Background of the Invention Field of the Invention

The present invention relates to a method for producing a-hydroxy acid(s), a-keto acid(s) and additionally ethylene glycol in eukaryotic microbial host cells. In particular, the present invention relates to a method, wherein a genetically engineered fungal microorganism metabolizes sugars to sugar acid intermediates via Dahms pathway and further to a-hydroxy acid(s). This invention relates also to an a-hydroxy acid or a-keto acid product obtained by using the said method and eukaryotic host described herein and to the use of eukaryotic cells in the production of ethylene glycol and α-hydroxy acid(s) or a-keto acid(s), in particular glycolic acid and co-production of glycolic and lactic acid. Description of Related Art

Glycolic acid is the smallest α-hydroxy acid and it is a widely used chemical. It has applications within cosmetic field and organic synthesis of polyglycolic acid (PGA) and other biocompatible polymers. In textile industry it is used as a dyeing and tanning agent and in food processing as a flavouring agent and as a preservative. Glycolic acid is usually derived from fossil resources and produced in larger scales by chemical synthesis involving toxic chemicals. It is not naturally produced by micro-organisms, at least not in feasible quantities.

Ethylene glycol and lactic acid are also widely used chemicals. They are used as polymer precursors and especially polylactic acid (PLA) is widely used in biodegradable plastics. Copolymer of glycolic acid and lactic acid poly(lactic-co-glycolic acid) (PLGA) has medical applications e.g. in drug delivery (Fredenberg et al, 201 1). Ethylene glycol is also used as an anti-freezing agent and coolant (Baudot and Odagescu, 2004). Lactic acid also finds uses in leather, tanning, cosmetics and pharmaceutical applications as well as in food industries. Lactic acid is produced naturally by a large number of micro-organisms (Miller et al.,2011). Other α-hydroxy acids and a-keto acids derived from D-xylose such as 3- deoxy pentanoic acid and 2-keto-3-deoxy pentanoic acid may have similar useful applications. To produce e.g. glycolic acid from carbohydrates in a fermentative way by using microorganisms requires genetically engineered micro-organisms. Previously Escherichia coli strains were described that were engineered to produce glycolic acid from D-glucose (WO 2007/140816 Al, WO 2007/141316 A2, WO2010/108909 Al and WO2011/036213). D- Glucose from cellulose can readily be converted to various products by micro-organisms, but conversion of D-xylose abundant in the hemicellulose fraction to useful chemicals is more challenging.

Using E. coli as a production host for acids has several drawbacks. E. coli requires a complex growth medium, which is in general more expensive than growth media for yeast or filamentous fungi. Another drawback is that E. coli is operating close to neutral pH. This increases the contamination risk and in the case of acid production requires base to neutralize the acid. Further, in neutral conditions the produced acid is in dissociated form, which is a disadvantage in downstream processing.

Kataoka et al. 2001 describes glycolic acid production from ethylene glycol by various yeast and bacteria but a pathway allowing use of more abundant carbon sources, preferably pentose sugars commonly originating from side and waste streams under normal bioprocessing conditions would be desirable. Compared to E. coli, yeasts, such as S. cerevisiae, are generally regarded industrially more amenable hosts as they are more robust and tolerant to low pH. Using eukaryotic microorganisms can also have other advantages, such as better acid and inhibitor tolerance, lack of glycolic acid consuming reactions and lower contamination risks. In the present invention one focus is on production of glycolic acid, ethylene glycol and co-production of glycolic and lactic acid from D-xylose by a genetically engineered fungal host via the so called Dahms pathway (Dahms, 1974) that ends up in glycolaldehyde and pyruvate through an oxidative D-xylose degradation. Glycolaldehyde can be either reduced to ethylene glycol or oxidised to glycolic acid. On the other hand, pyruvate can be further converted to lactic acid by lactate dehydrogenase. Moreover, Dahms pathway has an interesting intermediate 2-keto-3-deoxy pentanoic acid (2K3DX) that can be converted to potentially high- value products such as 1,2,4-butanetriol (US 2006/234363) (Niu et al, 2003). US 2006/234363 A and Niu et al. describe a method with genetically modified E. coli strains for production of 1,2,4-butanetriol via 2-keto-3-deoxy pentanoic acid including the production of other compounds. Dahms pathway, which is used in the present invention, is described in these publications. However, glycolic acid and lactic acid are not mentioned.

Liu et al. similarly describe production of ethylene glycol from D-xylose via Dahms pathway in E. coli. Production of glycolic acid is inhibited by deletion of aldehyde dehydrogenase encoding gene, in contrast to the present invention. The patent application US 2012/0005788 Al describes D-xylonic acid production with a eukaryotic microbe. Although D-xylonic acid is an intermediate in the pathway for production of glycolic acid, lactic acid, and ethylene glycol, this application and the present invention share only one common enzyme, i.e. D-xylose dehydrogenase.

WO 2012/033832 A2 describes a fermentative production of butanol and isomers thereof, by using recombinant host cells. In one embodiment of this application a step in a pyruvate-utilizing biosynthetic pathway is enhanced by deleting FRA2 gene in S. cerevisiae host. This deletion can also be applied to the present invention, in order to improve D-xylonate dehydratase activity.

Thus, no solutions exist in the prior art for the production of sugar acid intermediates, a- hydroxy acid(s), a-keto acid(s), and ethylene glycol from sugars, in particular from pentose sugar, originating from side and waste streams, with a microbial eukaryotic host, although such solutions would have commercial and industrial interest and also important benefits compared to the traditional production methods.

Summary of the Invention

It is an aim of the invention to provide a method and a eukaryotic host organism for producing sugar acid intermediates, a-hydroxy acid(s) and optionally alcohols.

It is a further aim of the invention to provide such a method and a host for producing 2- keto-3-deoxy-pentanoic acid, 3-deoxy-pentanoic acid, glycolic acid, co-producing glycolic and lactic acid, and optionally producing ethylene glycol. These and other objects are achieved by present invention as hereinafter described and claimed.

The first aspect of the invention is a method for producing a- hydroxy acid(s). According to the present invention the method comprises the steps of culturing a eukaryotic host encoding genes expressing at least the enzymes required for producing one or more a- hydroxy acid, and optionally one or more a-keto acid, and optionally ethylene glycol.

The second aspect of the invention is a eukaryotic microbial production host that is genetically modified to express D-xylose dehydrogenase, D-xylonate dehydratase, aldolase, glycolaldehyde dehydrogenase and lactate dehydrogenase genes, or some of these genes, to produce glycolic acid, and optionally 2-keto-3-deoxy-pentanoic acid or 3-deoxy- pentanoic acid, and optionally ethylene glycol, and/or to co-produce glycolic acid and L- lactic acid.

The third aspect of the invention is an acid product containing one or more a-hydroxy acids, and optionally one or more a-keto acids, examples of suitable acids being glycolic acid, lactic acid, 2-keto-3-deoxy-pentanoic acid, and 3-deoxy-pentanoic acid, which acid product is obtained by culturing the production host of the invention in a suitable medium, particularly by using the method of the invention. Further, the product can contain ethylene glycol.

The fourth aspect of the invention is the use of a microbial eukaryotic host in production of a-hydroxy acid(s), a-keto acids, and ethylene glycol.

More specifically, the method for producing α-hydroxy acid(s), a-keto acids and ethylene glycol according to the invention is characterized by what is stated in claim 1.

Further, the eukaryotic host of the present invention is characterized by what is stated in claim 10, the obtained acid product is characterized by what is stated in claim 21, and the use of said eukaryotic host in said production is described in claim 24.

Next, the invention will be described more closely with reference to the attached drawings and a detailed description. Brief Description of the Drawings

Figure 1 is a schematic presentation of the metabolic route leading to production of 2-keto- 3-deoxy pentanoic acid, 3-deoxy pentanoic acid, glycolic acid, or co-production of glycolic acid and L-lactic acid, and ethylene glycol. Enzymes are marked with letters A to G, wherein A) is D-xylose dehydrogenase, B) is lactonase, C) is D-xylonate dehydratase, D) is aldolase, E) is aldehyde dehydrogenase, F) is L-lactate dehydrogenase, G) is alcohol dehydrogenase and H) is 2-keto acid dehydrogenase.

Figure 2 a) shows the formation of 2-keto-3-deoxy-pentanoic acid and consumption of D- xylose in shake flask cultures on 10 g/L D-glucose, 20 g/L D-xylose and 10 g/L CaC0 ₃ with the control strains H4098 and H4097 and the strains H4096 and H4099 expressing xylB and xylD. D-Xylose consumption is shown by filled symbols and 2-keto-3-deoxy- pentanoic acid concentration by open symbols. Figure 2 b) shows the formation of 3- deoxy-pentanoic acid in shake flask cultures on 10 g/L D-glucose, 20 g/L D-xylose and 10 g/L CaC0 ₃ with the control strains H4098 and H4097 and the strains H4096 and H4099 expressing xylB and xylD.

Figure 3 shows the formation of ethylene glycol in shake flask cultures on 10 g/L D- glucose, 20 g/L D-xylose and 10 g/L CaC0 ₃ with the control strain H4096 and H4099 and the aldolase gene expressing strains H4078, H4079, H4081 and H4082.

Figure 4 shows plasmid pMLV151. The numerical codes in the feature table refer to the SEQ ID NO: 21 and C refers to complementary strand. Figure 5 shows plasmid pMLVlOOA. The numerical codes in the feature table refer to the SEQ ID NO: 22 and C refers to complementary strand.

Figure 6 shows plasmid pMLV175. The numerical codes in the feature table refer to the SEQ ID NO: 23 and C refers to complementary strand.

Figure 7 shows plasmid pMLV177. The numerical codes in the feature table refer to the SEQ ID NO: 24. Detailed Description of the Preferred Embodiments of the Invention

This invention relates to a method, wherein a carbon source, such as pentose sugar, is converted to sugar acid intermediates, a-hydroxy acid(s) and optionally to ethylene glycol by culturing the cells of a eukaryotic microbial host, which is selected from micro- organisms encoding D-xylose dehydrogenase activity, D-xylonate dehydratase activity, aldolase activity and glycolaldehyde dehydrogenase activity and L-lactate dehydrogenase activity in a suitable growth and production medium.

The most common α-hydroxy acids (AHAs) are glycolic acid, lactic acid, citric acid and mandelic acid. In the present invention, however, the focus is mainly on production of glycolic acid, and optionally also on co-production of glycolic and L-lactic acid.

Further to the α-hydroxy acid(s), ethylene glycol and 2-keto-3-deoxy pentanoic acid and 3- deoxy-pentanoic acid can be produced using this method.

Said method is performed via the Dahms pathway. This is an oxidative pathway, which here is implemented by first oxidizing D-xylose to D-xylo no lactone by a D-xylose dehydrogenase (XylB), followed by either a spontaneous hydrolysis or a lactonase aided hydrolysis to convert D-xylo no lactone to D-xylonate. In the next step D-xylonate dehydratase (XylD) dehydrates D-xylonate to 2-keto-3-deoxy pentanoic acid (2K3DX). Further, 2K3DX is converted to pyruvate and glycolaldehyde by an aldolase (YagE or YjhH).

The pathway is here preferably complemented by further steps, wherein glycolaldehyde is then oxidised to glycolic acid by an aldehyde dehydrogenase, or optionally by alcohol dehydrogenase to ethylene glycol. Optionally, the previously formed glycolaldehyde and the pyruvate are converted to glycolic acid and lactic acid, e.g. by lactate dehydrogenase.

In order to convert glycolaldehyde to glycolic acid and pyruvate to lactic acid, genes encoding putative glycolaldehyde dehydrogenase (aldA, E. coli) and L-lactate dehydrogenase (IdhL, Lactobacillus helveticus) are expressed together with xylose dehydrogenase encoding gene (xylB, Caulobacter crescentus), xylonate dehydratase encoding gene (xylD, Caulobacter crescentus) and aldolase encoding genes (yagE or yjhH from E. coli). In one embodiment of the invention the genes encoding glycolaldehyde dehydrogenase (aldA) from E. coli, L-lactate dehydrogenase (IdhL) from Lactobacillus helveticus, xylose dehydrogenase (pylB) from Caulobacter crescentus, xylonate dehydratase (xylD) from Caulobacter crescentus and aldolase (yagE or yjhH) from E. coli are expressed.

The Dahms pathway can alternatively be used for the steps of converting the pentose sugar to D-xylonolactone by D-xylose dehydrogenase, converting the D-xylo no lactone to D- xylonate spontaneously or with lactonase, converting the D-xylonate to 2-keto-3-deoxy- pentanoic acid by D-xylonate dehydratase, and converting the 2-keto-3-deoxy-pentanoic acid to 3-deoxy pentanoic acid by e.g. 2-ketoacid dehydrogenases.

Additionally, in one embodiment glycolaldehyde can be reduced to glycol or ethylene glycol by e.g. an alcohol dehydrogenase (ADH). In a further embodiment, the production of 3-deoxy pentanoic acid is decreased.

Herein 2K3DX is named as "2-keto-3-deoxy-pentanoic acid", but also terms 2-keto-3- deoxy pentonic acid, 2-dehydro-3-deoxy pentonic acid, 2-dehydro-3-deoxy-arabinonate, 2- dehydro-3-deoxy-arabinonic acid, 2-dehydro-3-deoxy-pentonate, 2-dehydro-3-deoxy- pentonic acid, 3-deoxy-glycero-pentulosonic acid, 3-deoxy-pent-2-ulosonic acid, pentanoic acid, 4,5-dihydroxy-2-oxo-, (R)- or 4,5-dihydroxy-2-oxopentanoic acid are used meaning the same substance.

Herein 3DX is named as "3-deoxy pentanoic acid", but also terms 3-deoxypentonic acid, 3-deoxypentanoic acid, 3-deoxy-pentonic acid, 3-deoxy pentonic acid, 2,4,5- trihydroxypentanoic acid, pentonic acid, erythro-3-deoxypentanoic acid and 3-deoxy-D- threo-pentonic acid are used meaning the same substance.

D-Xylose dehydrogenase (XylB) is an enzyme, which belongs to the family of oxidoreductases (EC 1.1.1.175). It catalyses the chemical reaction, where D-xylose is converted to D-xylonolactone, thus D-xylose + NAD ⁺ → D-xylonolactone + NADH + H ⁺ .

D-Xylonate dehydratase (XylD) is an enzyme, which belongs to the family of lyases (EC 4.2.1.82), specifically the hydro-lyases, which cleave carbon-oxygen bonds. It catalyses the chemical reaction of D-xylonate → 2-keto-3-deoxy pentanoic acid + H ₂ 0. C. crescentus D-xylonate dehydratase, designated here as XylD, is one example of class EC 4.2.1.82 enzymes. Aldolases (such as YagE and YjhH) are enzymes, which belong to the families of lyases, carbon-carbon lyases and aldehyde lyases (EC 4.1.2.20). In the present invention, the enzyme catalyses the chemical reaction, wherein 2-keto-3-deoxy-pentanoic acid is converted to glycolaldehyde and pyruvate. Glycolaldehyde dehydrogenase (such as aldA) is also a member of the family of oxidoreductases (EC 1.2.1.21). This enzyme participates in glyoxylate and dicarboxylate metabolism and catalyses the chemical reaction of glycolaldehyde + NAD ⁺ + H ₂ 0 → glycolate + NADH + 2H ⁺ . In one embodiment the aldehyde dehydrogenase enzyme is characterized by having a glycolaldehyde dehydrogenase activity and EC number EC 1.2.1.22. AldA enzyme from E. coli is an example of class EC 1.2.1.22 enzyme having a glycolaldehyde dehydrogenase activity and it catalyses oxidation of glycolaldehyde to glycolic acid.

Lactate dehydrogenase (such as ldhL) is an enzyme, which exist in four distinct enzyme classes. Two of them are cytochrome c -dependent enzymes, acting on either D-lactate (EC 1.1.2.4) or L-lactate (EC 1.1.2.3). The other two are NAD ⁺ -oxidoreductases (EC 1.1.1.27- 28), which catalyse a reversible reaction of pyruvate + NADH + H ⁺ → lactic acid + NAD ⁺ , which is used for example in the anaerobic glycolysis in many cells. Alcohol dehydrogenases (ADH) are a group of enzymes belonging to the family of EC 1.1.1.1 or EC 1.1.1.2. These enzymes facilitate the conversion between alcohol and aldehydes or ketones with the reduction of NAD ⁺ to NADH. They are used e.g. to break down alcohols and to generate useful aldehyde, ketone or alcohol groups. These enzymes are in particular useful in anaerobic conditions, when large amount of alcohol is produced. Some ADHs catalyse the opposite reaction to ensure a constant NAD ⁺ supply in various fermentations. Examples of alcohol dehydrogenases in S. cerevisiae are Adhl-Adh7.

In the present method (production process), the microorganism is selected preferably from fungi, such as yeasts (e.g. S. cerevisiae). According to an embodiment, the cells of the microorganism are cultivated in a culture medium, which particularly is a growth and production medium, that includes a carbon source and typical nutrients required by the particular host, including but not limited to a source of nitrogen (such as amino acids, proteins, inorganic nitrogen sources such as ammonia or ammonium salts), and various vitamins and minerals. Alternatively, more than one different carbon source can be used.

The carbon source is herein particularly a pentose sugar. Examples of pentose sugars are D-xylose, xylan or other oligomer of D-xylose, and preferably also other carbon containing compounds to provide for growth and energy. The medium may also contain hexoses such as e.g. D-glucose or other carbon sources as ethanol, glycerol, acetate, or amino acids, or any mixture thereof.

The carbon source (the substrates) may be provided as pure substrates or from complex sources. The D-xylose containing sugars are suitably hydrolysates of plant biomass e.g. hemicellulose-containing biomass, such as lignocellulose. In addition, the medium may contain complex, poorly defined elements, such as would be present in relatively inexpensive sources like corn steep liquor or solids, or molasses. In case of oligomeric sugars, it may be necessary to add enzymes to the fermentation broth in order to digest these to the corresponding monomeric sugar. It is also possible to use production hosts, such as filamentous fungus hosts, that secrete hydro lytic enzymes enhancing the production of fermentative sugars.

Other growth or fermentation conditions, such as temperature, cell density, selection of nutrients, and the like are not considered to be critical to the present invention and are generally selected to be suitable for the cell used and to provide an economical process. Temperatures during each of the growth phase and the production phase may range from above the freezing temperature of the medium to about 50°C, although the optimal temperature will depend somewhat on the particular micro-organism. A preferred temperature, particularly during the production phase, is from about 25 to 30°C.

The pH of the process may or may not be controlled to remain at a constant pH, but should be between 1.5 and 6.5, depending on the production organism. In one embodiment the culturing pH is below 6. Depending on the production organism the lower limit of the pH may vary between 1.5 and 4. Preferred pH of the culture media is 1.5 to 5, more preferably 2 to 4 and most preferably 2 to 3. In one embodiment the culture medium contains no buffering agent.

Suitable buffering agents for regulating or buffering pH are basic materials that neutralize glycolic acid and possible co-produced L-lactic acid as they are formed, and include, for example, calcium hydroxide, calcium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, ammonium carbonate, ammonia, ammonium hydroxide and the like. In general, those buffering agents that have been used in conventional fermentation processes are also suitable here. It is within the scope of the invention, however, to allow the pH of the fermentation medium drop from a starting pH that is typically 6 or higher, to below the pKa of the acid fermentation product. As stated above, the present process can be implemented at a low pH, even as low as 1.5.

During the fermentation the products, including the glycolic acid (and lactic acid), are excreted out from the cells into the medium from which it may be recovered without disrupting the cells.

In one embodiment of the invention the products, including the glycolic acid, can be recovered from the medium by e.g. ion exchange chromatography or reactive extraction or it can be polymerized in the fermentation medium and recovered thereafter.

This invention relates also to a eukaryotic host which is genetically modified to express D- xylose dehydrogenase, D-xylonate dehydratase, aldolase, glycolaldehyde dehydrogenase and L-lactate dehydrogenase genes or some of these genes and produce sugar acid intermediates and a-hydroxy acid(s), and optionally ethylene glycol.

The eukaryotic host may be any eukaryotic organism but most usually it is a cell, preferably a fungal eukaryotic microbe cell. In one embodiment the host is a yeast cell. Yeasts like Saccharomyces cerevisiae and Pichia kudriavzevii are naturally operating at very acidic pH.

Suitable yeasts are for example the genera Saccharomyces, Kluyveromyces, Candida, Scheffersomyces, Pachysolen, Pichia and Hansenula. Yeast species of particular interest include S. cerevisiae, S. exiguus, K. marxianus, K. lactis, K. thermotolerans, C. sonorensis, P. kudriavzevii (also known as /. orientalis and C. krusei), C. shehatae, Pachysolen tannophilus and Scheffersomyces stipitis.

In one embodiment the host is a filamentous fungus. One particular advantage of using filamentous fungi to produce glycolic acid is that it can be done in a consolidated process, meaning that the fungus produces the enzymes for biomass hydrolysis and ferments the resulting sugars in the same process.

Suitable filamentous fungi hosts are for example of the genera Aspergillus, Trichoderma, Monascus, and Penicillium. Fungal species of particular interest include A. niger, A. ficuum, A. phoenicis, T. reesei, T. harzianum, M. ruber, and P. chrysogenum. Filamentous fungi allow using only partially hydrolysed biomass as a carbon source which is benefit if some lignocellulosic waste is used as a carbon source. In a preferred embodiment the host includes those of the species S. cerevisiae and P. kudriavzevii.

In one embodiment the expression of the genes will result in an acid and inhibitor tolerant production strain for conversion of D-xylose to 2-keto-3-deoxy pentanoic acid, 3-deoxy pentanoic acid, glycolic acid, or optionally as a co-production to glycolic acid and lactic acid, and additionally to ethylene glycol.

In one embodiment of the invention the host is capable of producing glycolic acid or of co- producing glycolic and lactic acid in non-buffered culturing conditions. In other words the strain is tolerant to decreasing pH during the cultivation process. This simplifies the culturing process and thereby reduces costs. However, this characteristic naturally does not exclude possibility to regulate the pH conditions using bases (or acids) or even buffering agents. The host organism of this invention is genetically modified and may contain also other genetic modifications than those specifically described herein. Methods for making modifications of these types are generally well known and are described in various practical manuals describing laboratory molecular techniques. Phrases "genetically modified to express" or "genetically engineered to express" as used herein covers the cells where a protein-encoding polynucleotide has been transformed in such a manner that the host is capable of producing an active protein or where a promoter region of a cell has been modified to allow or enhance the expression of a heterologous or homologous gene encoding D-xylose dehydrogenase and D-xylonate dehydratase activities or other overexpressed genes.

It is understood by the skilled reader that the gene must be operably linked to the sequences regulating the expression of the gene. Two DNA sequences are operably linked when the function of the promoter results in transcription. An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence. In one embodiment the gene is genetically optimized. It is understood by a skilled person that heterologous gene obtained from a different organism may need genetic optimization in order to properly function in the host cell. In one embodiment the heterologous gene is genetically optimized to fit the host systems. Standard molecular biology methods can be used in the cloning of D-xylose dehydrogenase, D-xylonate dehydratase encoding genes or other overexpressed genes. The basic methods used like isolation and enzyme treatments of DNA, E. coli transformations made for plasmid constructions, the isolation of the vectors or fragments containing the said gene and amplification of fragments by PCR are described in the standard molecular biology handbooks e.g. Sambrook et al. (1989) and Sambrook and Russell (2001). Genetic modification of the host fungus is accomplished in one or more steps via the design and construction of appropriate vectors and transformation of the host fungus with those vectors. Electroporation, protoplast-PEG and/or chemical (such as calcium chloride- or lithium acetate-based) transformation methods can be used.

The heterologous gene can be stably introduced into the genome of the host cell. Stable transformation is obtained when the expression cassette is integrated to the chromosomal DNA of the host either randomly, or by homologous recombination to a targeted locus. Targeted integration can be accomplished by designing a vector having regions that are homologous to the upstream (5 '-) and downstream (3'-) flanks of the target gene. Either or both of these regions may include a portion of the coding region of the target gene.

The use of native (homologous to the host cell) or non-native (heterologous to the host cell) promoters and terminators, together with respective upstream and downstream flanking regions, can permit the targeted integration of the XylB or any other gene mentioned above, or any other gene further described below, into specific loci of the genome of the host cell, and for simultaneous integration of the said gene and deletion of a native gene.

For example the exogenous D-xylose dehydrogenase and D-xylonate dehydratase genes may be maintained on a self-replicating plasmid, integrated randomly into the genome of the host cell or inserted at one or more targeted locations. Examples of targeted locations include the locus of a gene that is desirably deleted or disrupted in S. cerevisiae.

Genes of the present invention, and in general, are under the control of a promoter and a terminator, both of which are functional in the modified fungal cell. As used herein, the term "promoter" refers to a sequence located upstream (e.g. 5') to the translation start codon of a structural gene and which controls the start of transcription of the structural gene. Similarly, the term "terminator" refers to a sequence located downstream (e.g. 3') to the translation stop codon of a structural gene and which controls the termination of transcription of the structural gene. A promoter or terminator is "operatively linked" to a structural gene if its position in the genome relative to that of the structural gene is such that the promoter or terminator, as the case may be, performs its transcriptional control function.

The genetically modified fungus may contain a single copy or multiple copies of the D- xylose dehydrogenase gene or any other gene mentioned above, or any other gene further described below. If multiple copies of the gene are present, from 2 to 10 or more copies may be integrated into the genome, or > 100 copies may be present on self-replicating plasmids. If multiple copies of the gene are integrated into the genome, they may be integrated at a single locus (so they are adjacent each other), or at several loci within the host's genome. It is possible for different genes to be under the control of different types of promoters and/or terminators. It is well known that different enzymes can have the same enzyme activity but have very different amino acid sequences. The XylD from Caulobacter crescentus (xylD ; GeneBank GenelD: 7329902, SEQ ID NO: 4) and the D-xylonate dehydratase from Haloferax volcanii HB27 (HVO B0038A; GeneBank GenelD: 8919180) are examples for this. Both enzymes show D-xylonate dehydratase activity but when the amino acid sequences are aligned in a CLUSTALW2 sequence alignment the identities are only 12.6%. Comparison of the identity percentages of different D-xylonate dehydratases is presented in Table 1. It is also noteworthy that D-xylonate dehydratases from different organisms may belong to different protein families. As an example XylD from C. crescentus belongs to a dihydroxy acid dehydratase/6-phosphogluconate dehydratase (ILVD/EDD) superfamily while B0038A from H. volcanii belongs to an enolase superfamily.

Table 1. Comparison of different D-xylonate dehydratase protein sequences done with ClustalW2 alignment (Cost matrix: BLOSUM, Gap open cost 10, Gap extend cost 0.1)

In one embodiment the host cell comprises the gene encoding protein characterized by SEQ ID NO: 4 or a sequence having at least 40%, 50%, 55%, 60% or identity, preferably at least 65%, 70%, 75% or 80% identity, 85% identity, more preferably at least 90% identity and most preferably at least 95% or even 98% identity to the polypeptide having SEQ ID NO: 4 or an active fragment thereof. In one embodiment the host cell comprises genes encoding proteins characterized by SEQ ID NOs: 1-6 or sequences having at least 40%, 50%, 55%, 60% or identity, preferably at least 65%, 70%, 75% or 80% identity, 85% identity, more preferably at least 90% identity and most preferably at least 95% or even 98% identity to the polypeptides having SEQ ID NOs: 1-6 or active fragments thereof.

"An active fragment" means a fragment having all the parts needed for completing the function typical for the protein. As used in the present context the term "identity" refers to the global identity between two amino acid sequences compared to each other from the first amino acid encoded by the corresponding gene to the last amino acid. For the purposes of the present invention identity is preferably determined by means of known computer programs using standard algorithms. An example of such a program is NCBI BLAST, BLASTp (comparison of known protein sequences, amino acids), BLASTn (comparison of nucleic acid sequences), BLASTx (comparison of translated nucleic acid sequences against know protein sequences).

In one embodiment the genes encoding proteins that have a negative effect for the desired product yield are deleted or attenuated. Such deletions include for example deletion of pyruvate decarboxylase (PDC) encoding gene in case of some pyruvate is lost as ethanol or 2-ketoacid dehydrogenases that reduce 2-keto-3-deoxy pentanoic acid 3-deoxy pentanoic acid and thus decrease the yield of other products. If the host organism chosen for glycolic acid production is capable of further utilizing glycolic acid it is necessary to also delete or attenuate the genes responsible for these enzyme reactions.

Based on the above, a further embodiment of the invention is the use of eukaryotic host cells or eukaryotic organisms in production of a-hydroxy acids, such as glycolic acid, or as a starting organism for preparation a production host suitable for production of such acids. In preferred embodiment the eukaryotic organism is a fungal cell, preferably a yeast or filamentous fungus. The eukaryotic organism may be modified as described earlier and is suitable for the method as also described earlier.

"Fungal", "fungus" and "fungi as used herein refer to yeast and filamentous fungi. In a specific embodiment the host cell and the cultivations have been further modified by any combination of the above described modifications.

The invention is illustrated by the following non-limiting examples. It should be understood, however, that the embodiments given in the description above and in the examples are for illustrative purposes only, and that various changes and modifications are possible within the scope of the invention.

Example 1. Construction of a S. cerevisiae strain able to produce 2-keto-3-deoxy- pentanoic acid, 3-deoxy-pentanoic acid, ethylene glycol, glycolic acid and L-lactic acid or glycolic acid

The genes expressed in S. cerevisiae were obtained as synthetic genes, codon optimized for expression in S. cerevisiae (GenScript, USA or Gene Art, Germany).

The genes were ligated either into the BgUl site (yjhH (GeneBank GenelD: 12931978, SEQ ID NO: 2)) and yagE (GeneBank GenelD: 12932832, SEQ ID NO: 3)) between the PGK1 promoter and terminator of B1184 (derived from the multicopy plasmid YEplacl81 + PGK1PT containing LEU2 (Gietz and Sugino, 1988)) or as EcoRl/BamHl -fragment (xylD (GeneBank GenelD: 7329902, SEQ ID NO: 4)) under the TPI1 promoter of B2159 (pYX212 (Yanisch-Perron et. al, 1985)). YagE containing plasmid was named B3800, yjhH containing plasmid B3803 and xylB containing plasmid B3585. B5054 used for the simultaneous expression of yagE, aldA (GeneBank GenelD: 945672, SEQ ID NO: 5) and IdhL (GeneBank GenelD: Z81318.1, SEQ ID NO: 6) was constructed by ligating pTEFl between Apal/Xhol, tADHI between SaWPstl, pTPII between Pstl/Xmal, tCYCl between BamHl/Spel, pPGKl between Spel/Notl and tPGKl between Sacll/Sacl of pRS425 (Christianson et al.,1992). All promoters and terminators were made by PCR from the genomic DNA of H2802 (CEN.PK113-17A (MATa, ura3-52 HIS3, leu2-3/112, TRP1, MAL2-8 ^C , SUC2)) with the primers SEQ ID NO: 7-18. The cloning of promoter and terminator sequences was mostly carried out by recombination cloning using S. cerevisiae strain FY834 (Winston, et al, 1995). In order to construct B5055, yagE was amplified with primers yagE fw and yagE rev (SEQ ID NO: 19 and 20) from B3800 and cloned under the TEF1 promoter of B5054 as XhollSall fragment. AldA and IdhL were subsequently cloned under the TPI1 and PGK1 promoters of B5054 as Xmal/BamHl and Notl/Sacll fragments, respectively, resulting in B5055.

XylB (SEQ ID NO: 1) was integrated into GRE3 locus of S. cerevisiae strain H2802 essentially as described earlier (Toivari et al, 2012). except that the xylB integration cassette was released from a plasmid pMLV82A where xylB expression cassette and kanMX expression cassette were in the same orientation. The deletion cassette for FRA2 gene contained 50 bp flanking regions homologous to 5'- and 3'- regions of the FRA2 gene and the gene encoding a Hygromycin resistance marker. The deletion cassette was transformed into the yeast strain H3817. The transformants were selected for growth in presence of Hygromycin (20(^g/ml; YPD agar) and the hygromycin-resistant clones were tested for successful deletion of the FRA2 gene by PCR.

The plasmids and integration cassettes described above were transformed into yeast by Gietz method (Woods and and Gietz, 2001) resulting in modified S. cerevisiae strains listed in Table 2.

Example 2. Construction of S. cerevisiae strains able to produce glycolic acid

Glycolic acid producing S. cerevisiae strains were constructed by transforming B3585 together with B5264 into H3817 and H4007 resulting in strains H4244 and H4245 (Table 2.) B5264 was constructed by removing IdhL gene from B5055. B5055 was digested with Xrnal + BamHl and the plasmid ends were blunted and ligated back together resulting in B5264. Table 2. Modified S. cerevisiae strains

Strain code Parent strain + modification or Description

plasmid

H2802 CEN.PK113-17A (MATa, ura3- parental strain

52 HIS3, leu2-3/112, TRP1,

MAL2-8 ^C , SUC2)

H3817 H2802 gre3::xylB parental strain with xylB integrated

H4007 H2802 gre3::xylB, fra2::HygR parental strain with Z\fra2 and xylB integrated

H4078 H3817+B3585+B3800 parental strain with xylB integrated with xylD and yagE

H4079 H3817+B3585+B3803 parental strain with xylB integrated with xylD and yjhH

H4081 H4007+B3585+B3800 parental strain with Z\fra2 and xylB integrated with xylD and yagE

H4082 H4007+B3585+B3803 parental strain with Z\fra2 and xylB integrated with xylD and yjhH H4096 H3817+B3585+B1184 parental strain with xylB integrated with xylD and empty plasmid

H4097 H4007 + B2159 + B1184 parental strain with Z\fra2 and xylB integrated with empty plasmids

H4098 H3817 + B2159 + B1184 parental strain with xylB integrated with empty plasmids

H4099 H4007+B3585+B1184 parental strain with Afra2 and xylB integrated with xylD and empty plasmid

H4177 H3817 + B3585 + B5055 parental strain with xylB integrated with xylD, yagE, aid A and IdhL

H4178 H3817 + B3585 + B5054 parental strain with xylB integrated with xylD and empty plasmid

H4179 H4007 + B3585 + B5054 parental strain with Afra2 and xylB integrated with xylD, yagE, aldA and IdhL

H4180 H4007 + B3585 + B5054 parental strain with Afra2 and xylB integrated with xylD and empty plasmid

H4244 H3817+B3585+B5264 parental strain with xylB integrated with xylD, yagE and aldA

H4245 H4007+B3585+B5264 parental strain with Z\fra2 and xylB integrated with xylD, yagE and aldA

Example 3. Production of 2-keto-3-deoxy-pentonic acid or 3-deoxy-pentanoic acid by modified yeast strains

S. cerevisiae strains H4096 and H4099 were grown in 50 ml modified synthetic complete medium lacking uracil and leucine for the selection (Sherman et al, 1983). Cultivations were carried out in 250 ml Erlenmeyer flasks, at 250 rpm, 30°C. The media were supplemented with 10 g/L D-glucose, 20 g/L D-xylose and 10 g/L CaC0 ₃ . 2-keto-3-deoxy- pentonic acid, 3-deoxy-pentanoic acid and D-xylose concentration from culture supernatants was quantified with GC-MS. Samples (100 μί) with arabitol as internal standard were evaporated to dryness and silylated by adding 100 μΙ_, pyridine, 100 μΙ_, chlorotrimethylsilane and 100 N,0-Bis(trimethylsilyl)trifiuoroacetamide (BSTFA). Derivatisation was performed at +70 °C for 60 min. Derivatized samples (1 μί) were subjected to GC/MS analysis (Agilent 6890 Series, USA combined with Agilent, 5973 Network MSD, USA and Combipal injector, Varian Inc., USA). Analytes were injected on split mode (30:1) (200°C) and separated on ZB-1HT INFERNO capillary column (30 m x 0.25 mm) with a phase thickness 0.25 μιη (Phenomenex, Denmark). Helium was used as carrier gas on constant flow mode 0.9 mL/min. The temperature program started at 70°C with 3 min holding time and then increased 10°C/min up to 320°C. MSD was operated in electron-impact mode at 70 eV, in the full scan m/z 40-550. The ion source temperature was 250°C and the interface was 280°C. Compounds were identified according to corresponding standards and with the Palisade Complete 600K Mass spectral library (Palisade Mass Spectrometry, USA). The formation of 2-keto-3-deoxy-pentanoic acid and consumption of D-xylose are shown in Fig. 2a and formation of 3-deoxy-pentanoic acid in Fig. 2b. The control strains H4098 and H4097 did not produce 2-keto-3-deoxy-pentanoic or 3-deoxy-pentanoic acid. Example 4. Production ethylene glycol by modified yeast strains

S. cerevisiae strains H4078, H4079, H4081 and H4082 were grown in 50 ml modified synthetic complete medium lacking uracil and leucine for the selection (Sherman et al, 1983). Cultivations were carried out in 250 ml Erlenmeyer flasks, at 250 rpm, 30°C. The media were supplemented either with 10 g/L D-glucose, 20 g/L D-xylose and 10 g/L CaC0 ₃ . Ethylene glycol concentration from culture supernatants was quantified with GC- MS as described above. The formation of ethylene glycol and consumption of xylose are shown in Fig. 3. The control strains H4096 and H4099 did not produce ethylene glycol.

Example 5. Production of glycolic acid and L-lactic acid by modified yeast strains S. cerevisiae strains H4177 and H4179 were grown in 50 ml modified synthetic complete medium lacking uracil and leucine for the selection (Sherman et al, 1983). Cultivations were carried out in 250 ml Erlenmeyer flasks, at 250 rpm, 30°C. The media were supplemented either with 10 g/L D-glucose, 20 g/L D-xylose and 10 g/L CaC0 ₃ or 20 g/L D-xylose and 10 g/L CaC0 ₃ . Glycolic and L-lactic acid concentrations from growth media were quantified with capillary electrophoresis as described earlier (Turkia et al, 2010). Only glycolic acid was produced when cells were cultivated on media without D-glucose or ethanol. The control strains H4180 and H4178 did not produce glycolic and L-lactic acid. Glycolic acid and L-lactic acid concentrations in growth media are shown in Table 3. Example 6. Production of glycolic acid by modified yeast strains

S. cerevisiae strains H4244 and H4245 were grown in 50 ml modified synthetic complete medium lacking uracil and leucine for the selection (Sherman et al, 1983). Cultivations were carried out in 250 ml Erlenmeyer flasks, at 250 rpm, 30°C. The media were supplemented with 20 g/L D-xylose and 10 g/L CaC0 ₃ . Glycolic and L-lactic acid concentrations from growth media were quantified with capillary electrophoresis as described earlier (Turkia et al, 2010). The control strains H4180 and H4178 did not produce acid. Glycolic acid concentrations in growth media are shown in Table 3. Table 3. L-lactic and glycolic acid production of selected yeast strains (n/m, not measured).

Strain Used L-Lactic acid mg/L Glycolic acid mg/L

carbon Day 1 Day 2 Day 1 Day 2 sources

H4178 20 g/L 0 0 0 0

xylose

10 g/L 0 0 0 0

glucose, 20

g/L xylose

H4180 20 g/L 0 0 0 0

xylose

10 g/L 0 0 0 0

glucose, 20

g/L xylose

H4177 20 g/L 0 0 76 117

xylose

10 g/L 332 228 170 185 glucose, 20

g/L xylose

H4179 20 g/L 0 0 154 167

xylose

10 g/L n/m 927 n/m 696 glucose, 20 g/L xylose

H4244 20 g/L 0 0 50 27

xylose

H4245 20 g/L 0 0 63 36

xylose

Example 7a. Expression of genes of Dahms pathway, aldA and ldhL in yeast P. kudriavzevii

In order to construct P. kudriavzevii able to produce glycolic acid, ethylene glycol or co- produce glycolic acid and L-lactic acid, or 2K3DX and 3DX, some or all codon optimised synthetic genes based on SEQ ID NO: 1-6 are cloned under constitutive, endogenous promoters and introduced into the genome of P. kudriavzevii by either homologous recombination into targeted gene loci or by random integration into the genome.

Example 7b. Expression of genes of Dahms pathway, aldA and ldhL in yeast P. kudriavzevii

C. crescentus xylD gene (SEQ ID NO: 4) was released from the plasmid B3585 with EcoRI/BamHI and the ends were made blunt with T4-polymerase. The blunt end fragment was ligated between P. kudriavzevii TDHI promoter and S. cerevisiae PGKl terminator in pMLV141 which was first cut with Pad and the ends made blunt with T4-polymerase. The final plasmid obtained was named pMLV151 (Figure 4). P.kudriavzevii GPD1 flanks and the TDHI promoter were PCR-cloned from the total DNA of the strain VTT C-79090T (ATCC 32196) and the primers (SEQ ID NO: 25, 26, 27, 28 and SEQ ID NO: 29, 30) were planned on the basis of the draft genome sequence of the same strain. The cloning of the P.kudriavzevii PGK promoter has been described in Toivari et al., 2013. The double expression cassette [loxP- P.k pPGK-hph-tMEL5-loxP]-[P.k pTDHl-xylD-SctPGK] with P.k G Di-fianks (SEQ ID NO: 21) for targeted integration into the GPD1 locus was released with Notl from a plasmid pMLV151 and transformed into Pichia kudriavzevii VTT C-79090T (ATCC 32196) using the lithium acetate transformation protocol (Gietz et al, 1992). The transformants were selected on YPD plates containing hygromycin (400μg/ml). The transformant containing a functional copy of the xylD gene, Io255/pMLV151 pes 1.1, was used as a host strain for introducing the C. crescentus xylB gene (SEQ ID NO: 1) into the genome.

For introducing the xylB gene (SEQ ID NO: 1) into the yeast genome a double expression cassette [loxP- P.k pPGK-MEL5-tMEL5-loxP]-[P.k pPGK-xylB-SctADHl] with P.k PDC1- flanks (SEQ ID NO: 22) for targeted integration into the PDC1 locus was released with NotI from a plasmid pMLVlOOA (Figure 5) and transformed into the Io255/pMLV151 pes 1.1 similarly as described in Toivari et al., 2013. The trans formants were selected based on the blue colour formation on YPD plates containing 5-bromo-4-chloro-3-indolyl-a-D- galactopyranoside (X-a-gal) 40μg/ml. Two transformants, Io255/pMLV151+pMLV100A pes 1.1.1 and 2.1.1, containing functional copies of both the genes, xylD and xylB, were cured of the marker genes hph and MEL5. The transformants were retransformed with a modified plasmid pKlNatCre (Steensma and Ter Linde, 2001), expressing the Cre recombinase. To enhance the recombinase activity in P.kudriavzevii the S. cerevisiae GAL1 promoter in pKlNatCre was replaced with P. kudriavzevii PGK1 promoter. White colonies which had lost the MEL5 gene were selected on the YPD plates containing X-a- gal (40 μg/ml) and ClonNat (300 μg/ml). The removal of the hph gene was shown as an inability to grow on YPD plates containing hygromycin (400μg/ml). The strains obtained, Io 255/ xyDH + xyDHT pesl .3 and pes 2.2, were used as host strains for introducing the E. coli YagE and aldA genes (SEQ ID NO: 3 and SEQ ID NO: 5) into the genome.

The YagE gene (SEQ ID NO: 3) was released from the plasmid B4182 (synthetic gene in a plasmid vector, GenScript, USA) with Bglll/BamHI and ligated into Bglll site between P. kudriavzevii PGK1 promoter and S. cerevisiae ADH1 terminator in pMLV135 and the resulting plasmid was named pMLV173. The aldA gene (SEQ ID NO: 5) was made by PCR from the plasmid B5264 with primers aldA atg frw and aldA taa rev (SEQ ID NO: 31 and 32). The PCR product was digested with Pad and ligated into Pad site between P.kudriavzevii TDH1 promoter and S. cerevisiae PGK1 terminator in pMLV173. The resulting double expression cassette [IopPGK- YagE- SctADHl]-[IopTDHl-aldA-SctPGK\ was released with BamHI and ligated into BamHI site in pMLV127. The resulting plasmid was named pMLV175 (Figure 6). The triple expression cassette [P.k pPGK-hph-tMEL5]- [P.k PGK-YagE- SctADHI]-[IopTDHI-aldA-SctPGK] with P.k DCi-fianks (SEQ ID NO 23) for targeted integration into the PDC1 locus was released with NotI from the plasmid pMLV175 and transformed into the strains Io 255/xyDH+xyDHT pes 1.3 and pes 2.2. Three transformants, 1B1, 2(19)1 and 3(14)1, that grew on hygromycin and didn't produce ethanol were shown by PCR from the total DNA to contain all the four transformed genes, xylB, xylD, aldA and YagE, and the deletion of both the PDC alleles. 1B1 and 2(19)1 were transformants of the strain Io 255/xyDH+xyDHT pesl .3 and 3(14)1 was a transformant of the strain Io 255/xyDH+xyDHT pes 2.2. The strains 1B1, 2(19)1 and 3(14)1 were used as host strains for introducing the L. helveticus IdhL gene (SEQ ID NO: 1) into the genome.

The IdhL gene (SEQ ID NO: 6) was released from the plasmid B5284 with Bglll and ligated into Bglll site between P.kudriavzevii PGK1 promoter and S. cerevisiae ADHl terminator in pMLV94 and the resulting plasmid was named pMLV176. The double expression cassette [P.k pPGK-MEL5-tMEL5J-[lopPGK-ldhL-SctADHl] was released from pMLV176 with BamHl and ligated into BamHl site in pMLV167. The resulting plasmid was named pMLV177 (Figure 7). The PGll flanks were PCR cloned from the total DNA of the strain VTT C-79090T (ATCC 32196) and the primers (SEQ ID NO: 33, 34, 35, 36) were planned on the basis of the own draft genome sequence of the same strain. The double expression cassette [P.k pPGK-MEL5-tMEL5]-[P.k pPGK-ldhL- Set ADHl] with P.k GMlanks (SEQ ID NO: 24) for targeted integration into the PGl locus was released with Notl from the plasmid pMLV177 and transformed into the strains 1B1, 2(19)1 and 3(14)1. The transformants were selected based on the blue colour formation on YPD plates containing X-a-gal (4(^g/ml). Three transformants, lBl/1.1, 2(19)1/1.1 and 3(14)1/2. l,were shown by PCR from the total DNA to contain all the five transformed genes, xylB, xylD, aldA, YagE and IdhL.

Example 8a. Deletion of PDC encoding genes of S. cerevisiae or P. kudriavzevii in order to increase co-production of glycolic acid and L-lactic acid

In S. cerevisiae, the structural genes PDC I, PDC5 and PDC6 each encode an active pyruvate decarboxylase. As a result of strong pyruvate decarboxylase activity in S. cerevisiae ethanol is competitively produced from pyruvate at expense of L-lactate production in IdhL expressing strain. In order to enhance co-production of glycolic acid and L-lactic acid PDC I, PDC5 and PDC6 are disrupted from H3817 and H4007 by replacing their coding region with IdhL gene of Lactobacillus helveticus or any other DNA that prevents formation of mRNA producing active pyruvate decarboxylase protein. In order to avoid homologous recombination during the replacement, the aforementioned genes are alternatively replaced by integration of different genes in each of the PDC locus, e.g. xylB, xylD, aldA, yagE or yjhH (SEQ ID NO: 1-6). The integration of either of these genes or any other DNA into PDC loci is carried out by using standard well-known molecular biology techniques with the aid of selectable markers. Alternatively, XylD, aldA, yagE or yjhH and IdhL (SEQ ID NO: 2-6) are expressed in H3817 or H4007 derivatives with the PDC deletions in a multicopy plasmid or plasmids under constitutive or inducible promoters. Alternatively, pyruvate decarboxylase encoding gene(s) is deleted as described above from P. kudriavzevii. In P. kudriavzevii PDC loci are deleted by targeted integration via homologous recombination of the expression cassettes producing some of the proteins based on genes SEQ ID NO: 1-6 or by integration of well-known selectable markers.

Example 8b. Deletion of PDC encoding genes of P. kudriavzevii to increase glycolic acid and L-lactic acid production

P. kudriavzevii strains containing genes coding for xylose dehydrogenase, xylonate dehydratase, aldolase, aldehyde dehydrogenase and lactate dehydrogenase were constructed as described in example 7b. These strains were made deficient in the pyruvate decarboxylase (PDC) activity by introducing genetic integrations into the PDC locus. The strains 1B1, 2(19)1, 3(14)1, lBl/1.1, 2(19)1/1.1 and 3(14)1/2.1 were cultivated in modified synthetic complete medium supplemented with 20g/l xylose and 2g/l CaC03, and in modified synthetic complete medium supplemented with lOg/1 glucose, 20g/l xylose and 2g/l CaC03, and analysed with GC-MS. Cultivations were carried out in 250 ml Erlenmeyer flasks, at 250 rpm, 30 C. The PDC-deficient P. kudriavzevii strains 1B1, 2(19)1, 3(14)1, lBl/1.1 produced more lactic acid (5,5 g/1) after 24 hours of incubation in the presence of lOg/1 glucose, 20g/l xylose compared to the S. cerevisiae strains with functional PDC in example 6. In addition, 0,5 g/1 glycolic acid and 0,6 g/1 ethylene glycol were produced.

Example 9. Enhancing flux from 2-keto-3-deoxy-pentonoic acid to products

2-keto-3-deoxy-pentanoic acid (2K3DX) may be reduced in the host cell to 3-deoxy- - pentonoic acid (3DX). This reaction competes with the conversion of 2KDX to glycolaldehyde and pyruvate by aldolase. In order to prevent the reduction of 2KDX to 3DPA, one to all or in any combination of the yeast genes GOR1, YGL185C, YPL113C, SER3 and SER33 encoding homologues of 2-keto acid dehydrogenase are deleted from H3817 and H4007, with or without the PDC1, PDC5 and PDC6 disrupted or attenuated. GOR1, YGL185C, YPL113C, SER3 and SER33 or any combination thereof are deleted by either replacing them with the selectable markers or with genes of SEQ ID NO: 1-6 under suitable constitutive or inducible promoters by using well-known molecular biology techniques. Alternatively 2-keto acid dehydrogenase encoding homologues are deleted from P. kudriavzevii as described above.

Thus, while there have been shown and described and pointed out fundamental novel features of the invention as applied to a preferred embodiment thereof, it will be understood that various omissions and substitutions and changes in the form and details of the method and device may be made by those skilled in the art without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps which perform substantially the same results are within the scope of the invention. Substitutions of the elements from one described embodiment to another are also fully intended and contemplated. It is also to be understood that the drawings are not necessarily drawn to scale but they are merely conceptual in nature. It is the intention, therefore, to be limited only as indicated by the scope of the claims appended hereto.

Citation list - patent literature:

1. WO 2007/140816 Al

2. WO 2007/141316 A2

3. WO 2010/108909 Al

4. WO 2011/036213

5. US 2006/234363 A

6. US 2012/0005788 Al

7. WO 2012/033832 A2

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