# Patent

## Title
Dicarboxylic acid production by fermentation at low pH

## Abstract

The present invention relates to a process for the production of a dicarboxylic acid. The process comprises fermenting a yeast in the presence of a carbohydrate-containing substrate and low amounts of oxygen at a pH value at which at least 50% of the dicarboxylic acid is in the acid form. The process of the present invention allows for high yields of the dicarboxylic acid product and is more cost-effective than existing processes in which the salt is produced which during recovery has to be converted to the acid. It also leads to a simpler and more convenient downstream processing.

## Background
This application is the U.S. national phase of International Application No. PCT/EP2009/056181 filed 20 May 2009, which designated the U.S. and claims priority to EP Application No. 08159891.4, filed 8 Jul. 2008, the entire contents of each of which are hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to a process for the production of dicarboxylic acids. In particular, it relates to the production of dicarboxylic acids by fermentation of a yeast.
BACKGROUND OF THE INVENTION
Dicarboxylic acids, such as fumaric acid and succinic acid, are important compounds which are used in the food industry for the preparation and preservation of food, in the medical industry for the formulation of medical products and other industrial uses, such as monomers for (bio)polymers. To meet the increasing need for dicarboxylic acids, more efficient and cost effective production methods are being developed. Traditionally, dicarboxylic acids are made by fermentation of bacteria, which can produce large amounts of dicarboxylic acids. This is for example described in U.S. Pat. No. 5,573,931 which describes a method for producing succinic acid in high concentrations by employing a bacterial strain. However, one major drawback associated with the use of bacteria for producing dicarboxylic acids is the formation of dicarboxylic acid salt. If bacteria are used, the pH during fermentation needs to be maintained in the range of pH 6-7, which is higher than the pKa values of all dicarboxylic acids. As a consequence, most acids will be produced in their salt form and the salts will have to be converted into the acid. This is not practical or efficient in large-scale production processes and raises production costs. Also microorganisms other than bacteria have been employed for the production of organic acids. EP 0 424 384 discloses an aerobic process for the production of organic acids by Rhizopus in a medium containing calcium carbonate. EP 1 183 385 discloses genetically manipulated yeast cells with a Crabtree negative phenotype and containing an exogenous nucleus acid molecule for the production of lactic acid.
DETAILED DESCRIPTION
The present invention relates to a process for the production of a dicarboxylic acid. The process comprises fermenting a yeast in the presence of a carbohydrate-containing substrate and low amounts of oxygen at a pH value which is below the pKa of the dicarboxylic acid. The process of the present invention allows for high yields of the dicarboxylic acid product, allows for a simpler downstream processing and is more cost-effective than existing processes in which the salt is produced which has then to be converted to the acid. Since dicarboxylic acids have more than one pKa value, the pH should be below the lowest pKa of the dicarboxylic acid. For most acids, the pH will typically be in the range of pH 1.0 to pH 5.5, preferably between pH 2.0 and pH 4.0. In one embodiment, succinic acid is produced at a pH value of 3.0. Another advantage is that due to the low pH the risk of contamination is reduced.
The acid production phase is preferably preceded by a biomass formation phase for optimal biomass production. In the biomass formation phase the pH is in the range of pH 2 to pH 7. Preferably, the pH is in the range of pH 3 to pH 6, more preferably, the pH is in the range of pH 4 to pH 5.
The process according to the present invention is more cost-effective and may lead to a 30% lower cost price. One of the reasons is that titrant costs are significantly reduced.
The process may be used for the production of any dicarboxylic acid. Suitable examples include adipic acid, fumaric acid, itaconic acid, succinic acid, malic acid, oxalic acid. Preferably, the dicarboxylic acid is succinic acid, fumaric acid or malic acid.
The yeast which is used in the process may be any suitable yeast. Suitable examples of yeasts include Saccharomyces, Schizosaccharomyces, Kluyveromyces, Candida, Pichia and Yarrowia, such as species of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyvermoces lactis, Candida sonorensis, Pichia stipidis and Yarrowia lipolytica. In one embodiment, the eukaryotic microorganism used in the process is a Saccharomyces cerevisiae, a microorganism which is a widely used industrially interesting microorganism.
In a preferred embodiment the yeast according to the present invention is a genetically modified yeast. As used herein, a genetically modified yeast in the process according to the present invention is defined as a yeast cell which contains, or is transformed or genetically modified with a nucleotide sequence or polypeptide that does not naturally occur in the yeast cell, or it contains additional copy or copies of an endogenous nucleic acid sequence. A wild-type yeast cell is herein defined as the parental cell of the recombinant cell.
Preferably, the yeast in the process according to the present invention is a genetically modified yeast comprising a nucleotide sequence encoding a heterologous enzyme selected from the group consisting of a phosphoenolpyruvate carboxykinase, fumarate reductase and a fumarase. Preferred embodiments of the heterologous enzymes are as defined herein below.
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.
Preferably the genetically modified yeast comprises a nucleotide sequence encoding a phosphoenolpyruvate carboxykinase. The PEP carboxykinase (EC 4.1.1.49) preferably is a heterologous enzyme, preferably derived from bacteria, more preferably the enzyme having PEP carboxykinase activity is derived from Escherichia coli, Mannheimia sp., Actinobacillus sp., or Anaerobiospirillum sp., more preferably Mannheimia succiniciproducens or Actinobacillus succinogenes. In one embodiment the PEP carboxykinase is derived from Actinobacillus succinogenes (PCKa), wherein the PCKa preferably has been modified to replace EGY at position 120-122 with a DAF amino acid sequence. Preferably, a yeast cell according to the present invention is genetically modified with a PEP carboxykinase which has at least 80, 85, 90, 95, 99 or 100% sequence identity with amino acid sequence of SEQ ID NO: 6.
In another preferred embodiment a genetically modified yeast in the process according to the present invention comprises a nucleotide sequence encoding a fumarate reductase. Preferably, the fumarate reductase is a heterologous enzyme, preferably a NAD(H)-dependent fumarate reductase, which may be derived from any suitable origin, for instance bacteria, fungi, protozoa or plants. Preferably, a yeast in the process according to the invention comprises a heterologous NAD(H)-dependent fumarate reductase, preferably derived from a Trypanosoma sp., for instance a Trypanosoma brucei. In a preferred embodiment the nucleotide sequence encoding a NAD(H)-dependent fumarate reductase is expressed in the cytosol. In the event that the nucleotide sequence encoding a NAD(H)-dependent fumarate reductase comprises a peroxisomal or mitochondrial targeting signal, it may be essential to modify or delete a number of amino acids (and corresponding nucleotide sequences in the encoding nucleotide sequence) in order to prevent peroxisomal or mitochondrial targeting of the enzyme. The presence of a peroxisomal targeting signal may for instance be determined by the method disclosed by Schlüter et al, Nucleic acid Research 2007, 35, D815-D822. Preferably, a yeast cell according to the present invention is genetically modified with a NAD(H)-dependent fumarate reductase, which has at least 80, 85, 90, 95, 99 or 100% sequence identity with SEQ ID NO: 7.
In another preferred embodiment a genetically modified yeast in the process according to the present invention comprises a nucleotide sequence encoding a fumarase, which may be a heterologous or homologous enzyme. A nucleotide sequence encoding a heterologous fumarase may be derived from any suitable origin, preferably from microbial origin, preferably from a yeast, for instance Saccharomyces cerevisiae or a filamentous fungus, for instance Rhizopus oryzae. Preferably, a yeast in the process according to the present invention overexpresses a nucleotide sequence encoding a fumarase that has at least 70%, preferably at least 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, or 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8.
In another preferred embodiment a genetically modified yeast in the process according to the present invention further comprises a nucleotide sequence encoding a malate dehydrogenase (MDH) which is active in the cytosol upon expression of the nucleotide sequence. Preferably, the MDH lacks a peroxisomal or mitochondrial targeting signal in order to localize the enzyme in the cytosol. A cytosolic MDH may be any suitable homologous or heterologous malate dehydrogenase. Preferably, a yeast cell according to the present invention comprises a nucleotide sequence encoding a malate dehydrogenase that has at least 70%, preferably at least 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, 99% sequence identity with the amino acid sequence of SEQ ID NO: 9.
In another embodiment, a genetically modified yeast in the process according to the invention comprises a nucleotide sequence encoding a dicarboxylic acid transporter protein, preferably a malic acid transporter protein (MAE). A dicarboxylic acid transporter protein may be a homologous or heterologous protein. Preferably the dicarboxylic acid transporter protein is a heterologous protein. A dicarboxylic acid transporter protein may be derived from any suitable organism, preferably from Schizosaccharomyces pombe. Preferably, a dicarboxylic acid transporter protein is a malic acid transporter protein (MAE) which has at least 80, 85, 90, 95 or 99% or 100% sequence identity with SEQ ID NO: 10.
Preferably, the yeast used in the process according to the present invention is a genetically modified yeast comprising a heterologous PEP-carboxykinase, a heterologous NAD(P)H-dependent fumarate reductase, a heterologous fumarase, a heterologous malic acid transporter protein and a cytosolic malate dehydrogenase. Preferred embodiments of these enzymes are as defined herein above.
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, sequence identities or similarities 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.
Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include BLASTP and BLASTN, publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894). Preferred parameters for amino acid sequences comparison using BLASTP are gap open 11.0, gap extend 1, Blosum 62 matrix.
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.
In a preferred embodiment, the yeast in the process according to the invention overexpresses the nucleotide sequences encoding any of the enzymes as defined herein above. There are various means available in the art for overexpression of nucleotide sequences encoding enzymes in a yeast in the process 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 carbohydrate-containing substrate may be any carbohydrate containing substrate including molasses, sugar cane juice, pentoses and hexoses, such as glucose, fructose, xylose, arabinose. Preferably, the carbohydrate-containing substrate is a glucose-containing substrate, such as maltose, sucrose, glucose or a glucose syrup. The carbohydrate content of the carbohydrate-containing substrate is preferably more than 50% w/w, more preferably more than 55%, 60%, 65%, 70%, 75%, 80% w/w, most preferably more than 85%, 90%, 95% or 99% w/w on the basis of dry matter content.
The process according to the present invention, preferably comprises fermenting a yeast under carbon (C)-limited conditions. C-limited conditions are defined herein as a concentration of dissolved carbohydrate of below 1 g/l, preferably below 0.9 g/l, 0.8 or below 0.5 g/l of dissolved carbohydrate. It was found that fermenting yeast under C-limited conditions resulted in an increased yield of succinic acid as compared to non-C-limited conditions.
The oxygen for the fermentation may be supplied in any suitable form. In one embodiment, the oxygen is supplied in the form of air. The oxygen should be supplied in low amounts. This is reflected in the oxygen uptake rate (OUR) and/or the specific oxygen uptake rate (qO2) of the yeast. The OUR in the present invention is lower than about 8.0 mmol oxygen/L/hour, preferably lower than about 5.0, 4.0, 3.0, or 2.0 mmol oxygen/L/hour, more preferably lower than about 1.0, or 0.5 mmol oxygen/L/hour, preferably above 0.01 mmol oxygen/L/hour.
The specific oxygen uptake rate (qO2) in the process of the invention ranges between 8 mmol oxygen/g biomass dry weight/hour to 0.5 mmol oxygen/g biomass dry weight/hour, preferably between 5, 4, 3, or 2 mmol oxygen/g biomass/hour to about 0.4, 0.3, or 0.2 mmol/oxygen/g biomass/hour.
The process according to the present invention may be carried out in batch, fed-batch or continuous mode. These fermentation modes are known to the skilled man in the art. Depending on the fermentation mode, the biomass concentration during fermentation may vary more or less during fermentation. In batch and fed-batch mode the biomass concentration usually increases. Consequently, the specific oxygen uptake rate usually decreases in a batch and fed-batch mode.
The temperature of the process is typically between 10 and 40 degrees C., preferably between 20 and 35 degrees C., more preferably between 30 and 35 degrees C.
In one embodiment of the process according to the invention, an extra electron donor is present in addition to the carbohydrate-containing substrate. The extra electron donor is preferably an organic electron donor. Suitable examples of organic electron donors include glycerol, formate and polyols, such as mannitol, sorbitol and xylitol.



FIGURES
FIG. 1. Effect of the applied OUR on the succinic acid production after 90 h at pH 3.



EXAMPLES
Example 1
Succinic Acid Production by Saccharomyces cerevisiae 
1.1. Construction Yeast Strain
1.1.1. Construction of Expression Constructs
Expression construct pGBS414PPK-3 was created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS414 (Sirkoski R. S, and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the phosphoenolpyruvate carboxykinase (origin Actinobacillus succinogenes) synthetic gene construct (SEQ ID NO: 1). The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PPK-1. Subsequently, pGBK414PPK-1 was restricted with AscI and NotI. To create pGBS414PPK-3, an AscI/NotI restriction fragment consisting of glycosomal fumarate reductase from T. brucei (FRDg) synthetic gene construct (SEQ ID NO: 2) was ligated into the restricted pGBS414PPK-1 vector. The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PPK-3.
The expression construct pGBS415FUM-3 was created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS415 (Sirkoski R. S, and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the fumarase (origin Rhizopus oryzae) synthetic gene construct (SEQ ID NO: 3). The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS415FUM-1. Subsequently, pGBK415FUM-1 was restricted with AscI and NotI. To create pGBS415FUM-3, an AscI/NotI restriction fragment consisting of peroxisomal malate dehydrogenase from S. cerevisiae (MDH3) synthetic gene construct (SEQ ID NO: 4) was ligated into the restricted pGBS415FUM-1 vector. The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS415FUM-3.
The expression construct pGBS416MAE-1 was created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS416 (Sirkoski R. S, and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the Schizosaccharomyces pombe malate transporter synthetic gene construct (SEQ ID NO: 5). The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS416MAE-1.
1.1.2. Construction S. cerevisiae Strain
Plasmids pGBS414PPK-3, pGBS415FUM-3 and pGBS416MAE-1 (described under 1.1.) were transformed by electroporation into S. cerevisiae strain RWB064 (MATA ura3-52 leu2-112 trp1-289 adh1::lox adh2::lox gpd1::Kanlox) to create strain SUC-200, overexpressing PCKa, MDH3, FUMR, FRDg and SpMAE1. All genes were codon pair optimized for expression in S. cerevisiae according to WO2008/000632.
1.2. Succinic Acid Production S. Cerevisiae at Low pH and Oxygen Limited Conditions
The yeast strain SUC-200 (MATA ura3-52 leu2-112 trp1-289 adh1::lox adh2::lox gpd1::Kanlox, overexpressing PCKa, MDH3, FUMR, FRDg and SpMAE1), was cultivated in shake-flask (2×300 ml) for 3 days at 30° C. and 220 rpm. The medium was based on Verduyn (Verduyn et. al., 1992, Yeast 8, 501-517), but modifications in carbon and nitrogen source were made as shown in Table 1.







TABLE 1







Preculture shake flask medium composition











Concentration



Compound
(g/l)







C6H12O6•H2O
20.0



(NH2)2CO
2.3



KH2PO4
3.0



MgSO4•7H2O
0.5




1




1











aVitamin solution











Concentration


Component
Formula
(g/kg)





Biotin (D−)
C10H16N2O3S
0.05


Ca D(+) panthothenate
C18H32CaN2O10
1.00


Nicotinic acid
C6H5NO2
1.00


Myo-inositol
C6H12O6
25.00


Thiamine chloride
C12H18Cl2N4OS•xH2O
1.00


hydrochloride


Pyridoxol hydrochloride
C8H12ClNO3
1.00


p-aminobenzoic acid
C7H7NO2
0.20










bTrace elements solution











Concentration



Formula
(g/kg)







C10H14N2Na2O8•2H2O (EDTA)
15.00



ZnSO4•7H2O
4.50



MnCl2•2H2O
0.84



CoCl2•6H2O
0.30



CuSO4•5H2O
0.30



Na2MoO4•2H2O
0.40



CaCl2•2H2O
4.50



FeSO4•7H2O
3.00



H3BO3
1.00



KI
0.10










Subsequently, the content of the shake-flasks was transferred to 10 L fermenter (Startweight 6 kg), which contained the following medium:








TABLE 2







Main fermentation medium composition













Concentration



Raw material
Formula
(g/l)















Ammonium sulphate
(NH4)2SO4
2.5



Potassium dihydrogen
KH2PO4
3.0



phosphate



Magnesium sulphate
MgSO4•7H2O
0.5



Trace element solution

1



Vitamin solution

1










The pH was controlled at 3.0 by addition of 6 N KOH. The temperature was controlled at 30° C. Glucose concentration was kept limited (<1 g/l) by controlling feed addition to the fermenter. Different oxygen uptake rates (OUR) were applied to the fermentation, which resulted in oxygen limitation (FIG. 1).
0.33 vvm of total gasflow was applied, including 10% CO2 to supply enough CO2 for efficient succinic acid production.
The results of different applied OUR's on the succinic acid production are shown in FIG. 1. A minimal amount of aeration was required to sustain succinic acid production at a pH of 3. An OUR above 5 mmol/L/h resulted in lower succinic acid production.
During the cultivation of 90 hours, growth occurred to a typical biomass concentration of 8 g dry weight/L. Consequently, the specific oxygen uptake rate (qO2) decreased constantly during the fermentation. An OUR of 10 mmol/L/h applied in one fermentation correlated with a qO2 decreasing from 10 to 1.25 mmol/g biomass dry weight/h and an OUR of 1 mmol/L/h correlated with a qO2 decreasing from 1 to 0.1 mmol/g biomass dry weight/h.

## Claims

1. A process for the preparation of succinic acid, comprising: fermenting a yeast in the presence of a carbohydrate-containing substrate and low amounts of oxygen at a pH value which is below the lowest pKa of the succinic acid, wherein the oxygen is supplied at a specific oxygen uptake rate ranging from 8 to 0.2 mmol oxygen/g biomass dry weight/hour, and wherein, in an acid production phase, said process produces more than 10 g/L succinic acid during cultivation for 90 hours.

2. The process according to claim 1, wherein the pH is in the range of pH 1.0 to pH 5.5.

3. The process according to claim 1, comprising fermenting yeast under carbon-limited conditions.

4. The process according to claim 1, wherein the process is carried out in the presence of an extra electron donor.

5. The process according to claim 1, wherein the yeast is a Saccharomyces cerevisiae.

6. The process according to claim 1, wherein the yeast is a genetically modified yeast.

7. The process according to claim 6, wherein the genetically modified yeast comprises a nucleotide sequence encoding a heterologous enzyme selected from the group consisting of a phosphoenol pyruvate carboxykinase, fumarate reductase and a fumarase.

8. The process according to claim 1, wherein the pH is in the range of pH 2.0 to pH 4.0.

9. The process according to claim 1, wherein the pH is 3.0.

10. The process according to claim 1, wherein the oxygen is supplied at a specific oxygen uptake rate ranging from 5 to 0.2 mmol oxygen/g biomass dry weight/hour.

11. The process according to claim 3, wherein the carbon-limited conditions comprise a dissolved carbohydrate concentration below 1 g/l.

12. The process according to claim 3, wherein the carbon-limited conditions comprise a dissolved carbohydrate concentration below 0.5 g/l.

13. The process according to claim 4, wherein the extra electron donor is an organic electron donor.

14. The process according to claim 1 which produces more than 12 g/L succinic acid during cultivation for 90 hours.