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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01026—Glyoxylate reductase (1.1.1.26)

Definitions

• the present invention relates to a method for a eukaryotic host and a method for producing glycolic acid using eukaryotic host cells, especially cells of a genetically modified fungal host.
• Glycolic acid is a widely used chemical. It has applications within cosmetic field and organic synthesis of polyglycolic acid 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. Traditionally glycolic acid is derived from fossil sources but it is possible to derive glycolic acid from biomass by using biochemical routes.
• Glycolic acid is the smallest a-hydroxy acid and it is not naturally produced by microorganisms at least in feasible quantities for industrial use. This is different to lactic acid which is also a small a-hydroxy acid, but this acid is produced naturally by a large number of microorganisms.
• To produce glycolic acid from carbohydrates in a fermentative way using microorganisms requires genetically engineered microorganisms. Previously Escherichia coli strains were described that were engineered to produce glycolic acid from glucose (WO 2007/141316 A2, WO2010/108909 Al and WO2011/036213).
• the glyoxylate cycle was disrupted by deleting malate synthase and overexpressing an endogenous glyoxylate reductase so that the glyoxylate produced by isocitrate lyase could be converted to glycolic acid. Also other modifications were done to channel more carbon to the glyoxylate cycle or to prevent the utilization of glyoxylic acid. E. coli strains engineered in this way were producing glycolic acid from glucose.
• E. coli and bacteria in general is that the inner space of the cell or the cytosol of the organism is not compartmentalized. This is different in eukaryotic organisms. Eukaryotic cells, including yeast and filamentous fungi, have different compartments such as vacuoles, mitochondria or peroxisomes that are separated by membranes. In difference to bacteria the cellular reactions take place in different compartments. For example the reactions of the TCA cycle (Krebs cycle, citric acid cycle) are located in the mitochondria, whereas the different reactions of the glyoxylate cycle are in separate compartments. According to current understanding isocitrate lyase and malate synthase are peroxisomal in filamentous fungi. In yeasts such as S.
• cerevisiae isocitrate lyase occurs in cytosol whereas malate synthase can be cytosolic or peroxisomal depending on sole carbon source.
• the citrate synthase is located in the peroxisomes and in the mitochondria, the aconitase can be in mitochondria and cytosol and the succinate dehydrogenase is exclusively in mitochondria.
• the distribution of the enzyme activities to different compartments and inability of the metabolites to freely travel between the compartments makes a fundamental difference to metabolic pathways and consequently the approaches to engineer these pathways by means of genetic engineering are essentially different.
• E. coli is requiring 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 it requires base to neutralize the acid. Further, in neutral conditions the produced glycolic acid is in dissociated form, which is a disadvantage in downstream processing.
• WO 2013/050659 concerns a eukaryotic host selected from micro-organisms, and a method for producing glycolic acid using said eukaryotic host cells, especially cells of a genetically modified fungal host.
• a eukaryotic host selected from micro-organisms
• a method for producing glycolic acid using said eukaryotic host cells especially cells of a genetically modified fungal host.
• glycolic acid occurs via ethanol.
• a micro- organism which is able to convert sugars to glycolic acid.
• the present technology describes a host and a method for producing glycolic acid directly from various sugars.
• a phrase "direct conversion of sugars to glycolic acid" as used herein is intended to mean that no ethanol is accumulated as an intermediate before glycolic acid production. In other words, cells are able to produce glycolic acid directly from sugars without first converting sugars into ethanol.
• a phrase "genetically modified 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 desired enzyme activity.
• 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.
• 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).
• An active fragment means a fragment having all the parts needed for completing the function typical for the protein.
• homologous means native and term “heterologous” means non- native gene, promotor, terminator etc. to the host cell.
• attenuating refers to partial or total deletion or knock-out of the said gene, modification of regulatory regions of the said gene in order to decrease its activity or silencing or decreasing the activity of the said gene by any methods without restricting to those mentioned above.
• 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.
• terminatator 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.
• FIGURE 1 illustrates the metabolic routes relating to the present invention.
• FIGURE 2 illustrates pUG-lox71-LEU2-lox66 plasmid used for constructing the promoter change cassettes
• FIGURE 3 illustrates pUG-lox71-HIS3-lox66 plasmid used for constructing the promoter change cassettes
• FIGURE 4 illustrates p4185 plasmid where GLYR1 encoding gene is expressed under S. cerevisiae TPI1 promoter.
• FIGURE 5 is a chart showing the production curves of gly colic acid and ethanol with different strains during 100 hours.
• a eukaryotic production host is selected from microorganisms, wherein the host is a fungal cell or a filamentous fungus cell, which has been genetically modified by transformation of a protein-encoding polynucleotide, or modification of a promoter region of the host cell, to overexpress gene encoding glyoxylate reductase activity and produce gly colic acid, and thus able to convert C5-C6 sugars directly to glycolic acid.
• the host is able to convert C5-C6 sugars to glycolic acid directly without first accumulating ethanol.
• promoters of the glycolic acid pathway genes aconitase and isocitrate lyase encoding genes of the host are changed under constitutive promoters (such as pTEFl or pFBAl) to avoid the regulation of transcription caused by the native promoters.
• promoters of the extended glyoxylate cycle recycling succinate formed from the isocitrate lyase reaction are changed into constitutive promoters (such as pTEFl or pFBAl) to avoid regulation of transcription caused by the native promoters.
• constitutive promoters such as pTEFl or pFBAl
• genes include cytoplasmic malate dehydrogenase, fumarase and mitochondrial succinate- fumarate transporter.
• one or several of the host's promoters of the glycolic acid pathway encoding genes selected from citrate synthase, aconitase and isocitrate lyase, or the extended glycolic acid pathway encoding genes selected from cytosolic malate dehydrogenase, fumarase and mitochondrial succinate- fumarate transporter, are changed into constitutive promoters.
• the host conducts the whole glycolic acid pathway in cytosol.
• Naturally most of the enzymatic reactions of the pathway are cytosolic but some of the reactions are peroxisomal or mitochondrial, which therefore requires a transport between the organelles and cytosol and for example slows down the production process in the cell.
• the host is able to express citrate synthase (CITl or CIT2) in cytosol.
• CITl citrate synthase
• the enzymatic reaction is taking place in the mitochondria or peroxisomes, respectively.
• the eukaryotic host may be any eukaryotic organism, but most usually it is a cell, preferably a micro-organism.
• the cell is a fungal cell.
• fungal cells When compared to for example E. coli, fungal cells are generally more tolerant to changing culture conditions such as low pH, lignocellulosic hydrolysates, impurities, degradation products of hydrolytic enzymes and toxins. They also have lower nutritional requirements compared to bacteria most commonly used in industrial processes. Thus fungal production is an economic approach to microbial production of glycolic acid.
• the host is a yeast cell.
• Yeasts like Saccharomyces cerevisiae or filamentous fungi like Aspergillus niger are naturally operating at very acidic pH.
• Yeast produced in fermentations is often used as cattle feed which benefits the economics of the process. Bacteria are generally not used as cattle feed but deemed a waste.
• Suitable yeasts are for example the genera Saccharomyces, Kluyveromyces, Candida, Scheffersomyces, Pachysolen and Hansenula.
• Yeast species of particular interest include S. cerevisiae, S. exiguus (also known as Kazachstania exigua), K. marxianus, K. lactis, K. thermotolerans, C. sonorensis, C. krusei (also known as Issachenkia orientalis and Pichia kudriavzevii), C. shehatae, Pachysolen tannophilus and Scheffersomyces stipitis.
• the host is a filamentous fungus.
• 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.
• A. niger is also a well- known citric acid producer and by metabolic engineering it can be made an efficient host for glycolic acid production.
• the host includes those of the species S. cerevisiae, S. exiguus, K. marxianus, K. lactis, C. krusei and A. niger.
• 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.
• the host is capable of producing glycolic acid at pH below 6, preferably below 5.5, below 5.0, below 4.5, below 4.0, below 3.5, below 3.0, below 2.5 and even below 2.0, until pH 1.5.
• Acidic or mildly acidic culture conditions reduce risk of contaminations and thus improve the process hygiene and safety. Each contaminated large scale fermentation results in direct loss of money.
• Glycolic acid has a pKa of 3.83 meaning that below a pH 3.8 it is mainly in the acid form which facilitates the separation.
• the host is capable of producing glycolic acid in non-buffered culturing conditions.
• the strain is tolerant to decreasing pH during the cultivation process. This simplifies the culturing process and thereby reduces costs.
• this characteristic naturally does not exclude possibility to regulate the pH conditions using bases (or acids) or even buffering agents.
• the glyoxylate reductase gene is a heterologous gene to a host. Especially when the Km value of the homologous gene is high it is beneficial to replace the gene by heterologous gene having lower Km value.
• the heterologous gene is preferably obtained from a plant source, gene obtained from Arabidopsis thaliana (Hoover et al. 2007) being the most preferred. It is also possible to use endogenous or native genes for production and modify the cell e.g. by changing its native promoter to a constitutive promoter to overproduce glyoxylate reductase.
• the glyoxylate reductase enzyme is characterized by having an EC number EC 1.1.1.79 (NADP+) or EC 1.1.1.26 (NAD+).
• Enzymes of class EC 1.1.1.79 (glyoxylate:NADP+ reductases, CAS 37250-17-2) catalyze reduction of glyoxylate using NADPH into glycolic acid and NADP or reduces hydroxypyruvate to glycerate.
• A. thaliana glyoxylate reductase designated here as "GLYR1" is one example of this class.
• Enzymes of class EC 1.1.1.26 reduces glyoxylate to glycolic acid or hydroxypyruvate to D-glycerate.
• the enzymes are defined according to the type of reaction they catalyze. Each enzyme is given a systematic name and an IUBMB (International Union of Biochemistry and Molecular Biology) number such as EC 1.1.1.26 (www, chem. qmul . ac . uk/iubmb/enzy me/) .
• 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.
• the gene is genetically optimized. It is understood by a skilled man that heterologous gene obtained by 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 glyoxylate reductase 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. Targeted integration can be accomplished by designing a vector or fragment having regions that are homologous to the upstream (5'-) and downstream (3'-) flanks of the target locus.
• the GLYR1 cassette (including suitable promoters and terminators if different from those of the target gene) and/or selection markers (with suitable promoters and terminators) will reside between the regions that are homologous to the upstream and downstream flanks of the target locus. Stable transformation is preferred as no selection pressure is needed during cultivation but alternatively also episomal plasmids and other non-integrated constructs are within this invention.
• native (homologous to the host cell) or non-native (heterologous to the host cell) promoters and terminators can permit the targeted integration of the GLYR1 or any other gene mentioned above, or any other gene further described below, into specific loci of the host cell's genome, and for simultaneous integration of the said gene and deletion of a native gene, such as, for example, a malate synthase (e.g. MLS1 or DAL7) encoding gene.
• a native gene such as, for example, a malate synthase (e.g. MLS1 or DAL7) encoding gene.
• the exogenous glyoxylate reductase gene may be maintained on a self- replicating plasmid, integrated randomly into the host cell's genome or inserted at one or more targeted locations.
• targeted locations include the locus of a gene that is desirably deleted or disrupted, such as the MLS1 encoding gene in S. cerevisiae or in A. niger.
• the cell has been further modified to overexpress the gene encoding glyoxylate reductase.
• the overexpressed genes such as glyoxylate reductase (e.g. GLYRl gene from A. thaliana), isocitrate lyase, aconitase, citrate synthase, fumarate reductase, acetyl- coenzyme A synthetase or a gene regulating glyoxylate cycle is under the control of a promoter and a terminator, both of which are functional in the modified fungal cell.
• glyoxylate reductase e.g. GLYRl gene from A. thaliana
• the genetically modified fungus may contain a single copy or multiple copies of the glyoxylate reductase gene (e.g. GLYRl gene) or any other gene mentioned above, or any other gene further described below. If multiple copies of the glyoxylate reductase 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 glyoxylate reductase 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 glyoxylate reductase genes to be under the control of different types of promoters and/or terminators .
• the production host has been further modified by increasing the flux towards the glyoxylate cycle.
• the flux towards the cycle can be increased e.g. by overexpressing isocitrate lyase, aconitase, citrate synthase, fumarase, malate dehydrogenase, mitochondrial succinate-fumarate transporter or fumarate reductase. Heterologous fumarate reductase would convert succinate into fumarate, which can be then metabolized further and used again in the glyoxylate cycle.
• Heterologous fumarate reductase would also by-pass the natural fumarate dehydrogenase which has a dual role in citric acid cycle and in oxidative phosphorylation. Hence, heterologous fumarate reductase would decouple glyoxylate production from oxidative phosphorylation. Increase of the glyoxylate flux may require also reducing activity of the enzymes consuming the intermediates of the cycle, e.g. attenuating expression of malate synthase(s), NAD and/or NADP dependent mitochondrial and/or cytosolic isocitrate dehydrogenase(s). Increase of flux towards the cycle results in more efficient production of gly colic acid.
• the yield of the glycolic acid can be increased by several ways. Increased yield saves the fermentation costs and enhances the down-stream processing.
• the production host has been further modified by modifying the genes regulating the glyoxylate cycle i.e. genes involved in negative regulation of glucose-repressible genes. This can be done for example by activating genes such as CATS in S. cerevisiae or in K. lactis by mutation or overexpression or by attenuating genes such as REG1 in S. cerevisiae.
• the production host has been further modified by improving NADPH availability.
• This can be done e.g. by overexpressing cytosolic aldehyde dehydrogenase such as ALD6 in S. cerevisiae or deleting phosphoglucose isomerase gene such as PGI1 in S. cerevisiae or K. lactis.
• Glyoxylate reductases are usually NADPH dependent and thus need NADPH for functioning.
• Glyoxylate reductase can be also NADH dependent and in these cases the improvement of NADPH availability is unnecessary.
• the cell has been further modified by attenuating genes involved in alcohol production in yeast or production of hydrolytic enzymes in filamentous fungi.
• Alcohol production competes with the glyoxylate cycle and it can be preferred to decrease it.
• This can be done e.g. by overexpressing one or more of pyruvate carboxylases such as PYC2, PYC1 in S. cerevisiae and acetyl-coenzyme A synthetase genes such as ACS1 in S. cerevisiae; or reducing expression of genes encoding alcohol dehydrogenases in S. cerevisiae.
• the host organism chosen for glycolic acid production is capable of further utilizing glycolic acid it is necessary to also attenuate the genes responsible for these enzyme reactions.
• some microbes are known to have glycolate oxidase which oxidases glycolic acid to glyoxylate.
• glycolic acid production can also be reduced if the host has enzymes other than glyoxylate reductase utilizing glyoxylate. Deletion of malate synthase was already described above as an example how glyoxylate cycle needs to be modified to produce glycolic acid.
• some fungi are known to have e.g. glyoxylate oxidase, which would need to be deleted in order to produce glycolic acid efficiently.
• One embodiment of this invention is also a method for producing glycolic acid.
• a eukaryotic cell encoding glyoxylate reductase is cultured in conditions allowing the expression the glyoxylate reductase gene, producing and secreting the glycolic acid and optionally recovering the glycolic acid from said medium.
• the fungus is cultivated in 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.
• Carbon source contains a sugar which can be hexose or pentose.
• Hexose can be e.g. glucose, fructose, mannose, or galactose and oligomers of glucose such as maltose, maltotriose, isomaltotriose, starch or cellulose.
• pentoses are xylose, xylan or other oligomer of xylose, and preferably also other carbon containing compounds to provide for growth and energy.
• the medium may also contain ethanol, glycerol, acetate, or amino acids, or any mixture thereof, preferably ethanol or acetate, most suitably ethanol, which further components can also function as carbon sources.
• the carbon substrates may be provided as pure substrates or from complex technical sources.
• the xylose containing sugars are suitably hydro lysates of plant bio mass e.g. hemicellulose-containing biomass, such as lignocellulose.
• the medium may consist of or contain complex, poorly defined elements, such as would be present in relatively inexpensive sources like black liquor, corn steep liquor or solids, or molasses.
• enzymes it may be necessary to add enzymes to the fermentation broth in order to digest these to the corresponding monomeric sugar.
• production hosts such as filamentous fungus hosts that secrete hydro lytic enzymes enhancing the production of fermentative sugars.
• 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 35 °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.
• the culturing pH is below 6, preferably below 5.5, below 5.0, below 4.5, below 4.0, below 3.5, below 3.0, below 2.5 and even below 2.0, until pH 1.5.
• 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.
• the culture medium contains no buffering agent.
• the pH is controlled to a constant pH of 3.5 to 5.5.
• Suitable buffering agents for regulating or buffering pH are basic materials that neutralize glycolic acid as it is 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, such as in the range of about 3 to about 4.
• the fermentation is conducted aerobically or micro aerobically. If desired, specific oxygen uptake rate, dissolved oxygen level or redox level can be used as a process control.
• the process of the invention can be conducted continuously, batch-wise, or some combination thereof.
• glycolic acid is excreted out from the cells into the growth medium from which it may be recovered without disrupting the cells.
• a cell described above is cultured and glycolic acid is recovered.
• Glycolic acid can be recovered from the fermentation medium by e.g. two-phase liquid-liquid extraction, ion exchange chromatography or reactive extraction or it can be polymerized in the fermentation medium and recovered thereafter.
• One embodiment of the invention is the use of eukaryotic host cells or eukaryotic organisms in production of glycolic acid or as a starting organism for preparation a production host suitable for production of glycolic acid.
• the eukaryotic organism is a fungal cell, preferably a yeast or filamentous fungus.
• the eukaryotic organism may be modified as described here and is suitable for the method as described here.
• Glycolic acid may for example be polymerized to polyglycolic acid (PGA). Glycolic acid is also used as a copolymer with lactic acid. Producing glycolic acid directly from renewable sources is a green alternative to be an industrially feasible production process.
• PGA polyglycolic acid
• GLYR1 glyoxylate reductase (heterologous gene from Arabidopsis thaliana)
• control i.e. the old glycolic acid production strain used for glycolic acid production in WO 2013/050659
• KL21 KL16 strain as backround + p4185 plasmid (GLYR1 under TPI1 promoter)
• K. lactis H3976 strain described in WO 2013/050659 was used as a progenitor strain for all further modifications.
• K. lactis aconitase (SEQ ID NO: 13) and isocitrate lyase (SEQ ID NO: 16) promoters were changed first.
• HIS3 and LEU2 marker genes were used respectively.
• Both cassettes contained loxP sites in front and after the marker genes.
• the loxP sites function as a specific target site for the Cre DNA recombinase enzyme (Guldener et al., 1996). Cre catalyzes DNA recombination leading up to the cleavage of loxP sites.
• Cre recombinase was expressed in K. lactis by transforming a Cre expression plasmid pKlNatCre into the yeast cell.
• the plasmid contained NAT marker gene and the transformants were selected on medium containing nourseotricin.
• This constructed KL6 strain is further transformed with the glyoxylate reductase expression vector, p4185 ( Figure 4) or uracil marker containing GLYRl integration fragment and the transformants selected on media containing uracil.
• the endogenous glyoxylate reductase encoding gene is put under a constitutive promoter in a similar manner as aconitase and isocitrate lyase genes.
• the KL6 strain described in Example 1 was further modified by introducing constitutive promoter in front of a truncated cytosolic malate dehydrogenase gene (SEQ ID NO: 19), fumarase encoding gene (SEQ ID NO: 22) and mitochondrial succinate fumarase transporter encoding gene (SEQ ID NO: 25).
• Constitutive S. cerevisiae promoters were amplified by PCR with the primer pairs listed in the Table 1. Fragments were cloned between the Spel site of the vector pUG-lox71-LEU2- lox66 with LEU2 marker gene ( Figure 2) or pUG-lox71-LEU2-lox66 with HIS3 marker gene ( Figure 3) containing the selection marker between the loxP sites. The resulting vector was then amplified with flanking primer pairs shown in the Table 2. Each primer has flanks -60-70 bp which overlap with the 5 ' UTR region of the target gene and the start of the ORF of the gene i.e. they introduce the constitutive promoter in front of the target gene.
• This strain was further transformed with the glyoxylate reductase expression vector, p4185 containing URA3 marker.
• the resulting strain, KL21 was tested for gly colic acid production.
• K. lactis strain H3986 and KL21 were pregrown in flasks in modified synthetic complete (SC) medium lacking uracil (Sherman F, Fink G, Hicks JB. (1983) Methods in Yeast Genetics. A Laboratory Manual. Cold Springs Harbor Laboratory, Cold Springs Harbor, N.Y.). Glucose (20 g L "1 ) was provided as carbon source. Pregrown cells were used for inoculating bioreactors.
• K. lactis H3986 and KL21 was grown in Biostat Qplus bioreactors (max working volume 1.1 L, Sartorius AG, Germany) in synthetic complete medium lacking uracil with glucose (10 g L "1 ) at pH 5, 30°C, 600-750 rpm and 0.8 L air per minute ( ⁇ 1 volume air per volume culture per minute).
• Clerol antifoaming agent (Cognis, France, 0.08-0.10 ⁇ 1-1) was used to prevent foam formation.
• the initial reactor volume was 800 mL. Bioreactors were inoculated to an initial OD 6 oo value of 0.23, from pre-cultures in synthetic complete medium (lacking uracil), which had grown overnight in shaken flasks.
• Cells were collected from the pre-cultures by centrifugation and re-suspended in a small volume of medium. Cultures were maintained in batch phase for 29.6 h and then fed with 51 g L-l glucose in synthetic complete medium (lacking uracil) at rates between 0.7 and 4.2 g h-1. The feed rate was periodically adjusted to prevent or reduce glucose or ethanol accumulation.
• the culture supernatant samples were analysed with Waters Alliance e2695 HPLC system (Waters, Milford, USA) where the injection volume was 10 ⁇ .
• An Aminex HPX-87H Organic Acid Column (300 mm x 7.8 mm) (Bio-Rad, USA) linked to a Fast Acid Analysis Column (100 mm x 7.8 mm) (Bio-Rad, USA) was used as a stationary phase in the HPLC. Columns were maintained at +55 °C and 5.0 mM H2S04 (Merck KgaA, Germany) was used as an eluent with the flow rate of 0.3 min "1 .
• strain KL6 described in Example 1 and KL16 described in Example 2 are further modified by introducing a constitutive S. cerevisiae TDH3 promoter in front of the K. lactis cytosolic malate dehydrogenase MDH2 (SEQ ID NO: 26). This replaces the native MDH2 promoter function.
• the KL6 strain is transformed with cassettes replacing the native promoters of the cytosolic malate dehydrogenase encoding gene (SEQ ID NO: 26), fumarase encoding gene (SEQ ID NO: 22) and mitochondrial succinate fumarase transporter encoding gene (SEQ ID NO: 25) in different combinations.
• the K. lactis strain KL16 is transformed with a plasmid or a fragment overexpressing cytosolic citrate synthase encoding gene e.g. K. lactis citrate synthase (SEQ ID NO: 27).
• Citrate synthase is targeted to cytosol by deleting or altering the mitochondrial or peroxisomal targeting signal of an endogenous citrate synthase encoding gene or introducing a heterologous citrate synthase gene which is targeted to cytosol naturally or is targeted to cytosol by deleting or altering mitochondrial or peroxisomal targeting signal of the gene e.g. S. cerevisiae CIT2 (YCR005C).

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