EP2791332A1 - Fungal strains with genetic modification relating to a carboxylic acid transporter - Google Patents

Abstract

The invention relates to fungal strains having at least one genetic modification which leads to a reduction of the activity of at least one fungal carboxylic acid transporter and to a method for producing or using said fungal strains.

Description

 Thyssen Krupp Uhde GmbH

Fungal strains with genetic modification concerning a carboxylic acid transporter

The present invention relates to fungal strains with genetic modification relating to a carboxylic acid transporter, and to processes for producing or using such fungi. FIELD OF THE INVENTION

Interest in biotechnologically produced carboxylic acids, such as the Citrate cycle, as a potential intermediate source material and as an alternative to petrochemical production processes in industry is steadily increasing. Many of these carboxylic acids have the potential to act as a so-called building block chemical. Important precursors for chemical synthesis, polyester production and other processes can be formed from said carboxylic acids. For example, currently important for industrial purposes succinate is still mainly obtained petrochemically from maleic anhydride starting from butane. The petrochemical production of succinate using heavy metal catalysts, organic solvents, high temperatures and pressures is costly.

Therefore, the market for succinate has been relatively small so far. Due to rising oil prices, but also because of the continuing need for synthetic products, the biotechnological production of carboxylic acids, in particular of succinate, and the development moves corresponding processes increasingly in the focus of the manufacturing industry.

Carboxylic acids such as succinate are naturally produced by many microorganisms, plants and animals as an intermediate in the central metabolism or as a metabolic end product. The majority of natural and optimized producers are bacterial strains that accumulate, for example, succinate, especially under anaerobic conditions. Due to the disadvantages of bacterial production methods, such. However, for example, the need for complex culture media, the potential pathogenicity, and especially the low acid tolerance, microbial fungi are increasingly becoming the focus of research into the development of processes for the production of organic acids such as succinate.

Various genetic modifications are discussed to affect especially in succinate formation in fungi. Thus, several genes of the citrate cycle were disrupted, whereby it was shown that the deletion of SDH1, which codes for a subunit of SDH (succinate dehydrogenase), to a 1.6-fold increase in succinate production, in combination with a FUM1 deletion (gene coding for Fumarase) even led to a 2.6-fold increase in succinate production (Arikawa et al., 1999b). The disruption of SDH genes, which consists of at least four subunits in S. cerevisiae (Ghezzi et al., 2009) in sake strains of the genus Saccharomyces, resulted in a reduction in SDH activity and an increase in succinate formation, with a maximum of 0.3 g L ^"1 succinate were formed (Kubo et al. 2000). using these findings S. cerevisiae strains with the aim of Succinatproduktion were subsequently designed for industrial use (Raab and cross-201 1). concomitant deletion of SDH1, SDH2 , IDH1 (the isocitrate dehydrogenase gene), and IDP1 (which encodes a mitochondrial isoenzyme of IDH), a maximum of 3.62 g of L ^{" 1} succinate were produced after 168 hours. However, ethanol, glycerol, acetate, α-ketoglutarate (AKG) and pyruvate were also formed as by-products. Other changes, such as the repressible promoter-controlled expression of IDHI and SDH2 or IDHI and DICI encoding a dicarboxylate transporter to transport succinate and malate from the cytosol into the mitochondria, have been described in the literature (Raab and Lang 2011).

In this context, an attempt was made to deregulate the expression of additional genes (PYC1, ACS1, CIT1, ACO1, ICLI, MLSI, MDH2 and CIT2) by control of a constitutive promoter (pADHI promoter of alcohol dehydrogenase) or genes of craptree-negative yeasts to replace (Raab and Lang 2011).

US20110104771 discloses that a S. cerevisiae strain with an ICL gene from K. lactis, the malate synthase gene from S. cerevisiae, with a gene for PEPCK from M. succiniciproducens, the gene MDH2 from S. cerevisiae and with genes for fumarase ( from Rhizopus oryzae) and fumarate reductase (from Trypanosoma brucei) can produce up to 6.3 g of L ^"1 succinate after 7 days.

SUMMARY OF THE INVENTION It is an object of the invention to provide methods or organisms by means of which the production of organic acids can be economically achieved.

Another object of the invention is to provide processes or organisms for the production of organic acids which have advantages over the bacterial processes.

DETAILED DESCRIPTION OF THE INVENTION These objects are achieved by the features of the independent claims. The Subclaims indicate preferred embodiments.

Accordingly, a fungal strain is provided having a genetic modification that results in a reduction in the activity of at least one fungal carboxylic acid transporter.

The carboxylic acid transporter in question is a membrane transport protein capable of transporting carboxylic acids through the cell membrane and possibly the cell wall. These are preferably transporters for dicarboxylic acids, preferably transporters for carboxylic acids of the citrate cycle or neighboring metabolic pathways. These may be symporters (co-transporters), but also uniporter or antiporter, most preferably transporters for at least one organic acid selected from the group comprising fumarate, lactate, pyruvate, malate, malonate and / or succinate.

For Kluyveromyces lactis and Schizosaccharomyces pombe already Dicarboxylatpermeasen have been described, which mediate the transport of succinate / malate / fumarate (KUen2p) or lactate / pyruvate (KLJenlp) and the transport of succinate / malate / malonate (SpMaelp). The first two belong to the group of JEN transporters, while the latter belong to the group of MAE transporters.

As used herein, the term "targeting" is intended to clarify that said carboxylic acid transporter is encoded in the genome of the fungus. Alternatively, the term "homologous" may also be used. It is particularly preferred that said fungus has increased extracellular carboxylic acid production.

The term "carboxylic acids" as used herein is identical to the term "organic acids" and refers to all those metabolic products of said fungus which may be referred to as carboxylic acids. The term preferably denotes the carboxylic acids of the citrate cycle and of neighboring metabolic pathways, in particular pyruvate, α-ketoglutarate, Malate, oxaloacetate, citrate, isocitrate, fumarate, malonate, lactate and / or succinate. Most of these acids are dicarboxylic acids.

Also provided is a method of genetically modifying a fungal strain to increase extracellular carboxylic acid production, said method resulting in a reduction in the activity of at least one fungal carboxylic acid transporter.

It is preferably provided that the reduction of the activity of the carboxylic acid transporter is achieved by at least one property or a step selected from the group comprising: a) inhibiting or reducing the expression of a gene coding for a carboxylic acid transporter

 b) expression of a dysfunctional or diminished active carboxylic acid transporter, and / or

 c) inhibiting or reducing the activity of the expressed carboxylic acid transporter is accomplished.

Inhibition or reduction of expression may be accomplished, for example, by inhibiting or reducing transcription of the coding gene or translation of the generated mRNA. Expression of a dysfunctional or depressed active carboxylic acid transporter can be accomplished, for example, by deletion, insertion, substitution or point mutation in the coding gene.

For example, the following methods are suitable: In gene silencing, gene regulation takes place by inhibiting the transfer (transcription) of genetic information from the DNA to the mRNA (transcriptional gene silencing) or the subsequent translation (translation) of the mRNA stored information into a protein (post-transcriptional gene silencing). Transcriptional gene silencing is a result of epigenetic changes in DNA, such as DNA methylation or histone modifications in particular. These modifications of the histone ends create a kind of heterochromatic state around the gene which prevents it from binding to the transcription engine (RNA polymerase, transcription factors, etc.). The classic example is the phenomenon called position effect variegation (PEV). It alters the chromatin state and thus controls the transcriptional activity of the gene or gene region concerned. Post-transcriptional gene silencing (PTGS) refers to the processes of gene silencing that occur only after the transcription of the genetic information from the DNA to the transmitting mRNA. Forms of post-transcriptional gene silencing include, in particular, nonsense-mediated mRNA decay (NMD) and RNA interference (RNAi). While nonsense-mediated mRNA decay serves primarily to prevent nonsense point mutations, RNA interference is a predominantly regulatory process involving specific RNA molecules, such as miRNA and siRNA. Post-transcriptional gene silencing can result in increased degradation of the mRNA of a particular gene. The degradation of the mRNA prevents translation and thus the formation of the specific gene product (usually a protein). In addition, gene-specific direct inhibition of translation as a result of post-transcriptional gene silencing is possible. Gene knockout is understood to mean the complete shutdown of a gene in the genome of an organism. Switching off the gene is achieved by gene targeting. A deletion, also gene deletion, in genetics is a variant of the gene mutation or in which a nucleotide sequence or a part up to the entire chromosome is missing. A deletion is therefore always a loss of genetic material. Any number of nucleobases may be deleted, from a single base (point mutation) to the chromosome. A distinction is made between the interstitial and the terminal deletion. The former describes a loss within the chromosome, the latter a loss of an end portion, ie a part of the telomeric region.

As a consequence of the deletion, after translation, the mRNA produced from the DNA can be transformed create a faulty protein. Because deletion of base pairs can cause a frame shift mutation if a number of base pairs that are not divisible by three are removed. In site-specific or targeted mutagenesis, a targeted modification of the DNA is made possible by means of a recombinant DNA. It can be used to selectively exchange individual nucleobases of a gene or whole genes are removed. Alternatively, by means of a deletion cassette that has been integrated into a vector, a deletion can be generated in the gene to be mutated.

Alternatively, the inhibition or reduction of expression of a gene may be by cloning a heterologous promoter which is inhibitable by an exogenous factor, for example a tetracycline-regulated tetO promoter. Likewise, the inhibition or reduction of the expression of a gene can be carried out by cloning a heterologous promoter which is weaker than the relevant homologous promoter, for example pPOT1.

The inhibition or reduction of the activity of the expressed carboxylic acid transporter may be accomplished, for example, by the administration of inhibitors or the synchronous expression of suitable heterologous inhibitors. The said fungus strain is preferably a strain of microbial fungi.

The term "microbial fungus" as used herein is intended to include in particular those fungi which can be cultured by biotechnological methods and are particularly suitable for fermentative production processes, for example in suspension cultures.

It is preferably provided that the fungus is a member of the group of ascomycetes (Ascomycota). Particularly preferably, it is a member of the group of Hemiascomycetes or Euascomycetes. In a further preferred embodiment, said fungal strain is um a yeast or a mold. Said fungus particularly preferably belongs to a genus selected from the group comprising Saccharomyces; Schizosaccharomyces; Wickerhamia; Debayomyces; Hansenula; Hanseniospora; Pichia; Kloeckera; Candida; Zygosaccharomyces; Ogataea; Kuraishia; Komagataella (= Pichia); Yarrowia; Metschnikowia; Wiüiopsis; Nakazawaea; Kluyveromyces; Cryptococcus; Torulaspora; Torulopsis; Bullera; Rhodotorula; Sorobolomyces; Pseudozyma; Saccharomycopsis; Saccharomycodes; Aspergillus; Penicillium; Rhizopus; Trichosporon and / or Trichoderma.

In a further preferred embodiment, it is provided that the at least one carboxylic acid transporter originates from the group of the JEN transporters and / or the MAE transporters.

For Kluyveromyces lactis and Schizosaccharomyces pombe already Dicarboxylatpermeasen have been described, which mediate the transport of succinate / malate / fumarate (KUen2p) or lactate / pyruvate (KLJenlp) and the transport of succinate / malate / malonate (SpMaelp). The first two belong to the group of JEN transporters, while the latter belong to the group of MAE transporters.

To date, 35 proteins encoded by putative orthologues of the K. lactis genes KlJEN1 and KUEN2 have been found in 17 different species of fungi (13 hemiascomycetes, 7 euassomycetes). The number of JEN orthologues varies from one (eg, BS cerevisiae) to six orthologous genes in Y. lipolytica. The following table provides an overview of the group of JEN transporters, as well as, in the last few lines, the known MAE transporters:

Type Type Work name EMBL Swiss-Prot Length Number of

 (aa) transmembrane domains

JEN K. lactis KLLA-E-KLJEN1 AJ585426 Q70DJ7 600 12

 KLLA-F-KLJEN2 AJ627630 Q701Q9 528 1 1

Sac. cerevisiae SACE-K-JEN1 U24155 P36035 616 12

Sac. paradox SAPA-X1 -J101 616 12

Sac. mikatae SAMI-X1-J101 616 12

Sac. kudriavzevii SAKU-X1 -J101 614 10

Sac. bayanus SABA-X1 -J101 615 12

K. thermotolerans KLTH-G-J201 542 1 1

 K. waltii KLWA-X1-J201 534 1 1

Sac. kluyveri SAKL-X1 -J101 598 10

 SAKL-X2-J201 523 1 1

SAKL-X3-J001 531 1 1

A. gossypii ASGO-A-J001 AAS50559 Q75E88 500 10

 ASGO-B-J201 AAS50561 Q75E76 478 9

ASGO-F-J101 AAS53704 Q753H9 500 9

D. hansenii DEHA-D-J202 CAG87482 Q6BR62 51 1 10

 DEHA-F-J101 CAG89500 Q6BL03 513 12

DEHA-F-J201 CAG89946 Q6BJV3 506 12

C. albicans CAAL-D-J001 EAK98054 Q5A5U2 513 10

 CAAL-C-J101 EAK97104 Q5A2W4 541 10

Y. lipolytica YALI-B-J101 CAG83351 Q6CE14 529 10

 YALI-C-J102 CAG82184 Q6CBU1 499 10

YALI-C-J201 CAG82410 Q6CB72 523 10

YALI-D-J204 CAG81250 Q6C8F4 502 10

YALI-D-J202 CAG81440 Q6C7X3 503 10

YALI-E-J203 CAG80310 Q6C3Q6 524 10

N. crassa NECR-X1 -J201 EAA33605 Q7SB47 557 1 1

 NECR-X2-J202 EAA32361 Q9P732 518 9

A. fumigatus ASFU-G-J202 EAL86916 Q4WGM5 501 1 1

 ASFU-H-J001 EAL85090 Q4WB22 520 10

ASFU-B-J201 EAL93241 Q4X1 M4 51 1 9

A. nidulans ASNI-A-J201 495 10

 ASNI-A-J202 514 1 1

A. oryzae ASOR-A-J201 508 1 1

 ASOR-G-J202 512 10

MAE S. pombe n / a n / a P50537 438 10

 Y. lipolytica n / a n / a n / a

Known carboxylic acid transporters from the group of JEN or MAE transporters in microbial fungal strains. A = Aspergillus; Sac = Saccharomyces; N = neurospora; Y =

Yarrowia; C = Candida; D = Debaromyces and K = Kluyveromyces It is preferably provided that the at least one carboxylic acid transporter originates from the group of JEN transporters and / or MAE transporters listed in the following table.

Experimentally, the gene in question was deleted by means of a deletion cassette integrated in a vector (SEQ ID Nos 1, 3, 5, 7, 9 and 11). The vector used is in each case pUCBM21, which was originally developed by Boehringer (SEQ ID Nos 2, 4, 6, 8, 10, 12). However, all other suitable vectors known in the art are also usable.

It is particularly preferred that the at least one carboxylic acid transporter is encoded by the gene YALI-D-J204 (EMBL-ID: CAG81250; Swiss-Prot ID: Q6C8F4). In the context of this application, said gene is always also referred to as "JEN4." It is likewise particularly preferred for said fungus to belong to the genus Yarrowia, particularly preferably to the species Yarrowia lipolytica.

As shown above, Y. lipolytica has a total of 6 JEW orthologs. This high number of genes for putative carboxylate transporters reflects the great potential of yeast Y. lipolytica for the production of organic acids, such as. Succinate, malate or fumarate. In addition, Y. lipolytica is extensively genetically and physiologically characterized, can utilize a broad spectrum of substrates and is tolerant to low pH.

Furthermore, it is preferably provided that said fungus contains at least one further genetic modification selected from the group comprising:

Reduction of the activity or expression of the succinate dehydrogenase (SDH), for example by replacement of the native promoter of SDH2 by a weak promoter, (in the case of glucose and glycerol as substrate, for example, pPOT1 is suitable) or by using a suitable deletion cassette,

 Increasing the activity or expression of Pyruvatcarboxylase (PYC), for example by overexpression of the corresponding gene PYC1, and / or

 Increasing the activity or expression of isocitrate lyase (ICL), for example by overexpression of the corresponding gene ICL1

 having.

All of these genetic modifications are related to the formation of carboxylic acids in the citrate cycle. The modifications mentioned are described in detail in the method section.

Other candidate genetic modification that can be used in combination with the above modifications are a reduction in the activity or expression of the genes SDH1, SDH3 and / or SDH4, the introduction of a heterologous gene selected from the group coding for phosphoenolpyruvate carboxykinase (PCK1), fumarate reductase and / or fumarase (FUM1), and / or the reduction of the activity or expression of the gene coding for isocitrate dehydrogenase (IDHI) and at least one the genes coding for the mitochondrial dicarboxylate carrier (DIC1) and / or SDH2, and optionally at least one of the genes FUMI, osmotic growth protein (OSMl), malate dehydrogenase (MDH3) and / or citrate synthase (CIT2). The reduction of expression can be effected, for example, by a heterologous promoter which is inhibitable by an exogenous factor, preferably a tetracycline-regulated tetO promoter. Alternative methods (gene silencing, gene knock out, deletion) are shown above

The genes mentioned are identified in the following table, taking as an example the UniProt entries for Saccharomyces cerevisiae. It is understood that the expert can easily find the corresponding genes in other fungal strains, in particular in Yarrowia, based on this information.

In a further embodiment of the invention, a process for the production of carboxylic acids is provided, wherein in the process a fungus strain according to one of the preceding claims is used. Particular preference is given to using hexoses, such as, for example, glucose or sucrose, or alcohols, for example glycerol, as carbon source. Glucose and sucrose are particularly suitable because they are inexpensive substrates. Glycerol is obtained in large quantities as a waste product, for example in the production of biodiesel and is therefore also a low-cost substrate. For glycerol also speaks that its absorption by the fungi is not affected by an impairment of a carboxylic acid transporter. Further, it is preferably provided that the fungal strains are cultured in a medium, and further wherein the oxygen uptake rate (OUR) is adjusted to a range between> 5 and <50 mmol 02 / l * h.

According to investigations of the applicant, the highest production rates are achieved in this area. Preferably, the oxygen uptake rate is adjusted to a range between> 10 and <40 mmol 02 / l * h. Particularly preferably, the oxygen uptake rate is adjusted to a range between> 20 and <30 mmol 02 / l * h. Most preferably, the oxygen uptake rate is controlled to a range between 25 + 3 mmol O 2 / l * h.

Technically, the oxygen input OTR in a fermenter can be influenced by the air aeration rate (1 min ^-1 ), the stirrer speed (U min ^-1 ), the pressurization of the fermenter and the gas composition (proportion O 2 in the gas mixture). The oxygen uptake rate OUR can be determined from well-known equations based on the known gas composition as well as the amount of gas and the measurement of the oxygen concentration in the exhaust gas.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 Schematic representation of the expression cassette for exchanging the SDH2

Promoter against the promoter of the genes ICL1, GPR1 or POT1. All expression cassettes contained URA3 as a selection marker flanked by the / ox sites and homologous regions of the SZ ⁾ H2 promoter and SDH2 ORF.

The SDH2 ORF was fused directly to the respective promoter. The pICL1-containing expression cassette was 4.9 kb in size, the pPOT1 cassette was 3.8 kb and the GPR1 B cassette was 4.2 kb in size. Figure 2 Plasmids pSpIvS-Ura, pSpPS-Ura and pSpGS-U r a m i t d e n

 Expression cassettes for replacing the SZ) H2 promoter against pICL1, pPOT1 or GPRIB. The selection marker is the marker gene URA3 flanked by the / ox sites. Fig. 3 Vector construction of the plasmid p64PIC starting from p64PYCl and p64ICL l. In addition to the desired genes with their own promoter and terminator region, p64PIC contained the URA3-AWe \ ura3d4 as multicopy marker gene and rDNA as integration sequence. Fig. 4 PYC and ICL activities of the wild-type strain Y. lipolytica Η222 and the

 Transformants Η222-ΑΖ8 T3, T4, T5 and Η222-ΑΖ9 T2 and T3 in MG medium. The strains were cultured in 100 ml of MG medium (growth medium) in 500 ml Erlenmeyer flasks at 28 ° C. and 220 rpm. The sampling for the activity measurement took place after 4 h.

Fig. 5 Schematic representation of the deletion of JEN 4 by homologous

Recombination in the promoter and terminator region with the deletion cassette contained in URA3. Fig. 6 Growth (a) and succinate production (b) of the wild-type strain Y. lipolytica

H222 and the transformants H222-AZ7 Tl 1 and T23 in YNB medium. The strains were cultured in 150 ml of culture medium in 500 ml Erlenmeyer flasks at 28 ° C. and 220 rpm with 5% glycerol. The succinate contents in the culture supernatant were determined by ion chromatography.

Figure 7 Strategy for construction of strain H222-AZ10.

Fig. 8 PYC activities of the wild-type strain Y. lipolytica H222 and the

 Transformants H222-AZ 10 T l, T5 and T9 in MG medium. The strains were cultured in 100 ml of MG medium (growth medium) in 500 ml Erlenmeyer flasks at 28 ° C. and 220 rpm. The sampling for the activity measurement took place after 4 h.

Fig. 9 Growth (a) and succinate production (b) of the wild-type strain Y. lipolytica

 H222 and the transformants H222-AZ7 TU, H222-AZ8 T3 and H222-AZ10 Tl, T5 and T9 in YNB medium. The strains were cultured in 150 ml of culture medium in 500 ml Erlenmeyer flasks at 28 ° C. and 220 rpm with 5% glycerol. The succinate contents in the culture supernatant were determined by ion chromatography.

10 maximum product amounts of the organic acids succinate, malate, α-ketoglutarate (AKG) and fumarate when cultivating the strains Y. lipolytica H222, H222-AZ8 T3, H222-AZ7 TU and H222-AZ10 T l, T5 and T9 in YNB medium. Cultivation in YNB medium with 5% glycerol as C source.

Fig. 11 Percent of the organic acids succinate (SA), malate (MA), α-ketoglutarate (AKG) and fumarate (FA) in the total acid product when cultivating the strains Y. lipolytica H222, H222-AZ8 T3, H222-AZ7 TU and H222-AZ10 Tl, T5 and T9 in YNB medium. Cultivation in YNB medium with 5% glycerol as C source.

Fig. 12 Maximum acid production of the MAE1 deleted transformants. Shown was the mean, maximum acid amount of the H222-SW2AMAE1 transformants TF3, TF7 and TF8, as well as the reference strain H222 from 3-fold cultivation in production medium according to Tabuchi et al. (1981).

 AKG = α-ketoglutarate, FU = fumarate, MA = malate, SUC = succinate

Fig. 13: Vector construction of plasmid p64PYCl starting from p64T.

Fig. 14: Southern blot analysis confirming the multiple integration of p64PYCl in Y. lipolytica H222-S4. The genomic DNA was completely digested with the EcoRV restriction enzyme. For the detection of PYCl, a specific probe was used for the PYC1-OKF (2, 1 kb), which was obtained by means of PCR and the primers PYC ORF for and PYC ORF rev. The black arrow indicates the 8.9 kb genomic (g) PYCl fragment and the red arrow indicates the 6.2 kb vectorial (v) PYC1 fragment. λ-molecular weight standard; 1-recipient strain H222-S4; 2- transformant H222-AK1-1; 3-transformant H222-AK1-2; 4 transformants H222-AK1-3; 5-transformant H222-AK1-4; 6-transformant H222-AK1-5; 7-transformant H222-AK1-6; 8 transformant H222-AK1-7.

FIG. 15 Kinetics of the specific pyruvate carboxylase activity of the transformants with a multiple integration of the PYCI expression cassette in comparison to the wild-type H222. The specific activities were determined during culture in 100 ml of minimal medium containing 1% glucose. The characteristic course of the specific activities resulted from the enzyme activity determinations of three independent cultivations. Growth (a) and succinate production (b) of strains Y. lipolytica H222 (wild-type) and H222-AM3 in Tabuchi medium. To assess the growth, the optical density of the culture was determined at 600 nm at certain times. The strains were in each 100 ml of production medium according to Tabuchi et al. (1981) in 500 ml Erlenmeyer flasks at 28 ° C and 220 rpm with 10% glycerol. The succinate contents were determined by ion chromatography. The course of the optical densities (OD ₆ oo) and the succinate production are each one of the experiments from several repetitions.

Composition of the culture medium

1. YNB medium

 Table la: Composition of the YNB culture medium 2. Trace elements YNB

3. Tabuchi medium

Table lb: Composition of the medium modified according to Tabuchi et al. (1981):

At the beginning of the work the Y. lipolytica wild-type strain H222 was selected for the investigation of succinate production. H222 had already proven to be a very good acid producer with respect to citrate or AKG production compared to other Y. lipolytica strains. H222 was cultured in a culture medium optimized for itaconic acid production by Candida strains. As C sources, glucose (10%), glycerol (10%) and sunflower oil (5%) were selected. Cultivation was carried out in 500 ml Erlmeyer flasks without baffling in 100 ml culture medium at 28 ° C. and 220 rpm. When using sunflower oil as C source, the liquid cultures were shaken in 500 ml Erlenmeyer flasks with baffles. The preculture was carried out in 50 ml of the same medium and was incubated for 2 to 3 days at 28 ° C and 220 rpm. The preculture was harvested by centrifugation (3,500 rpm, 5 min, RT) and taken up in production medium without C source, yeast extract and iron sulfate. After determination of the optical densities, the main cultures were inoculated with 100 ml each of the production medium at an optical density of 2 OD ml ^-1 , followed by cultivation at 28 ° C. and 220 rpm for up to 500 h The analysis of the concentrations during cultivation Organic acids secreted by Y. lipolytica were obtained by ion chromatography using the Ion Chromatography System ICS-2100 from Dionex (Sunnydale, USA). The ion chromatography systems contained the following components: Isocratic pump and conductivity detector IC20, eluent generator EG40, autosampler AS40 / autosampler AS50, self-regenerating suppressor ASRS, separation column IonPac AS 19 (2 x 250 mm) with pre-column IonPac AS 19 (2 x 50 mm), analysis software Chromeleon version 6.8. The separation parameters of the separation method are listed in Table 2. Preparation of Samples for Ion Chromatography: At certain times of culture, 1 ml each of the culture was taken and prepared according to the source of C contained therein. Apart from the cultures in which sunflower oil was used as the C source, the samples were centrifuged at maximum speed (5 min, 4 ° C) and the supernatant was used with appropriate dilution in bidistilled water for ion chromatography. If sunflower oil was used as the C source, it had to be removed before the analysis. For this purpose, the sample was mixed with 0.5 ml of n-hexane, shaken vigorously for 5 minutes and then centrifuged for 5 min at maximum speed and 4 ° C. The resulting aqueous lower phase was transferred to a new reaction vessel and added again with 0.3 ml of n-hexane, shaken and centrifuged again. Again, the aqueous phase was removed and used with appropriate dilution for the ion chromatographic analysis. Since the culture medium used here has not been optimized for Succinatproduktion and cultivating conditions such. B. pH and p0 ₂ , in Schüttelko Ibenmaß stab can not be kept permanently stable, was waived in this and in all subsequent cultivation experiments on the calculation of maximum productivity or maximum specific product formation rates.

Column IonPac AS 19

 Flow rate 0.3 ml min-1

 Injection volumes 10 μΐ

Eluent ddH ₂ 0

 KOH

 0-3 min: isocratic, 5 mM

 3-25 min: linear to 38 mM

 eluent

 25-36 min: isocratic 38 mM

 36-38 min: linear to 5 mM

 38-40 min: isocratic, 5 mM

 Pyruvate 7.5 min

 Malat 19.6 min

 Retention times (min) succinate 20.3 min

 α-ketoglutarate 23.4 min

 Fumarate 27.4 min

Table 2: Separation parameters of ion chromatography

 Retention times for the organic acids.

1. starting organisms

Further molecular biology of this strain required the introduction of auxotrophic / selection markers. The uracil-auxotrophic strain H222-S4 was already present at the beginning of the work (Mauersberger et al., 2001). Additional auxotrophic strains were constructed. Within this project only one uracil auxotrophic strain (H222-SW2) was used. H222-SW2 was generated by deletion of the Ku70 gene whose gene product plays a role in DNA recombination in H222-S4.

Expression cassettes were constructed which, in addition to the selection marker URA3, contained the corresponding promoter (pICL1, GPR1B or pPOT1) fused to the beginning of the SDH2-OKF and a homologous region of pSDH2 (FIG. 1). The selection marker URA3 was flanked by the so-called / ox sites, which enabled a possible later removal of this marker by means of the Cre / ox system. The homologous regions from pSDH2 and SDH2-OKF should serve for the homologous integration of this expression cassette.

The starting plasmid was pUCBM21 (Figure 2). In these, the 5DH2 promoter fragment pSDH2 (569 bp) amplified with the primers pSDH2-fw and pSDH2-rv was first integrated into the NcoI site. A 1242 bp fragment of the SDH2 ORF (primer: SDH2-fw and SDH2-rv) and a 776 bp pICL1 fragment (primer: pICL1-fw and pICL1-rv) were amplified by means of PCR and these were fused by means of overlap PCR (Primer: overlap-fw and overlap-rv). After digestion with the restriction enzymes BamHI and EcoRI, this pICL1-SDH2 'fragment was integrated into the vector containing pSDH2, likewise BamH1 and EcoRI. Into this construct was cloned the KpnI and BamRI digest from the plasmid p64PT (Gerber, 1999), the 2.16 kb / / / CZJ fragment between the KpnI and 5 amHI sites, and finally integration the loxR-URA J-ox cassette into this plasmid with the pSDH2-pICL1-SDH2 'cassette digested with XbaI and KpnI.

The 1.4 kb expression cassette was isolated from plasmid JMP1 13 (Fickers et al., 2003) using XbaI / KpnI digestion. Finally, the 8196 bp plasmid pSpIvS-Ura (Figure 2) was obtained which contained the expression cassette pSDH2-loxR-URA3-loxP-pICL1-SDH2 'to replace the native SZ ⁾ H2 promoter with the / CZJ promoter. This was isolated from the plasmid by KspMIMlul-V Qxdau and used for transformation by LiAc method into the uracil-auxotrophic recipient strain H222-SW2 (MATA ura3- 302 ku70A-1572) (Werner 2008). The pSDH2-loxR-URA3-loxP-pPOTI-SDH2 '-containing plasmid pSpPS-Ura (Figure 2) was constructed from pSpIvS-Ura. In contrast, p S p Iv S-Ura was digested with the restriction enzymes KpnI and Mlul and the 4773 bp fragment isolated (with pSDH2 and URA3). Then the PCR-derived SDH2 '- (838 bp, primer: SDH2-pPOTl-fw, SDH2-MluI-rv) and the pPOTI fragment (1538 bp, primer: Kpnl-pPOTl-fw and pPOTl-SDH2-rv ) in an overlap PCR with the primers KpnI-pPOTl-fw and SDH2-MluI-rv fused to a 2336 bp construct. This construct was digested with KpnI and Mlul and ligated to the 4773 bp fragment of pSpIvS-Ura. Finally, a 7.7 kb pSpPS-Ura plasmid was isolated which contained the expression cassette pSDH2-loxR-URA3-loxP-pPOT1-SDH2 'to replace the native 5DH2 promoter with the POT1 promoter. This expression cassette could also be isolated from the plasmid by means of ÄTspAI / M digestion and used for transformation into the uracil-auxotrophic recipient strain H222-SW2 (MATA ura3-302 ku70A-1572) (Werner 2008). Analogously to pSpPS-Ura, the pSpGS-Ura pSDH2-loxR-URA3-loxP-GPRIB-SDH2 '-containing plasmid was constructed (FIG. 2). GPR1B was isolated from plasmid pTBS1 by PCR (primers: KpnI-pGPR1-fw and pGPR1-SDH2-rv, fragment size: 1971 bp). The PCR-derived SDH2'- (838 bp, primer: SDH2-pGPR1-fw, SDH2-MluI-rv) was converted to a 2769 bp size in an overlap PCR with the primers KpnI-pGPR1-fw and SDH2-MluI-rv Construct merged. This construct was digested with KpnI and Mlul and ligated to the 4773 bp fragment of pSpIvS-Ura. Finally, a 7.5 kb pSpGS-Ura plasmid was isolated which contained the expression cassette pSDH2-loxR-URA3-loxP-GPR1B-SDH2 'to replace the native 5DH2 promoter with the Gijii promoter. To verify the sequence, the expression cassettes were sequenced.

These expression cassettes were transformed into the uracil-auxotrophic recipient strain H222-SW2 (MATA ura3-302 ku 70A-1572) (Werner 2008). Finally, the uracil prototrophic transformants H222-AZ 1 (pICL1-SDH2), and H222-AZ2 (pPOT1-SDH2) and H222-AZ3 (pGPR1-SDH2) were obtained. The integration of the corresponding expression cassettes was detected by PCR or Southern hybridization. 2. Combination of overexpression of PYCl and SDH2 promoter exchange or PYC1 + ICL1 and SDH2 promoter exchange

Initial strain was Η222-ΑΖ2. Furthermore, starting from Η222-ΑΖ2, strains were constructed in which both PYCl and the isocitrate lyase-encoding gene ICL1 are simultaneously overexpressed.

For the construction of a strain (Η222-ΑΖ8) in which both the 5DH2 promoter exchanged against the POT1 promoter and the pyruvate carboxylase-encoding gene PYCl are overexpressed, Η222-ΑΖ2 was used as starting strain. In order to be able to use this for further manipulation, uracil auxotrophy had to be generated in Η222-ΑΖ2. The expression cassette for the replacement of the 5DH2 promoter was constructed such that the marker gene URA3, which is essential for the cell's own uracil synthesis, was flanked by the so-called / ox sites. In order to remove this marker gene and thereby achieve the loss of uracil prototrophy, the so-called Crelox system was used. For this purpose, Η222-ΑΖ2 was transformed with the plasmid pUB4-Cre, which contained both a gene for the Cre recombinase and a hygromycin B resistance-mediating gene. The cells were then selected on hygromycin B-containing complete medium and then on minimal medium with or without uracil. Transformants in which recombination of the / ox sites occurred with the help of Cre recombinase could no longer produce uracil due to the loss of URA3 caused by it and therefore could no longer grow on uracil-free medium.

From these uracil-auxotrophic transformants, a transformant was selected and used as H222-AZ2U for further work. This was followed by the integrative multicopy plasmid p64PYCl, which contained the PYCl gene with its own promoter and terminator regions coding for pyruvate carboxylase in Y. lipolytica and the URA3 allele ura3d4 as multicopy marker gene and rDNA as integration sequence by means of lithium acetate method (Barth and Gaillardin 1996) into uracil auxotrophic strain H222-AZ2U. There was a selection of the uracil prototrophic transformants uracil-free minimal medium with glucose as C source. From the transformants obtained, three were selected and checked for multiple integration of p64PYCl by PCR and Southern blot. For the detection of the PYC activity, all three transformants as well as the wild type H222 were cultured in minimal medium with glucose. After 4 hours cells were harvested and mechanically disrupted. The determination of the PYC activity in the cell-free extract was carried out according to van Urk et al. (1989). Cultivation was carried out analogously to the described cultivation of strains H222-AZ1 to H222-AZ3.

For the construction of a strain (H222-AZ9), in which both the 5DH2 promoter replaced by the POT1 promoter and over-expressed PYC1 and ICL1, was analogous to the construction of H222-AZ8 and a uracil auxotrophic derivative of H222-AZ2 as Initial strain used. The plasmid p64PIC to be transformed was constructed by restriction and ligation from the plasmids p64PYCl and p64ICL1 (Kruse et al., 2004). For this purpose, the approximately 4.6 kb Sphl-Fsp AI fragment was isolated from p64ICLI and integrated into the Sphl-FspAI cut vector p64PYCl (FIG. 3). The resulting plasmid p64PIC had a size of 15.4 kb and was linearized for transformation in the SacII site. The integration was verified by means of PCR or Southern Blot. Two selected transformants were checked for PYC and ICL activity (Table 3) according to Dixon and Kornberg, (1959) and finally cultured in YNB medium.

Stock solution final concentration

 Tris-HCl, pH 7.0 100 mM 53.3 mM

 Cysteine-HCl 20 mM 2 mM

MgCl ₂ 50mM 5mM

 D, L-Na isocitrate 16.7 mM 1.67 mM

 phenylhydrazine

33mM 3.3mM

HC1 Table 3: Composition of the reaction mixture for determining ICL activity. The principle of the assay is based on the measurement of the increase in absorbance at 324 nm resulting from the formation of glyoxylate-phenylhydrazone (ε = 17 L mmol ^"1 cm ¹ )

 Glyoxylate and phenylhydrazines.

For characterization, three transformants of H222-AZ8 (T3 - T5) and two transformants of H222-AZ9 (T2 + T3) were selected. In all transformants the PYC activity was examined and in the H222-AZ9 transformants additionally the activity of the ICL was investigated. FIG. 4 shows the results of these activity measurements. Both transformants of H222-AZ8 and H222-AZ9 showed increased PYC activity (2.6-3.7 fold) compared to wild-type H222. In addition, transformants H222-AZ9 T2 and T3 showed a 24-25 fold increase in ICL activity compared to H222.

3. Deletion of JEN4

The inventors have conducted studies to characterize the proteins encoded by JEW orthologs in Y. lipolytica. These studies provided clues to the functioning of these JEW gene products as dicarboxylate importers with several specific substrates. In the process, strains were constructed in which individual JEN genes were deleted. When these strains were cultivated, larger amounts of extracellular succinate were detected in part, especially in the deletion of JEN4.

In this case, a JEN4 deletion cassette (approximately 4.5 kb) was transformed into the uracil-auxotrophic Yarrowia lipolytica strain H222-AZ2U. The J £ 7V ¥ deletion cassette (Figure 5) contained the promoter and terminator regions of JEN4, with the URA3 flanked by TcR sequences in between (Hübner 2010). The deletion cassette was transformed by lithium acetate method. Homologous recombination in the promoter and terminator regions should result in the deletion of JEN4 (Figure 5). Two uracil prototrophic transformants in which the deletion of JEN4 was detected by PCR and Southern blot were isolated. Both transformants were cultured in YNB. The two transformants studied, H222-AZ7 T II and T23, behaved similarly to H222-AZ2 in terms of growth (Figure 3). Both transformants also produced more succinate than the parent strains H222 and H222-AZ2 (Figure 6, Table 2).

 H222 3.6 ± 0.4 9.8 ± 0.6 0.60 ± 0.01

AZ2 15.0 ± 4.0 31.8 ± 7.9 2.50 ± 0.68

AZ7 T11 21.1 ± 0.4 50.7 ± 2.2 3.00 ± 0.09

 AZ7 T23 20.8 ± 1.1 54.7 ± 13.3 3.06 ± 0.07

Table 4: Maximum succinate levels, volume specific and biomass specific product formation rate of strains H222, H222-AZ7 T1 and T23 in YNB medium.

 Cultivation in YNB medium with 5% glycerol as C source.

4. Overexpression of PYCl

For the overexpression of pyruvate carboxylase (PYC) in Y. lipolytica, the integrative multicopy plasmid p64PYC was constructed by integration of the PCR-amplified ORF of the corresponding gene together with its own promoter and terminator regions of about 1 kb into the recipient vector p64T.

The pyruvate carboxylase is encoded in Y. lipolytica by a gene {PYCl) (Flores and Gancedo 2005). Since the region of the ORF of PYCl (3844 bp) together with the associated 1 kb promoter and 0.3 kb terminator region each had a size of 5149 bp, this expression cassette was amplified in two steps in order to introduce any sequence errors decrease by the polymerase. The oligonucleotides PYCa for and PYCa rev amplified the 2442 bp region, which included the 1 kb promoter region and part of the ORF of PYCl. The 2822 bp area, the remaining ORFs of PYCl together with the 300 bp large terminator region, was amplified by the oligonucleotides PYCb for and PYCb rev. For ease of integration into the expression vector p64T, oligonucleotides PYCa for Pael and PYCb rev added an i g / II site, and the Bgl II site included in the ORF was used for vector integration and fusion of the two fragments. The 2442 bp PYCa fragment (promoter region with part of the FC ORF) was digested with Pael and BglII and cloned into the P64 and BglII digested p64T vector. The resulting p64PYCla vector was sequenced. Sequencing of the plasmid of clone T22 yielded a defect-free 7C7 ORF, and an error-free promoter region was detected for the plasmid of clone T97. An error-free p64PYCla vector was obtained by the integration of the error-free aeI-i? AmHI promoter region from p64PYCla from clone T97 into the Pael and BamHI digested p64PYCla vector from clone T22. In the resulting p64PYCla vector, the i? G / II PYCb fragment (part of the FCi ORF and the terminator region) was cloned and then sequenced. The vector p64PYCl was obtained, which contained an error-free expression cassette (pPYCl-PYCl-PYClt) for the overexpression of the pyruvate carboxylase-encoding gene. For transformation into Y. lipolytica strains H222-S4 and H222-AK7 (scs2) this vector was linearized with SacII. Integration of the expression cassette into the obtained transformants H222-AK1 was checked by Southern hybridization (FIG. 9). For the parent strain H222-S4 a band corresponding to the genomic 7C7 ORF was detected at 8.9 kb. The additionally detected band at 6.2 kb in the transformants served as evidence for the integration of the multicopy vector p64PYC l. These bands showed an increased intensity compared to the genomic band of the reference strain H222-S4, whereby a multiple integration of the corresponding expression cassette was confirmed. The transformants H222-AK1-2, H222-AK1-5, and H222-AK1-7 were selected for further study because of their high vector band intensities. The gene-donor effect on pyruvate carboxylase activity was in the strains H222-AK1-2, H222-AK1-5 and H222-AK1-7 examined.

For the determination of PYC activity, all selected transformants were cultured in 100 ml of minimal medium containing 1% glucose. After harvesting a sample of the culture, the yeast cells were disrupted by means of glass beads. The PYC activity was determined in the cell-free extract according to the method of van Urk et al. (1989). The composition of the reaction mixture is listed in the following table. The photometric measurement was carried out at 340 nm.

Stock solution final concentration

 tris

HC1, pH 1 M 100 mM

 7.8

MgS0 ₄ 100 mM 6.7 mM

 Pyrazole 15 mM 1.5 mM

 NADH 1.5 mM 0.15 mM

 Acetyl-10 mM 90 μΜ

 CoA

 Pyruvate 100 mM 10 mM

 malate

6 U μΓ 6 U

 DH

ATP 33 mM 3.3 mM

The increased gene dose of the PYC-encoding gene showed a positive effect on the specific PYC activities of the strains studied. The specific activities for the transformants H222-AK1 -2 and H222-AK1 -7 reached a maximum after 6 h. In contrast, no detectable maximum of the specific enzyme activity was detected for the transformants H222-AK1-5 and for the wild-type H222. The transformants H222-AK1-2 and H222-AK1-7 showed the highest specific PYC activities at 0.48 ± 0.04 U / mg and 0.51 ± 0.02 U / mg compared to the other strains investigated, their enzyme activities at 0.42 ± 0. 1 U / mg (H222-AK1-5) and at 0.16 ± 0.06 U / mg (H222) were determined. The strain H222-AK1-7 showed in addition to the highest specific activity growth comparable to the wild type and was thus selected for further studies. This transformant H222-A 1-7 is referred to below as Transformande H222-AK1 (= H222-AM3).

Compared to the wild type strain H222, the strain with increased PYC activity showed moderate differences in growth and production behavior. The wild type H222 produced a maximum of 3.3 ± 0.02 g L ^"1 succinate with an average productivity of 5, 4 ± 0.6 mg L ^{" 1} h ^{"1. For} H 2 2 2-AM3 slightly increased amounts of succinate could be produced of 4.1 ± 0.1 g L ^"1 .

5. Combination of JETW deletion with overexpression of PYCl

 Since both overexpression of PYCl and the deletion of JEN 4 from H222-AZ2 (with SZ) H2 promoter exchange) resulted in increased succinate production, strains were constructed for succinate production combining these three modifications.

The construction of this new strain (H222-AZ10) was carried out starting from H222-AZ7 (JEN4). For this purpose uracil auxotrophy was introduced in H222-AZ7 using FOA-selection. The strategy is shown in FIG. FOA (5 'fluororotic acid) is converted to 5-fluorouracil by the oritidine 5'-phosphate decarboxylase encoded by URA3. 5-fluorouracil is toxic to the cell. Therefore, in the presence of FOA only cells can grow in which no URA3 is present or in which URA3 was removed by recombination of the flanking TcR sequences. 10 ³ cells of H222-AZ7 were streaked with glucose on FOA- and uracil-containing minimal medium and the colonies obtained were checked for uracil auxotrophy by means of a punch test. The loss of URA3 was also detected by PCR. From the colonies tested, a uracil auxotrophic was selected (H222-AZ7U) and transformed with the integrative PYCl multicopy vector p64PYCl. After one to two weeks transformants were isolated, which were checked by PCR for the integration of the vector. All tested Transformants were positive. From these positive transformants, 3 were selected and tested for PYC activity. For this purpose, these 3 transformants and the wild type were cultured in minimal medium with glucose. After 4 hours, a sample was taken for the determination of PYC activity and the activity of pyruvate carboxylase was determined.

 Three selected transformants were examined for PYC activity and succinate production. The results of the activity measurements are shown in FIG. All transformanden show a compared to the wild type two to three times increased PYC activity.

The three transformants tested were cultured in YNB medium to examine effects on succinate production.

FIG. 9 shows growth behavior and succinate production of H222 (wild type) and the new transformants H222-AZ 10 T1, T5 and T9. For comparison, the strains H222-AZ7 TU and H222-AZ 8 T 3 were also included. The average values from all experiments carried out are summarized in Table 5.

succinate

 Tribes Q

[g L ¹ ] [mg L ¹ h ¹ ]

 H222 3.0 ± 0.0 6.0 ± 0.1

 AZ10 T1 16.8 ± 1.0 34.8 ± 3.1

 AZ10 T5 17.7 ± 1.3 36.1 ± 3.4

 AZ10 T9 18.3 ± 1.3 36.8 ± 4.4

 AZ8 T3 19.3 ± 0.9 45.5 ± 2.7

 AZ7 T11 13.2 ± 2.4 36.6 ± 10.5

Table 5: Average maximum succinate levels, volume specific productivities and biomass specific product formation rates of strains H222 (wild-type), H222-AZ10 Tl, T5, T9, H222-AZ8 T3 and H222-AZ7 TU. Cultivation in shake flasks at 28 ° C., 220 rpm in 150 ml of YNB medium with 5% glycerol as C source (+ 3% after 168 h) in two

 Trial runs.

In addition to succinate production, the by-product spectrum also became considered organic acids malate, AKG and fumarate. However, all altered strains showed significantly reduced levels of malate, AKG and fumarate compared to the wild type (Table 6).

-MA _max AKGmax FA _max

[g L ¹ ] [g L ¹ ] [g L ¹ ]

 H222 7.5 ± 2.1 2.0 ± 0.7 2.8 ± 0.2

 AZ10 T1 3.2 ± 0.1 1.7 ± 0.1 1.7 ± 0.1

 AZ10 T5 1.3 ± 0.0 1.3 ± 0.2 1.3 ± 0.1

 AZ10 T9 1.5 ± 0.0 1.2 ± 0.0 1.3 ± 0.1

Table 6: Maximum product levels of the organic acids malate (MA), α-ketoglutarate (AKG) and fumarate (FA) when cultivating the strains Y. lipolytica H222, H222-AZ 10 T 1, T5 and T9 in YNB medium. Cultivation in YNB medium at 5%

 Glycerol as C source.

6. Deletion of MAE1

In a similar manner as described above, the gene of the carboxylate transporter Mael was deleted in Yarrowia lipolytica. The deletion cassettes and plasmids used can be found in the table presented at the beginning. In the deletion strain H222-SW2AMAE1, the deletion of the SpMAE1 homologous gene MAE1, coding for a putative dicarboxylate transporter, was performed. Cultures of the H222-SW2AMAE1 transformants 3, 7, and 8 were analyzed for the accumulated organic acids α-ketoglutarate, fumarate, malate, pyruvate, and succinate in comparison to the parent strain H222. In addition, during the cultivation, the determination of formed biosoluble masses continued to take place. The formed dry biomass of the ΔΜΑΕ1 transformants when cultivated in 10% glycerol was 40% lower compared to the reference strain H222, but showed little difference in comparison of the transformants with each other. This significant difference in growth may be due to a reduced uptake of the tricarboxylic acid cycle Intermediate α-ketoglutarate, fumarate, malate and succinate result, which are educts of many biosyntheses, such as for amino or fatty acids. When considering acid production, all transformants showed an increase in the production of the organic acids α-ketoglutarate, fumarate, malate and succinate studied. On average, a maximum of 30% more AKG, fumarate and succinate and 40% more malate (TF7 only 10%) were formed. The results are shown in Table 7. The mean values of the maximum amounts of malate, succinate, α-ketoglutarate and fumarate of transformants 3, 7, 8 of H222-SW2AMAE1 compared with reference strain H222 when cultivated in production medium according to Tabuchi et al , (1 98 1). SA ΔΜΑΕ 1 = standard deviation of the maximum values of the 3 trans formants:

LITERATURE

Arikawa et al. (1999) Isolation of Sake Yeast Strains Holding Various Levels of Succinate and / or Malate-producing Abilities by Gene Disruption or Mutation. Journal of Bioscience and Bioengineering Volume 87, Issue 3, 1999, Pages 333-339

Ghezzi D, Goffrini P, Uziel G, Horvath R, Klopstock T, Lochmüller H, DAdamo P,

Gasparini P, Strom ™, Prokisch H, Invernizzi F, Ferrero I, Zeviani M. (2009) SDHAF1, encoding a LYR complex-II specific assembly factor, is mutated in SDH-defective infantile leukoencephalopathy. Nat Genet. 2009 Jun; 41 (6): 654-6. Epub 2009 May 24. Kubo Y, Takagi H and Nakamori S, (2000). Effect of gene disruption of succinate dehydrogenase on succinate production in a yeast yeast strain. Journal of Bioscience and Bioengineering Volume 90, Issue 6, 2000, Pages 619-624 Raab A and Lang (2011), Oxidative versus reductive succinic acid production in the yeast saccharomyces cerevisiae, Bioengineered Bugs 2 (2) 2011, 120-123

Mauersberger, S., Wang, H.J., Gaillardin, C, Barth, G. and Nicaud, J.M. (2001). Yarrowia lipolytica: generation of tagged mutations in genes involved in hydrophobic substrate utilization. J Bacteriol 183 (17): 5102-5109.

Fickers, P., Le Dali, M.T., Gaillardin, C, Thonart, P. and Nicaud, J.M. (2003). Yarrowia lipolytica. New disruption cassettes for rapid gene disruption and marker. J Microbiol Methods 55 (3): 727-737.

Werner, S. (2008). Production and characterization of a yeast Yarrowia lipolytica strain with reduced heterologous recombination. Thesis. TU Dresden. Barth, G. and Gaillardin, C. (1996). Yarrowia lipolytica. Nonconventional Yeasts in

Biotechnology. K. Wolf. Berlin, Heidelberg, New York, Springer-Verlag van Urk, H., Schipper, D., Breedveld, G.J., Mak, P.R., Scheffers, W.A., and van Dijken, J.P. (1989). Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621. Biochim Biophys Acta 992 (1): 78-86.

Gerber, J. (1999) Studies on the optimization of the electron transport system for cytochrome P450-catalyzed steroids biotransformation in Yarrowia lipolytica.

Thesis. TU Dresden Dixon, HG and Kornberg, HL (1959). Assay method for key enzymes of the glyoxylate cycle. Biochem J 72: 3P

Kruse, K., Förster, A., Juretzek, T., Mauersberger, S. and Barth, G. (2004). Process for the biotechnological production of citric acid with a genetically modified yeast Yarrowia lipolytica. Patent: DE 10333144A1

Tabuchi, T., Sugisawa, T., Ishidori, T., Nakahara, T. and Sugiyama, J. (1981). Itaconic acid fermentation by a yeast belonging to the genus Candida. Agric Biol Chem 45: 745-479.

Claims

claims

A fungal strain having at least one genetic modification which results in a reduction in the activity of at least one fungal carboxylic acid transporter.

2. mushroom strain according to one of the preceding claims, characterized in that said fungus has an increased extracellular carboxylic acid production.

A method of genetically modifying a fungal strain to increase extracellular carboxylic acid production, said method resulting in a reduction in the activity of at least one fungal carboxylic acid transporter.

A fungal strain or method according to any one of the preceding claims, wherein the reduction of the activity of the carboxylic acid transporter is achieved by at least one property or step selected from the group comprising: a) inhibiting or reducing the expression of a gene encoding a carboxylic acid transporters

 b) Expression of a dysfunctional or diminished active carboxylic acid transporter

 c) inhibiting or reducing the activity of the expressed carboxylic acid transporter is accomplished.

5. mushroom strain or method according to one of the preceding claims, characterized in that said fungus strain is a member of the group of Ascomycota is acting.

A fungal strain or method according to any one of the preceding claims, wherein said fungal strain belongs to a genus selected from the group comprising Saccharomyces; Schizosaccharomyces; Wickerhamia; Debayomyces; Hansenula; Hanseniospora; Pichia; Kloeckera; Candida; Zygosaccharomyces; Ogataea; Kuraishia; Komagataella; Yarrowia; Metschnikowia; Williopsis; Nakazawaea; Kluyveromyces; Cryptococcus; Torulaspora; Torulopsis; Bullera; Rhodotorula; Sorobolomyces; Pseudozyma; Saccharomycopsis; Saccharomycodes; Aspergillus; Penicillium; Rhizopus; Trichosporon and / or Trichoderma.

7. fungal strain or method according to any one of the preceding claims, wherein the at least one carboxylic acid transporter from the group of Jen transporters and / or the Mae transporter comes from.

8. fungal strain or method according to one of the preceding claims, wherein said fungal strain containing at least one further genetic modification selected from the group

Reduction of the activity or expression of the succinate dehydrogenase (SDH), for example by replacement of the native promoter of SDH2 by an inducible promoter, for example pPOT1 or by use of a suitable deletion cassette,

 Increasing the activity or expression of Pyruvatcarboxylase (PYC), for example by overexpression of the corresponding gene PYC1, and / or

Increasing the activity or expression of isocitrate lyase (ICL), for example by overexpression of the corresponding gene ICL1.

A fungal strain or method according to any one of the preceding claims, wherein said fungal parent strain belongs to the species Yarrowia lipolytica.

10. A process for the production of carboxylic acids, wherein in the process a fungus strain according to one of the preceding claims is used.