl-coenzyme-A (eg. SEQ ID NO: 41 , 43 or 45 or a sequence having at least 50% sequence identity to any one of said sequences);

conversion of xylulose-5-phosphate and phosphate to acetyl-phosphate and glyceraldehyde 3-phosphate (eg. SEQ ID NO: 35 or 37 or a sequence having at least 50% sequence identity to either of said sequences);

conversion of 6-phosphogluconate and NADP to xylulose-5-phosphate, NADPH and carbon dioxide;

conversion of glucose-6-phosphate and NADP to 6-phosphogluconate and NADPH; and

conversion of cytosolic pyruvate and bicarbonate to oxaloacetate (eg. SEQ ID NO: 25 or a sequence having at least 50% sequence identity thereto).

Preferably, in pathway 3, nucleic acids encoding polypeptides having the following activities are overexpressed in a recombinant cell of the invention: transportation of cytosolic itaconate to extracellular itaconic acid (eg. SEQ ID NO: 1 , 3 or 5 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences);

conversion of cytosolic cis-aconitate to itaconate (eg. SEQ ID NO: 7, 9, 1 1 or 13 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity to any one of said sequences);

conversion of cytosolic citrate to cis-aconitate (eg. SEQ ID NO: 15, 17 or 19 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences);

conversion of cytosolic oxaloacetate and acetyl-coenzyme-A to citrate (eg. SEQ ID NO: 27, 29 or 31 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences);

conversion of cytosolic acetyl-phosphate to acetyl-coenzyme-A (eg. SEQ ID NO: 41 , 43 or 45 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences);

conversion of xylulose-5-phosphate and phosphate to acetyl-phosphate and glyceraldehyde 3-phosphate (eg. SEQ ID NO: 35 or 37 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90% or at least 95%, at least 98% or at least 99% sequence identity to either of said sequences); and conversion of cytosolic pyruvate and bicarbonate to oxaloacetate (eg. SEQ ID NO: 25 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

PATHWAY 4 comprises at least one or more of the following reaction(s), typically one or more of which are overexpressed:

transportation of cytosolic itaconate to extracellular itaconic acid (eg. SEQ ID NO: 1 , 3 or 5 or a sequence having at least 50% sequence identity to any one of said sequences); conversion of cytosolic cis-aconitate to itaconate (eg. SEQ ID NO: 7, 9, 1 1 or 13 or a sequence having at least 50% sequence identity to any one of said sequences); conversion of cytosolic citrate to cis-aconitate (eg. SEQ ID NO: 15, 17 or 19 or a sequence having at least 50% sequence identity to any one of said sequences);

conversion of cytosolic oxaloacetate and acetyl-coenzyme-A to citrate (eg. SEQ ID NO: 27, 29 or 31 or a sequence having at least 50% sequence identity to any one of said sequences);

conversion of cytosolic acetyl-phosphate to acetyl-coenzyme-A (eg. SEQ ID NO: 41 , 43 or 45 or a sequence having at least 50% sequence identity to any one of said sequences);

conversion of cytosolic acetate and ATP to acetyl-phosphate, ADP, and phosphate (eg. SEQ ID NO: 39 or a sequence having at least 50% sequence identity thereto);

conversion of cytosolic pyruvate to acetaldehyde and carbon dioxide; and conversion of cytosolic pyruvate and bicarbonate to oxaloacetate (eg. SEQ ID NO: 25 or a sequence having at least 50% sequence identity thereto).

Preferably, in pathway 4, nucleic acids encoding polypeptides having the following activities are overexpressed in a recombinant cell of the invention:

transportation of cytosolic itaconate to extracellular itaconic acid (eg. SEQ ID NO: 1 , 3 or 5 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity to any one of said sequences);

conversion of cytosolic cis-aconitate to itaconate (eg. SEQ ID NO: 7, 9, 1 1 or 13 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences);

conversion of cytosolic citrate to cis-aconitate (eg. SEQ ID NO: 15, 17 or 19 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences);

conversion of cytosolic oxaloacetate and acetyl-coenzyme-A to citrate (eg. SEQ ID NO: 27, 29 or 31 or a sequence having at least 50 , at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences); conversion of cytosolic acetyl-phosphate to acetyl-coenzyme-A (eg. SEQ ID NO: 41 , 43 or 45 or a sequence having at least 50% , at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of said sequences);

conversion of cytosolic acetate and ATP to acetyl-phosphate, ADP, and phosphate (eg. SEQ ID NO: 39 or a sequence having at least 50% , at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto); and

conversion of cytosolic pyruvate and bicarbonate to oxaloacetate (eg. SEQ ID NO: 25 or a sequence having at least 50% , at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto).

Each of the pathways described above may be defined in terms of the polypeptides that are overexpressed. Thus, the pathways may be defined in terms of the polypeptides encoded by the nucleic acids defined above (see Tables 4 to 6) and sequences having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to such polypeptides.

Accordingly, in a method of the invention, a genetically modified yeast comprising one or more of these metabolic pathways may be used, whereby overexpression of one or more enzymes of these metabolic pathways confers yeast cell the ability to produce elevated levels of itaconic acid.

Also, in a method of the invention, a cell which is capable of producing one or more of 4-methyl itaconate or 1 -methyl itaconate may be used. Typically, such a recombinant cell is one in which one or more nucleic acid sequences encoding a polypeptide are overexpressed, said polypeptide(s) being capable of catalyzing one or more of the conversions:

a. cis-aconitate to itaconate (eg. SEQ ID NOs: 7, 9, 1 1 or 13 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any of said sequences);

b. itaconate to 4-methyl itaconate (eg. SEQ ID NO: 69 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto);

c. itaconate to 1 -methyl itaconate (eg. SEQ ID NO: 68 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto);

d. cis-aconitate to trans-aconitate (eg. SEQ ID NO: 70 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto);

e. trans-aconitate to (E)-3-carboxy-2-pentenedioate 5-methyl ester (eg.

SEQ ID NO: 69 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto);

f. trans-aconitate to (E)-3-(methoxycarbonyl)pent-2-enedioate (eg. SEQ ID NO: 68 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto);

g. (E)-3-carboxy-2-pentenedioate 5-methyl ester to 4-methyl itaconate (eg.

SEQ ID NOs: 7, 9, 1 1 or 13 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any of said sequences); and

h. (E)-3-(methoxycarbonyl)pent-2-enedioate to 1 -methyl itaconate (eg. SEQ ID NOs: 7, 9, 1 1 or 13 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any of said sequences).

Typically, such a recombinant cell is one in which one or more polypeptides are overexpressed, said polypeptide(s) being capable of catalyzing one or more of the conversions:

a. cis-aconitate to itaconate (eg. SEQ ID NOs: 8, 10, 12 or 14 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any of said sequences); b. itaconate to 4-methyl itaconate (eg. SEQ ID NO: 66 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto);

c. itaconate to 1 -methyl itaconate (eg. SEQ ID NO: 65 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto);

d. cis-aconitate to trans-aconitate (eg. SEQ ID NO: 67 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto);

e. trans-aconitate to (E)-3-carboxy-2-pentenedioate 5-methyl ester (eg. SEQ ID NO: 66 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto);

f. trans-aconitate to (E)-3-(methoxycarbonyl)pent-2-enedioate (eg. SEQ ID NO: 65 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto);

g. (E)-3-carboxy-2-pentenedioate 5-methyl ester to 4-methyl itaconate (eg. SEQ ID NOs: 8, 10, 12 or 14 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any of said sequences); and h. (E)-3-(methoxycarbonyl)pent-2-enedioate to 1 -methyl itaconate (eg. SEQ ID NOs: 8, 10, 12 or 14 or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any of said sequences).

In a method of the invention, a recombinant cell may be used which is capable of producing 1 -methyl itaconate may comprise one or more nucleic acid sequences encoding polypeptides capable of catalyzing the conversions:

a and c; or

d, f and h. Such a recombinant cell may may be defined in terms of the polypeptides that are overexpressed. Thus, the pathways may be defined in terms of the polypeptides encoded by the nucleic acids defined above (see Tables 4 to 6) and sequences having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identical to such polypeptides.

In a method of the invention, a recombinant cell may be used which is capable of producing 4-methyl itaconate may comprise one or more nucleic acid sequences encoding polypeptides capable of catalyzing the conversions:

a and b; or

d, e, and g.

Such a recombinant cell may may be defined in terms of the polypeptides that are overexpressed. Thus, the pathways may be defined in terms of the polypeptides encoded by the nucleic acids defined above (see Tables 4 to 6) and sequences having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to such polypeptides.

The conversions identified above are defined with reference to specific nucleic acids or polypeptides. These nucleic acids and polypeptides are given merely be way of example and should not be seen as limiting. Any suitable nucleic acid can be used which encodes a polypeptide having the desired activity or any polypeptide having the desired activity may be used. Sequences related to those specifically set out herein may be used in the invention.

A suitable nucleic acid may encode a polypeptide as encoded by one of the nucleic acids identified above or a polypeptide shared at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% sequence identity with a polypeptide encoded by one of the nucleic acids identified herein.

That is to say, nucleic acids and polypeptides suitable for use in the herein may be have 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%, at least 97%, at least 98%, at least 99% sequence identity with a nucleic acid or polypeptide specifically identified herein.

Accordingly, in a method of the invention, a recombinant cell may comprise a metabolic pathways comprising reactions catalyzed by the amino acid sequences listed in Table 4, whereby overexpression of one or more of those amino acid sequences within the same metabolic pathway in a genetically modified yeast cell confers on the yeast cell the ability to produce elevated levels of itaconic acid or ester of itaconic acid specifically when cultivated under anaerobic conditions.

Expression levels of these amino acid sequences in a recombinant cell may be controlled by constitutive strong promoters conferring on a recombinant cell the ability to produce elevated levels of itaconic acid and/or an ester of itaconic.

In a method of the invention, there is thus further provided that a genetically modified yeast cell may overexpressing of the metabolic pathways as mentioned above may comprise a deletion of pyruvate decarboxylase, alcohol dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, or succinyl-CoA ligase whereby the deletion confers yeast cell the ability to produce elevated levels of itaconic acid and itaconate methyl ester specifically when cultivated under anaerobic conditions.

As used herein, a recombinant cell or recombinant yeast cell suitable for use in a method of the present invention is defined as a cell which contains, or is transformed or genetically modified with one or more nucleotide sequence and/or protein that does not naturally occur in the yeast, or it contains additional copy or copies of an endogenous nucleic acid sequence (or protein). A wild-type cell or yeast cell is herein defined as the parental cell or yeast cell of the recombinant cell or yeast cell.

The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain.

The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but have been obtained from another cell or synthetically or recombinantly produced.

Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Usually, sequences are compared over the whole length of the sequences compared. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.

The parameter "identity" as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences. For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et a/., 2000, Trends in Genetics 16: 276-277; http://emboss.org), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

A nucleotide sequence encoding an enzyme which catalyses a conversion as set out herein may also be defined by its capability to hybridise with the nucleotide sequences encoding an enzyme capable catalyzing the reaction, under moderate, or preferably under stringent hybridisation conditions.

Stringent hybridisation conditions are herein defined as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and most preferably of about 200 or more nucleotides, to hybridise at a temperature of about 65°C in a solution comprising about 1 M salt, preferably 6 x SSC (sodium chloride, sodium citrate) or any other solution having a comparable ionic strength, and washing at 65°C in a solution comprising about 0.1 M salt, or less, preferably 0.2 x SSC or any other solution having a comparable ionic strength. Preferably, the hybridisation is performed overnight, i.e. at least for 10 hours and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridisation of sequences having about 90% or more sequence identity.

Moderate conditions are herein defined as conditions that allow a nucleic acid sequence of at least 50 nucleotides, preferably of about 200 or more nucleotides, to hybridise at a temperature of about 45°C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength. Preferably, the hybridisation is performed overnight, i.e. at least for 10 hours, and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridisation of sequences having up to 50% sequence identity. The person skilled in the art will be able to modify these hybridisation conditions in order to specifically identify sequences varying in identity between 50% and 90%.

The term "gene", as used herein, refers to a nucleic acid sequence containing a template for a nucleic acid polymerase, in eukaryotes, RNA polymerase II. Genes are transcribed into mRNAs that are then translated into protein.

The term "nucleic acid" as used herein, includes reference to a deoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, in either single-or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single- stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

The term "enzyme" as used herein is defined as a protein which catalyses a (bio)chemical reaction in a cell, such as a yeast cell. To increase the likelihood that the introduced enzyme is expressed in active form in a yeast of the invention, the corresponding encoding nucleotide sequence may be adapted to optimise its codon usage to that of the chosen yeast cell. Several methods for codon optimisation are known in the art. A preferred method to optimise codon usage of the nucleotide sequences to that of the yeast is a codon pair optimization technology as disclosed in WO2008/000632. Codon-pair optimization is a method for producing a polypeptide in a host cell, wherein the nucleotide sequences encoding the polypeptide have been modified with respect to their codon-usage, in particular the codon-pairs that are used, to obtain improved expression of the nucleotide sequence encoding the polypeptide and/or improved production of the polypeptide. Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence.

Usually, the nucleotide sequence encoding an enzyme introduced into a cell of the invention is operably linked to a promoter that causes sufficient expression of the corresponding nucleotide sequence in the cell according to the present invention to confer on the cell the ability to the enzyme.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements (or coding sequences or nucleic acid sequence) in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences known to a person skilled in the art. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation.

A promoter that could be used to achieve the expression of a nucleotide sequence coding for an enzyme may be not native to the nucleotide sequence coding for the enzyme to be expressed, i.e. a promoter that is heterologous to the nucleotide sequence (coding sequence) to which it is operably linked. Preferably, the promoter is homologous, i.e. endogenous to the host cell. Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art. Suitable promoters in eukaryotic host cells may be GAL7, GAL10, or GAL 1 , CYC1 , HIS3, ADH1 , PGL, PH05, GAPDH, ADC1 , TRP1 , URA3, LEU2, ENO, TPI, and AOX1. Other suitable promoters include PDC, GPD1 , PGK1 , TEF1 , and TDH.

Usually a nucleotide sequence encoding an enzyme comprises a terminator. Any terminator, which is functional in the cell, may be used in the present invention. Preferred terminators are obtained from natural genes of the host cell. Suitable terminator sequences are well known in the art. Preferably, such terminators are combined with mutations that prevent nonsense mediated mRNA decay in the host cell of the invention (see for example: Shirley et al., 2002, Genetics 161 :1465-1482).

In the invention, the nucleotide sequence encoding an enzyme that catalyses a conversion as described herein may be overexpressed to achieve increased production of that enzyme in a recombinant cell according to the present invention.

There are various means available in the art for overexpression of nucleotide sequences encoding enzymes in the yeast cell of the invention. In particular, a nucleotide sequence encoding an enzyme may be overexpressed by increasing the copy number of the gene coding for the enzyme in the cell, e.g. by integrating additional copies of the gene in the cell's genome, by expressing the gene from a centromeric vector, from an episomal multicopy expression vector or by introducing an (episomal) expression vector that comprises multiple copies of the gene. Preferably, overexpression of the enzyme according to the invention is achieved with a (strong) constitutive promoter.

The nucleic acid construct may be a plasmid, for instance a low copy plasmid or a high copy plasmid. The yeast according to the present invention may comprise a single or multiple copies of a nucleotide sequence encoding an enzyme encoding a given conversion, for instance by multiple copies of a nucleotide construct.

The nucleic acid construct may be maintained episomally and thus comprise a sequence for autonomous replication, such as an autosomal replication sequence. A suitable episomal nucleic acid construct may e.g. be based on the yeast 2μ or pKD1 plasmids (Gleer et al., 1991 , Biotechnology 9: 968-975), or the AMA plasmids (Fierro et al., 1995, Curr Genet. 29:482-489). Alternatively, each nucleic acid construct may be integrated in one or more copies into the genome of the yeast cell. Integration into the cell's genome may occur at random by non-homologous recombination but preferably, the nucleic acid construct may be integrated into the cell's genome by homologous recombination as is well known in the art (see e.g. WO90/14423, EP-A-0481008, EP-A- 0635 574 and US 6,265,186).

With the exception of transporter polypeptides, in the invention, it is preferred the enzyme or enzymes expressed in a recombinant cell of the invention is/are active in the cytosol upon expression of the encoding nucleotide sequence(s). Cytosolic activity of the enzyme(s) is/are preferred for a high productivity of itaconic acid or an itaconic acid ester by the cell.

A nucleotide sequence encoding an enzyme that catalyses a conversion as described herein, may comprise a peroxisomal or mitochondrial targeting signal, for instance as determined by the method disclosed by Schluter et al, Nucleic acid Research 2007, Vol 25, D815-D822. In the event the enzyme comprises a targeting signal, it may be preferred that the yeast according to the invention comprises a truncated form of the enzyme, wherein the targeting signal is removed.

A recombinant cell of the invention may be a yeast cell. The yeast according to the present invention preferably belongs to one of the genera Saccharomyces, Pichia, Kluyveromyces, or Zygosaccharomyces. More preferably, the yeast cell may be Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Pichia stipidis, Kluyveromyces marxianus, K lactis, K thermotolerans, or Zygosaccharomyces bailii.

In a preferred embodiment, the yeast according to the present invention may be able to grow on any suitable carbon source known in the art and convert it to itaconic acid or an itaconic acid ester. The yeast may be able to convert directly plant biomass, celluloses, hemicelluloses, pectines, rhamnose, galactose, fructose, maltose, maltodextrines, ribose, ribulose, or starch, starch derivatives, sucrose, lactose and glycerol. Hence, a preferred yeast cell expresses enzymes such as cellulases (endocellulases and exocellulases) and hemicellulases (e.g. endo- and exo-xylanases, arabinases) necessary for the conversion of cellulose into glucose monomers and hemicellulose into xylose and arabinose monomers, pectinases able to convert pectines into glucuronic acid and galacturonic acid or amylases to convert starch into glucose monomers. The ability of a yeast to express such enzymes may be naturally present or may have been obtained by genetic modification of the yeast. Preferably, the yeast is able to convert a carbon source selected from the group consisting of glucose, fructose, galactose, xylose, arabinose, sucrose, lactose, raffinose and glycerol. The process of the invention for the preparation of itaconic acid or an itaconic acid ester comprises fermenting a recombinant cell, for example a yeast cell, as described herein in the presence of a suitable fermentation medium under anaerobic conditions. Suitable fermentation media are known to the skilled man in the art. Preferably, the itaconic acid ester produced in the process according to the present invention is 4-methyl itaconate or 1 -methyl itaconate.

For the purposes of this invention, an anaerobic fermentation process may be herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5, 1 , 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05 mmol 0 ₂ /L/h, more preferably 0 mmol 0 ₂ /L/h is consumed, and preferably wherein organic molecules serve as both electron donor and/or electron acceptors.

Therefore in one embodiment of the invention in the method for the production of itaconic acid or an ester of itaconic acid, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced, the anaerobic conditions are established when in the fermentation broth substantially no oxygen is consumed, preferably less than 5, 2.5, 1 , 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05 mmol 0 ₂ /L/h is consumed, more preferably 0 mmol 0 ₂ /L/h is consumed. In one embodiment organic molecules, typically sugars, serve as electron donor and/or electron acceptors, preferably organic molecules, typically sugars serve as both electron donor and electron acceptor. The skilled person will know which type of organic molecules can serve as elector donor and/or electro acceptors in the anaerobic fermentation process. Typical organic molecules which can be used as donor and/or electron acceptors during the fermentation are sugars. Sugars which typically can be used may be selected from monosaccharides (e.g. fructose, glucose), disaccharides (e.g. maltose, sucrose), oligosaccharides (e.g. maltodextrine), or polysaccharides (e.g. cellulose, starch). The fermentation process according to the present invention may also first be run under aerobic conditions and subsequently under anaerobic conditions. Anaerobic conditions are typically used in the production phase (production of the itaconic acid or ester thereof).

The fermentation process of the invention may also be run under oxygen- limited, or micro-aerobical, conditions which are, for the purposes of this invention, considered to be anaerobic processes. Alternatively, the fermentation process may first be run under aerobic conditions and subsequently under oxygen-limited conditions. An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The degree of oxygen limitation is determined by the amount and composition of the ingoing gasflow as well as the actual mixing/mass transfer properties of the fermentation equipment used.

The process for the production of itaconic acid or an itaconic acid ester according to the present invention may be carried out at any suitable pH between 1 and 9. Preferably, the pH in the fermentation broth is between 2 and 7, preferably between 3 and 5. It was found advantageous to be able to carry out the process according to the present invention at a low pH, since this prevents bacterial contamination. In addition, since the pH drops during itaconic acid production, a lower amount of titrant is needed to keep the pH at a desired level.

A suitable temperature at which the process according to the present invention may be carried out is between 5 and 60°C, preferably between 10 and 50°C, more preferably between 15 and 35°C, more preferably between 18°C and 30°C. The skilled man in the art knows which optimal temperatures are suitable for fermenting a specific yeast cell.

Preferably, the itaconic acid or itaconic acid ester is recovered from the fermentation broth by a suitable method known in the art, for instance by crystallisation.

Preferably, the itaconic acid or an ester of itaconic acid that is prepared in the process according to the present invention is further converted into a desirable product, such as a pharmaceutical, cosmetic, food, feed or chemical product. In particular, itaconic acid or an ester of itaconic acid may be further converted into a polymer.

Standard genetic techniques, such as overexpression of enzymes in the host cells, genetic modification of host cells, or hybridisation techniques, are known methods in the art, such as described in Sambrook and Russel (2001 ) "Molecular Cloning: A Laboratory Manual (3 ^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987). Methods for transformation, genetic modification etc of fungal host cells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671 , WO90/14423, EP-A- 0481008, EP-A-0635 574 and US 6,265,186.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

EMBODIMENTS OF THE INVENTION

1 . A method for the production of itaconic acid or an ester of itaconic acid, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced.

2. A method according to embodiment 1 , wherein the anaerobic conditions are established when in the fermentation broth substantially no oxygen is consumed, preferably less than 5, 2.5, 1 , 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05 mmol 0 ₂ /L/h is consumed, more preferably 0 mmol 0 ₂ /L/h is consumed.

3. A method according to embodiment 2, wherein organic molecules, preferably sugars, serve as electron donor and/or electron acceptors.

4. A method according to any one of embodiments 1 to 3, wherein the recombinant cell is capable of producing 4-methyl itaconate or 1 -methyl itaconate.

5. A method according to embodiment 4, wherein the recombinant cell is one in which one or more nucleic acid sequences encoding a polypeptide are overexpressed, said polypeptide(s) being capable of catalyzing one or more of the conversions:

a. cis-aconitate to itaconate;

b. itaconate to 4-methyl itaconate;

c. itaconate to 1 -methyl itaconate;

d. cis-aconitate to trans-aconitate;

e. trans-aconitate to (E)-3-carboxy-2-pentenedioate 5-methyl ester; f. trans-aconitate to (E)-3-(methoxycarbonyl)pent-2-enedioate; g. (E)-3-carboxy-2-pentenedioate 5-methyl ester to 4-methyl itaconate; and h. (E)-3-(methoxycarbonyl)pent-2-enedioate to 1 -methyl itaconate. A method according to embodiment 5, wherein the recombinant cell is capable of producing 1 -methyl itaconate and comprises one or more nucleic acid sequences encoding polypeptides capable of catalyzing the conversions:

a and c; or

d, f and h. A method according to embodiment 5 or 6, wherein the recombinant cell is capable of producing 4-methyl itaconate and comprises one or more nucleic acid sequences encoding polypeptides capable of catalyzing the conversions: a and b; or

d, e, and g. A method according to any one of the preceding embodiments, wherein the recombinant cell is a yeast cell. A method according to embodiment 1 , wherein the recombinant cell is a yeast cell which is capable of producing itaconic acid and which overexpresses:

a nucleic acid encoding a polypeptide having cis-aconitate decarboxylase activity; and

one or more nucleic acids encoding polypeptides which separately or together catalyze a reaction towards acetyl CoA. A method according to embodiment 9, wherein the nucleic acid encoding a polypeptide which catalyzes a reaction towards acetyl CoA is

nucleic acid sequences encoding polypeptides which together have pyruvate dehydrogenase activity;

one or more nucleic acid sequences encoding one or more polypeptides having pyruvate decarboxylase activity, acetaldehyde dehydrogenase activity and/or acetyl-CoA synthetase activity;

a nucleic acid sequence encoding a polypeptide having acetylating acetaldehyde dehydrogenase activity; a nucleic acid sequence encoding a polypeptide having pyruvate: NADP oxidoreductase activity;

a nucleic acid encoding a polypeptide having acetate:CoA ligase (ADP- forming) activity;

a nucleic acid encoding a polypeptide ATP:acetate phosphotransferase activity and a nucleic acid encoding a polypeptide having acetyl-CoA:Pi acetyltransferase activity/phosphate acetyltransferase activity. A method according to any one of the preceding embodiments, wherein the recombinant cell overexpresses:

a nucleic acid encoding a polypeptide catalyzing conversion of citrate to cis- aconitate; and/or

a nucleic acid encoding a polypeptide having citrate synthase activity. A method according to any one of the preceding embodiments, wherein the recombinant cell overexpresses:

a nucleic acid encoding a polypeptide having pyruvate carboxylase; and/or a nucleic acid encoding a polypeptide having PEP carboxykinase activity; and/or

a nucleic acid encoding a polypeptide having PEP carboxylase. A method according to any one of the preceding embodiments, wherein the recombinant cell overexpresses:

a nucleic acid sequence encoding a mitochondrial membrane citrate transporter. A method according to any one of the preceding embodiments, wherein the recombinant cell comprises:

a nucleic acid sequence encoding a itaconic acid transporter, a 4-methyl itaconate transporter or a 1 -methyl itaconate transporter. A method according to any one of the preceding embodiments, wherein the recombinant cell comprises a genetic modification resulting in reduced expression and/or activity of pyruvate decarboxylase, alcohol dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, or succinyl-CoA ligase in the cell as compared to a cell without the genetic modification. A method according to any one of the preceding embodiments, wherein the recombinant cell is a S. cerevisiae cell. A method for the production of itaconic acid or an ester of itaconic acid, optionally according to any one of embodiments 1 to 16, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced and wherein the recombinant cell comprises, for example overexpresses polypeptides catalysing the following reactions:

transportation of cytosolic itaconate to extracellular itaconic acid;

conversion of cytosolic cis-aconitate to itaconate;

conversion of cytosolic citrate to cis-aconitate;

conversion of cytosolic oxaloacetate and acetyl-coenzyme-A to citrate; conversion of cytosolic acetaldehyde, NAD, and coenzyme-A to acetyl- coenzyme-A and NADH; and

conversion of cytosolic pyruvate and bicarbonate to oxaloacetate. A method for the production of itaconic acid or an ester of itaconic acid, optionally according to any one of embodiments 1 to 16, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced and wherein the recombinant cell comprises, for example overexpresses polypeptides catalysing the following reactions:

transportation of cytosolic itaconate to extracellular itaconic acid;

conversion of cytosolic cis-aconitate to itaconate;

conversion of cytosolic citrate to cis-aconitate;

transportation of mitochondrial citrate to the cytosol;

transportation of cytosolic oxaloacetate to the mitochondria; and conversion of cytosolic pyruvate and bicarbonate to oxaloacetate. A method for the production of itaconic acid or an ester of itaconic acid, optionally according to any one of embodiments 1 to 16, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced and wherein the recombinant cell comprises, for example overexpresses polypeptides catalysing the following reactions:

transportation of cytosolic itaconate to extracellular itaconic acid;

conversion of cytosolic cis-aconitate to itaconate;

conversion of cytosolic citrate to cis-aconitate;

conversion of cytosolic oxaloacetate and acetyl-coenzyme-A to citrate; conversion of cytosolic acetyl-phosphate to acetyl-coenzyme-A;

conversion of xylulose-5-phosphate and phosphate to acetyl-phosphate and glyceraldehyde 3-phosphate; and

conversion of cytosolic pyruvate and bicarbonate to oxaloacetate. A method for the production of itaconic acid or an ester of itaconic acid, optionally according to any one of embodiments 1 to 16, which method comprises fermenting a recombinant cell capable of producing itaconic acid or an ester of itaconic acid in a suitable fermentation medium under anaerobic conditions, wherein the itaconic acid or ester of itaconic acid is produced and wherein the recombinant cell comprises, for example overexpresses polypeptides catalysing the following reactions:

transportation of cytosolic itaconate to extracellular itaconic acid;

conversion of cytosolic cis-aconitate to itaconate;

conversion of cytosolic citrate to cis-aconitate;

conversion of cytosolic oxaloacetate and acetyl-coenzyme-A to citrate; conversion of cytosolic acetyl-phosphate to acetyl-coenzyme-A;

conversion of cytosolic acetate and ATP to acetyl-phosphate, ADP, and phosphate; and

conversion of cytosolic pyruvate and bicarbonate to oxaloacetate. 21 . A method according to any one of embodiments 17 to 20 wherein the recombinant cell is a yeast cell, such as a Saccharomyces cerevisiae cell.

22. A process according to any one of the preceding embodiments, wherein the itaconic acid or ester of itaconic acid is further converted into a pharmaceutical, cosmetic, food, feed or chemical product.

23. A fermentation broth comprising a itaconic acid and/or an ester of itaconate obtainable by a process according to any one of embodiments 1 to 21 .

The present invention is further illustrated by the following Examples:

EXAMPLES

Example 1 : Overexpression of enzymes for different metabolic pathways for itaconic acid and itaconate methyl ester production in Saccharomyces cerevisiae

1 .1 Expression constructs

The nucleotide sequences of SEQ ID NOs 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, and 47 are obtained by the codon-pair optimization method as disclosed in PCT/EP2007/05594 for S. cerevisiae were synthesized. The nucleotide sequences of SEQ ID NOs 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63 and 64 were synthesized. From these sequences (promoter, open reading frame and terminators) expression cassettes were built according to the methods described in the co-pending patent application no. US61/616254 and WO2013/144257. The formed expression cassettes (cassette 1 17 - cassette 149) were used as a template to PCR amplify the DNA fragments used in the transformation.

1 .2 Preparation and purification of PCR fragments for transformation

Assembly and integration of the itaconic acid pathways is done according to the described methods in the co-pending patent application no. US61/616254 and WO2013/144257. Amplification of expression cassettes with connector sequences from the plasmids was carried out with a standard set of primers binding to the connectors. The primers are set out in SEQ I D NOs: 87 to 1 10 of the co-pending patent application no. US61/616254 and WO2013/144257 and named after the connector and the direction of amplification. For example "con 5 fw" was the forward primer on connector 5. Only a subset of the primers was used in this experiment. Table 1 shows the primers used with the corresponding PCR templates used in the PCR reactions. PCR reactions were performed with Phusion polymerase (Finnzymes) according to the manual.

Table 1 : Overview of all cassettes, the content of the cassettes and the primer combinations for generating expression cassettes equipped with connectors used in the transformation of S. cerevisiae

cassette Nos forward reverse PRO ORF TER BBN

CAS117 Sc Actl .pro SEQ I D NO: 1 ADH1 terminator Sc 5a.bbn

CAS118 con5 forw conA rev Sc Actl .pro SEQ I D NO: 3 ADH1 terminator Sc 5a.bbn

CAS119 Sc Actl .pro SEQ I D NO: 5 ADH1 terminator Sc 5a.bbn

CAS120 Sc TDH3.pro SEQ I D NO: 7 TDH1 terminator Sc bc.bbn

CAS121 Sc TDH3.pro bc.bbn conB forw conC rev SEQ I D NO: 9 TDH1 terminator Sc

CAS122 Sc TDH3.pro SEQ ID NO: 11 TDH1 terminator Sc bc.bbn

CAS123 Sc TDH3.pro SEQ ID NO: 13 TDH1 terminator Sc bc.bbn

CAS133 Sc FBAl.pro SEQ ID NO: 15 GPM1 terminator Sc cd.bbn

CAS134 Sc FBAl.pro SEQ ID NO: 17 GPM1 terminator Sc cd.bbn

CAS135 Sc FBAl.pro

conC forw conD rev SEQ ID NO: 19 GPM1 terminator Sc cd.bbn

CAS144 Sc PRE3.pro SEQ ID NO: 15 GPM1 terminator Sc cd.bbn

CAS145 Sc PRE3.pro SEQ ID NO: 17 GPM1 terminator Sc cd.bbn

CAS146 Sc PRE3.pro SEQ ID NO: 19 GPM1 terminator Sc cd.bbn

CAS136 con D forw con E rev Sc PGKl.pro SEQ ID NO: 25 TPI1 terminator Sc de.bbn

CAS124 Sc Tefl.pro SEQ ID NO: 21 PDC1 terminator Sc ef.bbn

CAS125 Sc Tefl.pro SEQ ID NO: 47 PDC1 terminator Sc ef.bbn

CAS137 Sc Tefl.pro SEQ ID NO: 27 PDC1 terminator Sc ef.bbn

CAS138 Sc Tefl.pro C1 terminator Sc ef.bbn conE forw conF rev SEQ ID NO: 29 PD

CAS139 Sc Tefl.pro SEQ ID NO: 31 PDC1 terminator Sc ef.bbn

CAS147 Sc TDHl.pro SEQ ID NO: 27 PDC1 terminator Sc ef.bbn

CAS148 Sc TDHl.pro SEQ ID NO: 29 PDC1 terminator Sc ef.bbn

CAS149 Sc TDHl.pro SEQ ID NO: 31 PDC1 terminator Sc ef.bbn

CAS126 Sc EN02.pro SEQ ID NO: 23 TALI terminator Sc f3.bbn

CAS130 Sc EN02.pro SEQ ID NO: 41 TALI terminator Sc f3.bbn

CAS131 conF forw con3 rev Sc EN02.pro SEQ ID NO: 43 TALI terminator Sc f3.bbn

CAS132 Sc EN02.pro SEQ ID NO: 45 TALI terminator Sc f3.bbn

CAS140 Sc EN02.pro SEQ ID NO: 33 TALI terminator Sc f3.bbn

CAS141 Sc EN02.pro SEQ ID NO: 41 TALI terminator Sc fg.bbn

CAS142 FG FG Sc EN02.pro SEQ ID NO: 43 TALI terminator Sc fg.bbn

CAS143 Sc EN02.pro SEQ ID NO: 45 TALI terminator Sc fg.bbn

CAS127 Sc PGIl.pro SEQ ID NO: 35 TDH3 terminator Sc g3.bbn

CAS128 G3 G4 Sc PGIl.pro SEQ ID NO: 37 TDH3 terminator Sc g3.bbn

CAS129 Sc PGIl.pro SEQ ID NO: 39 TDH3 terminator Sc g3.bbn

The dominant marker KanMX is amplified using a standard plasmid containing the fragments as template DNA. The 5' and 3' INT1 deletion flanks were amplified by PCR using CEN.PK1 13-7D genomic DNA as template. The dominant marker, integration flanks and the primers used are the same as used in the methods described in the co-pending patent application no. US61/616254 and WO2013/144257. Size of the PCR fragments was checked with standard agarose electrophoresis techniques. PCR amplified DNA fragments were purified with the NucleoMag® 96 PCR magnetic beads kit of Macherey-Nagel, according to the manual. DNA concentration was measured using the Trinean DropSense® 96 of GC biotech.

1 .3 Transformation of the fragments to S. cerevisiae

Transformation of S. cerevisiae was done as described by Gietz and Woods (2002; Transformation of the yeast by the LiAc/SS carrier DNA/PEG method. Methods in Enzymology 350: 87-96).

CEN.PK1 137D {MATa URA3 HIS3 LEU2 TRP1 MAL2-8 SUC2) and the PDC1 KO strain were transformed with 1 μg of each of the amplified and purified PCR fragments. Each transformation will result in a "itaconic acid pathway" with the itaconic acid cassettes and KanMX marker integrated into the INT1 locus on the genome. Transformation mixtures were plated on YEPhD-agar (BBL Phytone peptone 20.0 g/l, Yeast Extract 10.0 g/l, Sodium Chloride 5.0 g/l, Agar 15.0 g/l and 2% glucose) containing G418 (400 g/ml). After 3 days of incubation at 30 °C, colonies appeared on the plates, whereas the negative control (i.e., no addition of DNA in the transformation experiment) resulted in blank plates. Table 2 shows an overview of the transformations that were done to both CEN.PK1 137D and the PDC1 KO strain.

Table 2 Overview of the cassettes transformed in each transformation

Transformation # Position 1 Position2 Position3 Position4 Position5 Position6 Position7

1 .4 Cultivation of the transformants Single colonies were picked and transferred to a MTP agar well containing 200 μΙ YEPhD-agar containing 400 μg ml G418. For each transformation 2 to 4 colonies were used for further analysis. After 3 days of incubation of the plate at 30 °C, good grown colonies were inoculated by transferring some colony material with a pin tool in a MTP plate with standard lid containing in each well 200 μΙ_ Verduyn medium (Verduyn et al., Yeast 8:501 -517, 1992, where the (NH4)2S04 was replaced with 2 g/l Urea) with a C-source based on starch and an enzyme providing release of glucose during cultivation. The MTP was incubated in a MTP shaker (INFORS HT Multitron) at 30 °C, 550 rpm and 80% humidity for 72 hours. After this pre-culture phase a production phase was started by transferring 80 μΙ of the broth to 4 ml Verduyn media (again with the urea replacing (NH4)2S04) with a C-source based on starch and an enzyme providing release of glucose during cultivation. After 7 days growth in the shaker at 550 rpm, 30 °C and 80% humidity the plates were centrifuged for 10 minutes at 2750 rpm in a Heraeus Multifuge 4. Supernatant was transferred to MTP plates and itaconic acid levels in the supernatant were measured with a hereafter described LC-MS method.

1 .5 Detection of itaconic acid and itaconate methyl ester

UPLC-MS/MS analysis method for the determination of itaconic acid, and other compounds of the Krebs cycle. A Waters HSS T3 column 1 .7 μηη, 100 mm ^* 2.1 mm was used for the separation of itaconic, succinic, citric, iso-citric, malic and fumaric acid, as well as the possible methyl- and ethyl ester of itaconic acid with gradient elution. Eluens A consists of LC/MS grade water, containing 0.1 % formic acid, and eluens B consists of acetonitrile, containing 0.1 % formic acid. The flow-rate was 0.35 ml/min and the column temperature was kept constant at 40 °C. The gradient started at 95% A and was increased linear to 30 % B in 10 minutes, kept at 30 % B for 2 minutes, then immediately to 95% A and stabilized for 5 minutes. The injection volume used was 2 ul. A Waters Xevo API was used in electrospray (ESI) in negative ionization mode, using multiple reaction monitoring (MRM). The ion source temperature was kept at 130 °C, whereas the desolvation temperature is 350 °C, at a flow-rate of 500 L/hr.

For itaconic acid and the other compounds of the Krebs cycle the deprotonated molecule was fragmented with 10 eV, resulting in specific fragments from losses of H20 and C02. The standards of reference compounds spiked in blank fermentation broth were analyzed to confirm retention time, calculate a response factor for the respective ions, and was used to calculate the concentrations in fermentation samples. All samples were diluted appropriately (5-25 fold) in eluens A to overcome ion suppression and matrix effects during LC-MS analysis. Accurate mass analysis of itaconic acid and esters of itaconic acid. To confirm the elemental composition of the compounds analyzed accurate mass analyses was performed with the same chromatographic system as described above, coupled to a LTQ orbitrap (ThermoFisher). Mass calibration was performed in constant infusion mode, using a NaTFA mixture (ref), in such a way that during the experimental set-up the accurate mass analyzed could be fitted within 2 ppm from the theoretical mass, of all compounds analyzed.

1 .6 Itaconic acid and itaconate methyl ester concentrations

Itaconic acid concentrations per pathway group and per strain group are shown in Table 3. The concentrations in the table are median values per strain or pathway group. The LC-MS analysis also detected 4-methyl itaconate in the samples and confirmed the mass and retention time with the standard. Concentrations found in the samples of 4-methyl itaconate range between 100 and 200 mg/l.

Table 3: Itaconic acid concentration results

Table 4: Description of sequence listing

Nucleic acid Amino acid Id* UniProt Organism

SEQ ID NO: 1 SEQ ID NO: 2 ITE_01 Q0C8L2 A. terreus

SEQ ID NO: 3 SEQ ID NO: 4 ITE_02 A. terreus

SEQ ID NO: 5 SEQ ID NO: 6 ITE_03 Orfl6 A. terreus

SEQ ID NO: 7 SEQ ID NO: 8 CAD_01 mCAD3 A. terreus

SEQ ID NO: 9 SEQ ID NO: 10 CAD_02 mCAD2 A. terreus

SEQ ID NO: 11 SEQ ID NO: 12 CAD_03 Q0C8L3 A. terreus

SEQ ID NO: 13 SEQ ID NO: 14 CAD_04 Q9Y7D9 A. terreus

SEQ ID NO: 15 SEQ ID NO: 16 ACO_01 A7A1I8 S. cerevisiae

SEQ ID NO: 17 SEQ ID NO: 18 ACO_02 PRPD_ECOLI E. coli

SEQ ID NO: 19 SEQ ID NO: 20 ACO_03 ACON2_ECOLI E. coli

SEQ ID NO: 21 SEQ ID NO: 22 CTP_01 Q04013 S. cerevisiae

SEQ ID NO: 23 SEQ ID NO: 24 OTP_01 P32332 S. cerevisiae

SEQ ID NO: 25 SEQ ID NO: 26 PYC_01 P32327 S. cerevisiae Nucleic acid Amino acid Id* UniProt Organism

SEQID NO: 27 SEQID NO: 28 CSc_01 CISY_YEAST 5. cerevisiae

SEQID NO: 29 SEQID NO: 30 CSc_02 CISY_PIG Sus scrofa

SEQID NO: 31 SEQID NO: 32 CSc_03 C9ROQ1_ECOD1 E. coli

SEQID NO: 33 SEQID NO: 34 ACDH67 Q92CP2 Listeria innocua

SEQID NO: 35 SEQID NO: 36 XFP_01 Q6UPD8 Lactobacillus paraplantarum.

SEQID NO: 37 SEQID NO: 38 XFP_02 Q9AEM9 Bifidobacterium animalis subsp. lactis

DSM 10140

SEQID NO: 39 SEQID NO: 40 ACK_01 Q1R9B8 E. coli

SEQID NO: 41 SEQID NO: 42 PTA_01 F5ZUJ6 S. enter ica

SEQID NO: 43 SEQID NO: 44 PTA_02 P41790 S. enter ica

SEQID NO: 45 SEQID NO: 46 PTA_03 P39646 Bacillus subtilis

SEQID NO: 47 SEQID NO: 48 CTP_03 Orfl4 A. terreus

Table 5: Description of sequence listing

SEQID SEQ NAME

SEQID NO: 49 Sc Actl.pro

SEQID NO: 50 Sc TDH3.pro

SEQID NO: 51 Sc Tefl.pro

SEQID NO: 52 Sc EN02.pro

SEQID NO: 53 Sc PGIl.pro

SEQID NO: 54 Sc FBAl.pro

SEQID NO: 55 Sc PGKl.pro

SEQID NO: 56 Sc PRE3.pro

SEQID NO: 57 Sc TDHl.pro

SEQID NO: 58 Sc ADHl.ter

SEQID NO: 59 Sc TDHl.ter

SEQID NO: 60 Sc PDCl.ter

SEQID NO: 61 Sc TALl.ter

SEQID NO: 62 Sc TDH3.ter

SEQID NO: 63 Sc GPMl.ter

SEQID NO: 64 Sc TPIl.ter Table 6 Description of sequence listing

Example: Itaconic acid and itaconate methyl ester production in Saccharomyces cerevisiae under aerobic and anaerobic cultivation conditions

2.1 Strains used in this experiment

Strain ITA06 is the strain obtained from PDC1 KO strain using transformation #6 as described in Example 1 . Strain ITA07 is the strain obtained from PDC1 KO strain using transformation #16 as described in Example 1 . Strain ITA09 is obtained by transforming CEN.PK1 137D strain with cassette CAS1 17, CAS121 , CAS133, CAS136, CAS139, and CAS140 as described in patent Example 1.

2.2 Aerobic and anaerobic cultivation of the transformants

One loop of cells from a culture on a YEPhD-agar plate were transferred to a 20ml YEPhD medium in 100ml shake flask. After 36 hours of incubation at 30 °C and 280 rpm, 8 ml was transferred to 1 L Dasgip fermenter containing 300 ml 2x concentrated Verduyn medium (Verduyn, C, Postma, E., Scheffers, W. A. and Van Dijken, J. P. (1992) Effect of benzoic acid on metabolic fluxes in yeasts: a continuous- culture study on the regulation of respiration and alcoholic fermentation. Yeast 8, 501 - 517) with a C-source based on starch and an enzyme providing release of glucose during cultivation. To control the pH, a buffer of 30 g/l MES hydrate was added and the pH was set at 5.4 with 4N KOH. The medium was supplemented with 0.01 g/l ergosterol (Andreasen, A. A. and Stier, T. J. (1953) Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a defined medium. J. Cell Physiol. 41 , 23-36) and 0.36 g/l Tween 80 9Andreasen, A. A. and Stier, T. J. (1954) Anaerobic nutrition of Saccharomyces cerevisiae. II. Unsaturated fatty acid requirement for growth in a defined medium. J. Cell Physiol. 43, 271 -281 ) dissolved in ethanol and 8 mg/l biotin. Samples were taking at 40.5 and 106 hours of fermentation at 30 °C, 500 rpm stirring speed and a gasflow of 10 l/h N ₂ or 10 l/h air. Itaconic acid and itaconate methyl ester levels in the supernatant were measured with the LC-MS method described as in patent 29140-WO-PCT. Biomass dry weight was determined by centrifuging 10 ml of whole broth for 10 minutes at 6.000 rcf in a table centrifuge, the pellet was washed with demineralized water and dried at 105 °C for 24 hours and weighed.

2.3 Detection of itaconic acid and itaconate methyl ester

UPLC-MS/MS analysis method for the determination of itaconic acid, and other compounds of the Krebs cycle. A Waters ACQUITY UPLC HSS T3 column 1 .7 μηι, 150 mm ^* 2.1 mm was used for the separation of itaconic, succinic, citric, iso-citric, malic and fumaric acid, as well as the possible methyl- and ethyl ester of itaconic acid with gradient elution. Eluents A consists of LC/MS grade water, containing 0.1 % formic acid, and eluents B consists of acetonitrile, containing 0.1 % formic acid. The flow-rate was 0.35 ml/min and the column temperature was kept constant at 40 °C. The gradient started at 95% A, was increased linear to 21 .6 % B in 6 minutes, then increased to 40 % B and was constant for 1 .3 minutes (wash), then immediately decreased to 95% A and stabilized for 1 .4 minutes (equilibration). The injection volume used was 5 ul.

A Waters Xevo API was used in electrospray (ESI) in negative ionization mode, using multiple reaction monitoring (MRM). The ion source temperature was kept at 150 °C, whereas the desolvation temperature is 600 °C, at a flow-rate of 700 L/hr.

For itaconic acid and the other compounds of the Krebs cycle the deprotonated molecule was fragmented with 13 eV, resulting in specific fragments from losses of H20 and C02. The standards of reference compounds were analysed to confirm retention time, calculate a response factor for the respective ions, and was used to calculate the concentrations in fermentation samples. All samples were diluted appropriately (5-25 fold) in water to overcome ion suppression and matrix effects during LC-MS analysis. Accurate mass analysis of itaconic acid and esters of itaconic acid. To confirm the elemental composition of the compounds analyzed accurate mass analyses was performed with the same chromatographic system as described above, coupled to a LTQ orbitrap (ThermoFisher). Mass calibration was performed in constant infusion mode, using a NaTFA mixture (ref), in such a way that during the experimental set-up the accurate mass analysed could be fitted within 2 ppm from the theoretical mass, of all compounds analysed.

2.4 Itaconic acid and itaconate methyl ester concentrations

Itaconic acid and itaconate methyl ester concentrations per pathway group and growth condition aerobic or anaerobic are shown in 7. The overall specific productivity of itaconic acid and itaconate methyl ester per biomass per hour at 106 hours is shown in 8.

Table 7: Itaconic acid (IA) and methyl itaconate (Ml) concentration results

Table 8: Specific productivity of Itaconic acid (IA) and methyl itaconate (Ml) at 106 hours

Biomass Specific Specific

Pathway Strain Condition dry weight productivity IA productivity Ml

[g/kg] [mg/g/h] [mg/g/h]

1 ITA06 aerobic 20.2 0.15 0.15

3 ITA07 aerobic 16.3 0.23 0.20

2 ITA09 aerobic 16.4 0.42 0.20

1 ITA06 anaerobic 1.6 0.15 0.18

3 ITA07 anaerobic 1.6 0.20 0.19

2 ITA09 anaerobic 2.6 0.46 0.31