Production of dicarboxylic acids with genetically modified candida maltosa

Abstract

The present invention relates to a Candida maltosa having gene coding for enzymes involved in the ω-oxidation of monocarboxylic acids to dicarboxylic acids and subjected to at least one genetic modification for increasing the productivity of dicarboxylic acids by fermentation and a process for the production of medium-to-long chain dicarboxylic acids from monocarboxylic acids, esters of monocarboxylic acids and their mixtures, using said Candida maltosa.

Description

[0001]“Production of dicarboxylic acids with genetically modified Candida maltosa” Cross-Reference to Related Applications This Patent Application claims priority from Italian Patent Application No. 102023000028089 filed on December 27, 2023, the entire disclosure of which is incorporated herein by reference. Technical Field The present invention relates to a Candida maltosa subjected to at least one genetic modification aimed at increasing the productivity of dicarboxylic acids by fermentation and to a process for the production of medium-to-long-chain dicarboxylic acids from monocarboxylic acids, esters of monocarboxylic acids and mixtures thereof, using said Candida maltosa. Background of the Invention Medium-to-long-chain dicarboxylic acids (in the meaning of the present invention starting from 10 carbon atoms) have a wide range of applications: from use as monomers for the synthesis of polymers, to use in sectors as cosmetics, pharmaceuticals and phytopharmaceuticals. For example, sebacic acid (C10) is used for the synthesis of polyesters that find applications in fibres, films, resins, plasticisers, synthetic lubricants and adhesives. The industrial production of dicarboxylic acids (hereinafter DCAs or diacids) is typically carried out by chemical synthesis, but while short-chain dicarboxylic acids (in the meaning of the present invention, with less than 10 carbon atoms) can be synthesised with high yields, the costs of producing medium-to-long-chain DCAs are significantly higher due to the generation of by- products that may require complex purification processes. Production by chemical means is therefore technically difficult, environmentally unsustainable and economically expensive. Over the past 25 years, research has therefore turned its attention to the production of dicarboxylic acids by means of biotechnological processes, with the aim of achieving sustainable and low-cost processes. These processes are typically mediated by microorganisms such as yeasts, including those belonging to the Candida genus, such as C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. maltosa, C. parapsilosis, C. sake, C. tropicalis and C. viswanathii. Microorganisms belonging to the Candida maltosa species, for example, have genes coding for enzymes involved in the ω-oxidation metabolic pathway that enables the conversion of monocarboxylic acids to dicarboxylic acids and are therefore potentially capable of oxidising substrates such as alkanes, oils or monocarboxylic fatty acids to dicarboxylic acids. However, such microorganisms often produce no or very poor yields of dicarboxylic acids, and to obtain significant yields it is necessary to subject them to genetic modification. The genetic modifications typically applied are intended to favour the ω-oxidation metabolic pathway, for example by inhibiting the β-oxidation catabolic pathway, thereby reducing the degradation of the substrates and of the dicarboxylic acids produced. Using this approach, for example, complete blocking of the β-oxidation pathway in Candida tropicalis has been achieved by deletion of the POX genes (US 5,254,466). Another approach used is partial or complete inactivation of the function of the CAT gene, the gene coding for the enzyme carnitine acetyltransferase, obtained by deletion, where “partial inactivation of CAT gene function” means that only one allele of the CAT gene has been rendered non-functional, while “complete inactivation” means that both alleles of the CAT gene have been rendered non-functional. This enzyme is not directly involved in the β-oxidation catabolic pathway but is involved in the movement of acetyl-CoA within the cell. In fact, it catalyses transport of the acetyl group from acetyl-CoA to carnitine, with the consequent production of acetyl-carnitine, which can then be transported across the membrane. This enzyme thus enables the acetyl-CoA produced during β- oxidation to be transported from peroxisomes to mitochondria. Inactivation of CAT gene function results in reduction or absence of synthesis of the enzymes encoded by it (peroxisomal and mitochondrial CAT enzymes are generally encoded by a single gene). In the absence of these enzymes, acetyl-CoA produced by β-oxidation cannot be transported outside the peroxisomes and accumulates there, causing β-oxidation inhibition. CAT gene deletion in Candida has been described by Cao Z. et al. (Biotechnology Journal, 2006, 1: 68-74), Zhang et al. (Appl. Microbiol. Biotechnol., 2016, 100: 9567-9580) and in WO 2019 / 030652. Cao Z. et al. describes the deletion of one or two alleles of the CAT gene in Candida tropicalis. The microorganism heterozygous for the deletion is able to produce dicarboxylic fatty acids from alkanes. However, as β-oxidation is still partly active, some of the DCAs produced and of the substrate supplied are degraded. Conversely, the homozygous microorganism (with deletion on both alleles) is unable to convert alkanes into dicarboxylic acids and grows very slowly, even in the presence of glucose as a carbon source. Zhang et al. describes a strain of Candida tropicalis, subjected to the deletion of one or both alleles of the CAT gene, that can produce dicarboxylic acids from alkanes. Both the heterozygote and the homozygote for the deletion are capable of converting dodecane into the corresponding dicarboxylic acid in quantities not exceeding 13 g / l after 192 hours of fermentation. However, the microorganism homozygous for the deletion shows a low growth rate, even in a culture medium containing glucose, and lower biomass production than the microorganism with no deletion and the heterozygote. WO 2019 / 030652 describes the deletion of both alleles of the CAT gene in wild type Candida maltosa. The microorganism obtained, however, produces only very small amounts of dicarboxylic acids, amounting to 1.1 g / L, after 78 hours of fermentation. In order to overcome the limitations of microorganisms in the known art, the Applicant has surprisingly identified a microorganism of the species Candida maltosa having β-oxidation inhibited by (i) inactivation of the function of the CAT gene and / or (ii) inactivation of the function of the MFE2 gene (multifunctional enzyme type 2), with very good results in terms of amount of dicarboxylic acids produced, productivity and selectivity, for example with a productivity of more than 1 g / h / linit. This microorganism is therefore suitable for the production of dicarboxylic acids on an industrial scale. For the purpose of the invention, the term “Candida maltosa”' is used in its usual meaning and is therefore distinguished from Candida tropicalis. These are in fact two distinct species in the field of yeast taxonomy that show significant differences at both molecular and biochemical levels (Meyer et al., 1975, Arch. Microbiol., 104: 225-231; Kanekok et al., 1977, Agric. Biol. Chem., 41 (11): 2269-2275; C.P Kurtzman et al., 2011, 'Yeasts, a taxonomic study', Volume 2, Fifth Edition, 1135-1136 and 1256-1258). Summary of the Invention According to a first aspect, the present invention thus relates to a Candida maltosa having the function of the CAT gene (carnitine acetyltransferase) and / or of the MFE2 gene (multifunctional enzyme type 2) inactive, characterised by a dicarboxylic acid productivity of more than 1 g / h / linitat an optical density (OD625) of at least 90 and after incubation at 30°C for at least 6 hours in a culture medium fed with an oleic acid, in a quantity to maintain its concentration of at least 13 g / l, and 1.5 g / l / h glucose. The production of dicarboxylic acids may be quantified by gas chromatography (GC) or high- performance liquid chromatography (HPLC). Those skilled in the art know that the result remains the same regardless of the method of analysis used. In the present invention, the production of dicarboxylic acids is quantified by gas chromatography analysis using a gas chromatograph equipped with a ZB-5MS plus column (30 m x 0.25 mm x 0.25 µm) and a flame ionisation detector (GC-FID), using N,O-bis-trimethylsilyl- trifluoroacetamide (BSTFA) as a derivatising agent. In the present invention, optical density (OD625) is measured using a Perkin Elmer Lambda 35 spectrophotometer. The inactivation of CAT or MFE2 gene function is achieved by rendering non-functional one or both alleles of the CAT or MFE2 gene and / or the product of their transcription. Preferably one or both - even more preferably both - alleles of the CAT gene or the MFE2 gene are rendered non-functional through their deletion. The Candida maltosa according to the invention therefore has one or both alleles of the CAT gene and / or the product of its transcription non-functional. Preferably, the Candida maltosa according to the invention has both alleles of the CAT gene non-functional. Even more preferably, the Candida maltosa according to the invention has one or both, preferably both, alleles of the CAT gene deleted. Alternatively, the Candida maltosa according to the invention has one or both alleles of the MFE2 gene and / or the product of its transcription non-functional. Preferably, the Candida maltosa according to the invention has both alleles of the MFE2 gene non-functional. Even more preferably, the Candida maltosa according to the invention has one or both, preferably both, alleles of the MFE2 gene deleted. A second aspect of the invention relates to a process for producing dicarboxylic acids from a substrate selected from monocarboxylic acids, monocarboxylic acid esters and mixtures thereof, comprising fermenting a Candida maltosa in a culture medium. Description of Embodiments of the Invention The present invention will be described in more detail below. The present invention relates to a Candida maltosa having enzymes involved in the ω-oxidation of monocarboxylic acids to dicarboxylic acids and capable of ω-oxidising monocarboxylic acids to dicarboxylic acids. The ω-oxidation metabolic pathway is therefore active. For the purposes of the present invention, “productivity” means the ratio of grams of DCA produced at the end of the process to the time elapsed since the addition of the substrate compared to one litre of initial medium, g / h / linit. For the purposes of the present invention, “quantity of DCA produced” means the quantity (grams) of dicarboxylic acids produced in a fermentation process conducted from 1 litre of initial broth (g). For the purposes of the present invention, “selectivity” means the ratio of moles of DCA produced to moles of substrate consumed (mol / mol). Said Candida maltosa may advantageously have undergone genetic modification and / or selection techniques aimed at increasing dicarboxylic acid productivity, such as non-specific mutagenesis. According to a preferred embodiment, the Candida maltosa according to the invention was subjected to random mutagenesis, has the function of the CAT (carnitine acetyltransferase) gene and / or the MFE2 (multifunctional enzyme type 2) gene inactive and is characterised by a dicarboxylic acid productivity of more than 1 g / h / linit at an optical density (OD625) of at least 90 and after incubation at 30°C for at least 6 hours in a culture medium fed with oleic acid, in a quantity to maintain its concentration of at least 13 g / l, and 1.5 g / l / h glucose. Said Candida maltosa advantageously produces dicarboxylic acids with better results compared to Candida maltosa not subjected to random mutagenesis and / or with β-oxidation not inhibited, in particular having the CAT and MFE2 gene function active. In a process for the production of dicarboxylic acids, because β-oxidation is inhibited by inactivation of the function of (i) the CAT gene and / or (ii) the MFE gene, the Candida maltosa according to the invention advantageously produces dicarboxylic acids with a productivity of 1 g / h / linit or more, preferably 1.3 g / h / linit, more preferably 1.6 g / h / linit, more preferably 1.8 g / h / linit, more preferably 2.2 g / h / linit, even more preferably above 2.6 g / h / linit, depending for example on the type of substrate fed. For example, in a fermentation process conducted using a mixture of monocarboxylic acids comprising at least 70% oleic acid as substrate, the microorganism according to the invention produces dicarboxylic acids with a productivity that is at least 30%, preferably at least 40% higher than that obtainable with the microorganism subjected to random mutagenesis but not having β-oxidation inhibited, and in particular having the function of the CAT gene and the MFE2 gene active. In addition, the amount of DCA produced by the microorganism according to the invention is advantageously at least 35 g of dicarboxylic acids in a fermentation process conducted from 1 litre of initial broth (g), preferably at least 60 g, more preferably at least 80 g, more preferably at least 90 g, more preferably at least 110 g, even more preferably at least 130 g, depending for example on the type of substrate fed. For example, in a fermentation process conducted using a mixture of monocarboxylic acids comprising at least 70% oleic acid as substrate, the microorganism according to the invention is able to produce an amount of dicarboxylic acids at least 30%, preferably at least 40% higher than that obtainable with the microorganism subjected to random mutagenesis but having β-oxidation not inhibited, in particular having the function of the CAT gene and the MFE2 gene active. Furthermore, the microorganism according to the invention, in a process for the production of dicarboxylic acids, advantageously produces dicarboxylic acids with a selectivity of more than 0.35 mol / mol, preferably 0.5 mol / mol, more preferably 0.7 mol / mol, more preferably 0.8 mol / mol, even more preferably 0.9 mol / mol, depending for example on the type of substrate fed. For example, in a fermentation process conducted using a mixture of monocarboxylic acids comprising at least 70% oleic acid as substrate, the microorganism according to the invention is capable of producing dicarboxylic acids with selectivity at least 50%, preferably at least 60%, higher than that obtainable with the microorganism subjected to random mutagenesis but having β-oxidation not inhibited, in particular having the CAT and MFE2 gene function active. The production of dicarboxylic acids may be quantified by gas chromatography (GC) or high- performance liquid chromatography (HPLC). Those skilled in the art know that the result remains the same regardless of the method of analysis used. In the present invention, the production of dicarboxylic acids is quantified by gas chromatography analysis using a gas chromatograph equipped with a ZB-5MS plus column (30 m x 0.25 mm x 0.25 µm) and a flame ionisation detector (GC-FID), using N,O-bis-trimethylsilyl- trifluoroacetamide (BSTFA) as a derivatising agent. According to a preferred embodiment, the Candida maltosa according to the invention was subjected to the operations of: a) random mutagenesis; b) selection of one or more strains producing dicarboxylic acids from monocarboxylic acids; c) inhibition of β-oxidation by inactivation of the function of (i) the CAT gene, or (ii) the MFE2 gene, or a combination thereof. Among operations c), inactivation of the CAT gene function is preferred. Operations a), b) and c) may be performed in any order. Preferably, random mutagenesis operations a) precede β-oxidation inhibition operations c). Selection operations b) are performed to identify, from among the strains of the Candida maltosa microorganism and / or among the mutants obtained by operation a) and / or among the strains obtained by operation c), one or more strains producing dicarboxylic acids from monocarboxylic acids with better results in terms of productivity and / or quantity of DCA produced and / or selectivity. Such selection operations b) may for example be performed by inoculating the strains in a culture medium containing a sufficient quantity of a monocarboxylic acid and verifying, by appropriate analytical techniques, that dicarboxylic acids are present in the fermentation broth after incubation; for example, selection operations may be carried out by inoculating the strains at an optical density (OD625) of 1.5 in culture medium containing 10-20% by weight of oleic acid and incubating them at 30°C for 24 hours. The production of dicarboxylic acids may, for example, be quantified by gas chromatography analysis using a gas chromatograph equipped with a ZB-5MS plus column (30 m x 0.25 µm) and flame ionisation detector (GC-FID), using N,O-bis-trimethylsilyl trifluoroacetamide (BSTFA) as a derivatising agent. Operations b) of selecting one or more strains producing dicarboxylic acids from monocarboxylic acids may precede and / or follow random mutagenesis a). Preferably, selection operations b) follow random mutagenesis a). Operations b) of selecting one or more strains producing dicarboxylic acids from monocarboxylic acids may precede and / or follow the operations of inhibiting β-oxidation c). Preferably, selection operations b) precede β-oxidation inhibition operations c). According to a preferred aspect, a Candida maltosa was subjected to the following operations: a) random mutagenesis; b) selection from among the mutant strains obtained in step a) of one or more strains producing dicarboxylic acids with a productivity greater than 0.7 g / h / linit, c) inhibition of β-oxidation in one or more strains selected in b) by inactivating the function of the (i) CAT gene, or (ii) MFE2 gene, or a combination thereof resulting in a Candida maltosa characterised by a productivity of 1 g / h / linit or more, more preferably 1.3 g / h / linit, more preferably 1.6 g / h / linit, more preferably 1.8 g / h / linit, more preferably 2.2 g / h / linit, even more preferably 2.6 g / h / linit, at an optical density (OD625) of at least 90 and after incubation at 30°C for at least 6 hours in a culture medium fed with oleic acid, in a quantity to maintain its concentration of at least 13 g / l of oleic acid, and 1.5 g / l of glucose. Advantageously, these results are also obtained by subjecting a Candida maltosa that does not produce dicarboxylic acids or produces them with a yield of less than 0.015 g / h / linitto random mutagenesis a). Advantageously, through selection operations b) are identified one or more strains from mutants obtained by mutagenesis operation a) that produce dicarboxylic acids from monocarboxylic acids with a productivity more than 0.7 g / h / linit, preferably more than 0.9 g / h / linit, more preferably more than 1.3 g / h / linit, even more preferably more than 1.4 g / h / linit. Alternatively, or in combination, by means of selection operations b) are identified one or more strains from among mutants obtained by mutagenesis operation a) that produce an amount of DCAs of more than 15 g, preferably more than 30 g, more preferably more than 60 g, more preferably more than 70 g, from monocarboxylic acids. Alternatively, or in combination, one or more strains that produce dicarboxylic acids from monocarboxylic acids with a selectivity greater than 0.15 mol / mol, preferably greater than 0.2 mol / mol are identified from among the mutants obtained by the mutagenesis operation a). Preferably, among the strains identified by selection operations b), the one that produces dicarboxylic acids with higher productivity and / or in greater quantity and / or with greater selectivity is subsequently subjected to the inhibition of β-oxidation. Preferably, among the strains identified from selection operations b), the one that produces dicarboxylic acids with higher productivity is subsequently subjected to the inhibition of β-oxidation. Random mutagenesis operations a) may be carried out by any technique known to those skilled in the art. They preferably include one or more techniques chosen from among: X-rays, ultraviolet (UV) radiation, treatment with chemical mutagens (such as, for example, nitrosoguanidine (NTG), 4-nitroquinolone-1-oxide, methyl methane sulfonate (MMS), ethyl methane sulfonate (EMS), hydroxylamine (HA), methyl nitrosoguanidine (MNNG or MNG) and combinations thereof. Treatments with ethyl methane sulfonate and / or ultraviolet radiation are preferred. According to a preferred embodiment of the invention, operations c) comprise inactivating the function of the CAT gene, which may be done by any known technique that renders one or both alleles of the CAT gene or the product of its transcription or the product of its translation non- functional. Preferably, CAT gene function is inactivated by any known technique that renders both alleles of the CAT gene non-functional. The genetic technique of inactivation by deletion is particularly preferred. Within the meaning of the present invention, deletion means a gene mutation consisting of the loss of one or more nucleotides in a DNA sequence. Even more preferred is the genetic technique of deletion carried out by a homologous recombination process, in particular by using a deletion cassette. A plasmid containing a gene for resistance to an antibiotic under the control of a constitutive promoter as well as a gene for a recombinase under the control of an inducible promoter has proved to be particularly preferred. CAT gene alleles may for instance be deleted as described in the example on pages 4 to 6 in WO 2019 / 030652, which is therefore intended to form part of this description. According to another embodiment of the invention, operations c) comprise inactivating the function of the MFE2 gene, which may be done by any known technique that renders one or both alleles of the MFE2 gene or the product of its transcription or the product of its translation non-functional. Preferably, the operations for inactivating the function of the MFE2 gene are performed by any known technique that renders both alleles of the MFE2 gene non-functional. The genetic technique of inactivation by deletion is particularly preferred. A second aspect of the invention therefore relates to a process for producing dicarboxylic acids from a substrate selected from monocarboxylic acids, monocarboxylic acid esters and mixtures thereof, comprising fermenting Candida maltosa according to the invention in a culture medium. In the process according to the invention, fermenting preferably comprises a phase of Candida maltosa cell growth and a subsequent phase of production of dicarboxylic acids. Said monocarboxylic acids are preferably C12-C24 acids, more preferably C16-C22 acids. Said monocarboxylic acids may be used alone or in the form of mixtures of acids of different chain lengths and unsaturation. Examples of monocarboxylic acids are lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, oleic acid, stearic acid, linoleic acid, linolenic acid, ricinoleic acid, erucic acid and arachidic acid. Preferably, these monocarboxylic acids are unsaturated, more preferably monounsaturated. Oleic acid is preferred. Said mixtures of acids preferably comprise at least 70%, more preferably at least 80%, even more preferably at least 90% by weight of unsaturated monocarboxylic acids. Said unsaturated monocarboxylic acids may be monounsaturated or polyunsaturated. Preferably said unsaturated monocarboxylic acids comprise more than 65%, preferably more than 70%, more preferably more than 80% by weight of monounsaturated monocarboxylic acids. Said monounsaturated monocarboxylic acids preferably comprise more than 80%, more preferably more than 90% by weight oleic acid. An example of a particularly preferred mixture includes at least 70% by weight of oleic acid (preferably 80-90% by weight), up to 20% by weight of linoleic acid (preferably 4-12% by weight), up to 5% by weight of stearic acid (preferably 1.5-4% by weight) and up to 6% by weight of palmitic acid (preferably 2-5% by weight). Said mixtures are obtained, for example, through the hydrolysis of vegetable oils or a mixture of vegetable oils or waste oils. By vegetable oils are meant either the unmodified product of pressing or an oil that has undergone chemical or chemical-physical modifications such as purification treatments, enzyme enrichment or hydrogenation, for example oils that have been selectively hydrogenated to increase the monounsaturated fatty acid content. Examples of vegetable oils are thistle oil, soybean oil, olive oil, castor oil, sunflower oil, peanut oil, maize oil, palm oil, safflower oil, jatropha oil, cuphea oil, oils from Brassicaceae such as Crambe abyssinica, Brassica carinata, Brassica napus (rapeseed), Lesquerella, and other oils with a high monounsaturated acid content. Sunflower, thistle, safflower, palm and Brassicaceae oils are preferred, preferably with a high monounsaturated acid content. Particularly preferred is the use of mixtures of monocarboxylic acids from the hydrolysis of sunflower oil, thistle oil, safflower oil, Crambe abyssinica, Brassica carinata, Brassica napus (rapeseed) with a high monounsaturated acid content. Even more preferred is the use of mixtures of monocarboxylic acids from one or more oils with a high oleic acid content and / or a high erucic acid content. Such mixtures may contain geometric or positional isomers of said monocarboxylic acids, which may result from the chemical or physical-chemical modification of polyunsaturated acids. An example of an oleic acid isomer is 12-octadecenoic acid. Monocarboxylic acid esters are carboxylic acids whose carboxylic group forms an ester bond, for example by reaction with alcohols, preferably a monoalcohol or polyalcohol. Preferred monoalcohols include C1-C9 alkyl groups; more preferred are methyl, ethyl, propyl and butyl alcohols. An example of a preferred polyalcohol is glycerol. Methyl and ethyl esters of unsaturated carboxylic acids may be obtained by the transesterification of methanol and ethanol with the triglycerides contained in vegetable oils. The production of dicarboxylic acids with the Candida maltosa microorganism according to the invention is preferably performed by means of biphasic fermentation, in which the cell growth phase is followed by a dicarboxylic acid production phase. Advantageously, the growth and production phases are conducted in the same culture medium. In the cell growth phase, the microorganism may for example be inoculated in a culture medium at an optical density (OD625) of 1.5 to 4 and maintained at a temperature of 25 to 35°C, at a pH of 5 to 8, in the presence of 5 to 40% dissolved oxygen. According to the present invention, "culture medium" means an aqueous solution comprising one or more components selected from: salts, trace elements, vitamins, nitrogen sources, preferably inorganic, carbon sources. For example, said culture medium may comprises glucose from 60 to 80 g / l, phosphate salts from 2 to 10 g / l, inorganic nitrogen from 1.4 to 1.6 g / l, organic nitrogen from 1.5 to 1.8 g / l, magnesium salt from 0.5 to 0.7 g / l, vitamins from 0.5 to 2 µg / l, trace elements from 0.2 to 8 mg / l, weak organic acid from 1.0 to 2 g / l. The production step is preferably a fed-batch process intended to keep the metabolism of the cell biomass active and catalytically performing in producing dicarboxylic acids. Advantageously, this phase has a dual feed: a sugar, to keep the cells active, and a substrate for biotransformation selected from monocarboxylic acids, monocarboxylic acid esters and their mixtures. The sugar may be at least one of glucose, sucrose, fructose. Preferably, the sugar is glucose. Sugar is preferably supplied in quantities of 0.5 to 4.0 g / L / h, more preferably 1 to 3 g / L / h. The amount of sugar supplied depends on the amount of biomass obtained in the previous growth phase. The production phase is typically conducted at a temperature of 25 to 35°C, more preferably between 28 and 32°C. The pH of the medium is preferably maintained at 5 to 8, more preferably 6 to 7. To maintain the pH in the ranges indicated, an aqueous solution of at least one inorganic base may be added, such as, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide or mixtures thereof. An aqueous solution of potassium hydroxide is preferred. Dissolved oxygen is preferably maintained at 5 to 50%, more preferably at 10 to 40%, through the control of agitation and aeration. The process according to the invention, preferably carried out by biphasic fermentation, makes it possible to obtain the corresponding dicarboxylic acids from a mixture of monocarboxylic acids with a high monounsaturated acid content. According to a preferred embodiment of the invention, through the process according to the invention, conducted using as a substrate a mixture comprising at least 70% by weight of oleic acid (preferably 80-90% by weight), up to 20% by weight of linoleic acid (preferably 4-12% by weight), up to 5% by weight of stearic acid (preferably 1.5-4% by weight) and up to 6% by weight of palmitic acid (preferably 2-5% by weight), dicarboxylic acids are advantageously obtained: - with a productivity of 1 g / h / linitor more, preferably 1.3 g / h / linit, more preferably 1.6 g / h / linit, more preferably 1.8 g / h / linit, even more preferably more than 2.6 g / h / linit, and / or - in quantities exceeding 35 g from an initial litre of substrate, preferably at least 60 g, more preferably at least 80 g, more preferably at least 90 g, more preferably at least 110 g, even more preferably at least 130 g of dicarboxylic acids, and / or - with selectivity higher than 0.35 mol / mol, preferably 0.5 mol / mol, more preferably 0.7 mol / mol, more preferably 0.8 mol / mol, even more preferably 0.9 mol / mol. The process according to the invention, conducted using the microorganism of the invention and the substrate above mentioned, advantageously allows to obtain a composition of dicarboxylic acids with a content of saturated aliphatic dicarboxylic acids having a number of carbon atoms one unit less than the monocarboxylic acids fed to fermentation (namely for example C15, C17 and C19 acids in a composition of C16, C18 and C20 acids, i.e. one-carbon shorter saturated aliphatic dicarboxylic acids), that is less than 500 ppm, preferably less than 450 ppm, more preferably 350 ppm and even more preferably from 1 ppm to 200 ppm. Said saturated aliphatic dicarboxylic acids can be obtained as fermentation by-product. More preferably, the content of said saturated aliphatic dicarboxylic acids having a number of carbon atoms one unit less is individually less than 150 ppm. This makes the composition particularly suitable for use as a monomer in polymerisation reactions, e.g. in the synthesis of polyesters, polyamides and polyurethanes. In particular, said composition is useful for the synthesis of polyesters from diacid-diols. Moreover, at the end of a fermentation process wherein the pH is maintained at values of 5-8 by means of KOH, the viscosity of the fermentation broth, measured by using viscosimeter set up at 50 rpm, is advantageously low, in particular is less than 500 cST, preferably less than 400 cST. Such a low viscosity allows to obtain homogeneous broths that therefore show a better dissolution of nutrients and substrates and require reduced agitation in order to maintain the required level of dissolved oxygen in fermentation process. The kinematic viscosity of the fermentation broth, measured in centi Stokes, can be evaluated for example by Fungilab™ Alpha Series Rotational Viscometer at different rpm. The Applicant also surprisingly found that, in a process for the production of dicarboxylic acids by biphasic fermentation with the microorganism Candida maltosa according to the invention, an improvement in production performance can be achieved by reducing the amount of phosphate supplied during preparation of the culture medium. Reducing the phosphate content promotes better emulsification of the substrate added at the beginning of the fermentation phase. It also has the added advantage of decreasing the costs associated with preparation of the culture medium and the eventual removal of phosphates during purification of the product of interest, as the residual phosphate content at the end of the fermentation process is reduced. According to a preferred embodiment, the process for the production of dicarboxylic acids with the microorganism Candida maltosa according to the invention is advantageously conducted in a culture medium comprising an amount of phosphate of less than 5 g / l, preferably less than 4.5 g / l, more preferably less than 4 g / l, even more preferably less than 3 g / l. Such a culture medium includes an amount of phosphate greater than 1 g / l, preferably greater than or equal to 2 g / l. With the presence of phosphate in quantities of more than 1 g / l and less than 5 g / l in the medium, advantageously an improvement in performance in the production phase of at least 2%, preferably at least 3%, even more preferably at least 5%, is achieved compared to the process in a medium with phosphate quantities of more than 5 g / l. Production performance refers to productivity and / or grams of dicarboxylic acids produced and / or selectivity. In said process for the production of dicarboxylic acids, an amount of phosphate below 5 g / l may advantageously be achieved by providing the culture medium with dibasic potassium phosphate in amounts of less than 7 g / l, preferably 3 to 6.5 g / l, more preferably 3.5 to 5 g / l, and by not introducing monobasic potassium phosphate or supplying it in amounts of less than 1.3 g / l. Thus, at the end of the production phase, the phosphate content is advantageously less than 2 g / l, preferably less than 1 g / l. According to a preferred embodiment, the process for producing dicarboxylic acids with the microorganism Candida maltosa according to the invention is conducted in a culture medium comprising 3.5 to 5 g / l potassium dibasic phosphate and in the absence of monobasic phosphate. According to a preferred aspect, the process for the production of dicarboxylic acids with the microorganism Candida maltosa according to the invention, preferably subjected to random mutagenesis, comprises the addition of one or more compounds selected from fatty acids, methyl or ethyl esters of fatty acids, fatty acid salts, alkanes and vegetable oils at the beginning of the cell growth phase. Preferably, said compounds are selected from fatty acids, methyl or ethyl esters of fatty acids, salts of fatty acids and alkanes. More preferably, said compounds are selected from fatty acids and alkanes. However, fatty acids are preferred. Among fatty acids, oleic acid is preferred. Said compounds are advantageously added in quantities of 0.01 to 5% by weight, preferably 0.05 to 5% by weight, relative to the initial volume of culture medium. Said compounds are preferably added to the culture medium before inoculation of the microorganism or during the first 8 hours, more preferably during the first 7 hours after inoculation. According to a preferred embodiment, said compounds are added to the culture medium before inoculation of the microorganism. In fact, the Applicant surprisingly found that, with the addition of these compounds before inoculation of the microorganism or during the first 7 hours after inoculation, the duration of the cell growth phase is shortened in comparison with not adding them or with adding them after 7 hours from inoculation of the microorganism, for example after 9 hours. The duration of the cell growth phase may, for example, be assessed by monitoring the Oxygen Uptake Rate (OUR). The OUR, i.e. the amount of oxygen consumed, may for example be measured using the following formula: OUR = (Pair in x O2in) - (Pair out x O2out) where Pair inis the flow rate of the incoming air (for example measured in mmol / h), O2inis the percentage concentration of oxygen in the incoming air, Pair out is the flow rate of the outgoing air, O2out is the percentage concentration of oxygen in the outgoing air. The cell growth phase begins when the microorganism is inoculated into the culture medium and is considered to have ended when the OUR reaches its maximum value. The duration of the cell growth phase is therefore the time it takes for the microorganism that has just been inoculated into the growth medium to reach the maximum OUR value. During the cell growth phase, the microorganism according to the invention reaches the maximum OUR value in at least 15 minutes less time, preferably 20 minutes less time, more preferably at least 30 minutes less time, even more preferably at least 40 minutes less time than if said compounds were not added. Indeed, the Applicant has identified a method for accelerating the growth of the Candida maltosa microorganism according to the invention, preferably subjected to random mutagenesis, comprising the addition of one or more compounds selected from fatty acids, methyl or ethyl esters of fatty acids, alkanes and vegetable oils at the beginning of cell growth, preferably before inoculation of the microorganism. A further advantageous effect of adding one or more of the above-mentioned compounds to a dicarboxylic acid process at the beginning of the cell growth phase is to slow down foam formation and reduce the amount of foam that typically forms during the production phase. Foam formation is indeed a common problem in fermentation processes for the production of dicarboxylic acids, especially when conducted on a large scale. Foam typically forms when the substrate to be converted is added to the culture medium, causing acidification of the fermentation medium, which must be brought to the appropriate pH for the process conditions by the addition of a base, resulting in the formation of a soap and consequently foam. This foam is generally reduced by adding an antifoaming agent. However, despite addition of the antifoaming agent, in many cases the foam will form again within minutes of addition. In the process according to the invention, on the other hand, by the addition of 0.05 to 5% by weight of one or more compounds selected from fatty acids, fatty acid salts, methyl or ethyl esters of fatty acids, alkanes and vegetable oils, respect to the initial volume of the culture medium, foam formation is slowed down when the substrate to be converted is added to the culture medium. Advantageously, foam formation also slows down after addition of the defoaming agent during the production phase. This defoaming agent may be chosen from silicone and non-silicone, preferably the defoaming agent is silicone. “Slowing down of foam formation” means a delay in foam formation of at least 5 minutes, preferably at least 10 minutes, from the time the substrate to be converted is added and / or at least one hour after addition of the defoaming agent. In addition, in fermentation processes for the production of dicarboxylic acids, addition of the above-mentioned compounds makes it possible to decrease the quantity of antifoaming agent required to contain foam formation by at least 30%, compared to the quantity required in processes conducted without addition of the above-mentioned compounds, thus also reducing the adverse effects of the antifoaming agent on oxygen transport during the production phase and on the product purification process. Accordingly, the applicant has identified a method for slowing down and decreasing foam formation in a process for the production of dicarboxylic acids by Candida maltosa according to the invention, preferably subjected to random mutagenesis, comprising the step of adding one or more compounds selected from fatty acids, fatty acid salts, methyl or ethyl esters of fatty acids, alkanes and vegetable oils at the beginning of the cell growth phase. Preferably, these compounds are selected from fatty acids and vegetable oils. Said compounds are advantageously added in amounts of 0.05 to 5%, preferably 0.1 to 5%, more preferably 0.6 to 5% by weight, respect to the initial volume of the culture medium. Said compounds are preferably added to the culture medium prior to inoculation of the microorganism. The following examples illustrate the present invention for non-limiting purposes. METHODS used in the Examples Sample preparation and analysis of monocarboxylic (MCA) and dicarboxylic acids (DCA) by GC- FID. A volume of 0.5 ml of 8M NaOH was added to 5 ml of culture broth at the end of fermentation and the sample was incubated at 50°C for 5 minutes.1.1 ml of this solution (equal to 1 ml of the original sample) was taken and diluted 5-fold with 10 mM NaOH.100 μl of 37% HCl was added to 1 ml of the sample. After stirring, the fatty acids were extracted with 5 ml diethyl ether. Following centrifuging at 4000 rpm for 3 min, the (lower) organic phase was recovered and made up to 10 ml with diethyl ether. 1 ml of the extract was taken and brought to dryness, then redissolved in 1 ml of acetonitrile. A ZB5MS plus column (30 m x 0.25 mm x 0.25 µm), Phenomenex, and BSTFA (Supelco) as derivative agent was used for GC-FID analysis of samples containing free fatty acids. 1 μl of sample was injected and the 18-min run was performed according to the following parameters: split 1:4; He flow 1.25 ml / min; T 300°C. All standards used for the quantification of fatty acids were prepared from a stock solution obtained by dissolving the compound in acetonitrile (w / v), with pyridine added initially where necessary, in a flask with a volume of at least 25 ml. Standard solutions were prepared by successive dilutions in acetonitrile (v / v) at different concentrations, in a range from 0.00625 to 1 mg / ml, depending on the fatty acid used. EXAMPLES EXAMPLE 1 A strain of Candida maltosa, subjected to random mutagenesis and selected on the basis of its ability to produce dicarboxylic acids from monocarboxylic acids (with higher productivity than the original strain), referred to as strain A, was subjected to deletion of both alleles of the CAT gene using the protocol described in the example on pages 4-6 of WO 2019 / 030652, resulting in the strain referred to as strain B. EXAMPLE 2 Strain B obtained in Example 1 was streaked onto a plate with YPD medium (glucose 20 g / l, yeast extract 10 g / l, tryptone 20 g / l, agar 20 g / l) and incubated at 30°C for 24 hours. Subsequently, a quantity of biomass was taken from the plate and inoculated into an Erlenmeyer flask containing liquid YPD medium. After a period of growth at 30°C with agitation, the culture was transferred into a fermenter containing 1 litre of medium. The process was conducted by biphasic fermentation, with decoupling of the biomass cell growth phase from the DCA production phase. When the biomass reached a steady state of growth (after about 960 minutes, when the OUR reached its maximum value), the substrate was added and the DCA production phase began, which lasted for about 50 hours. The DCA production phase involved the feeding of 1.5 g / l / h glucose and, as substrate, a mixture of monocarboxylic acids with a high oleic acid content (oleic acid 91.1%, linoleic acid 4.5%, stearic acid 2.6%, palmitic acid 1.8%). The supply of substrate was adjusted to maintain the substrate concentration at approximately 20 g / l. The medium used in fermentation for cell growth and production had the following composition: phosphate buffer, nitrogen source (3.81 g / l), vitamins (1 mg / l), magnesium salts (0.6 g / l), trace elements (1.25 ml), glucose (70 g / l). The process temperature in the two phases was maintained at 30°C. The pH was kept constant at about 6.4 by the addition of KOH. Dissolved oxygen was kept at 30%. The viscosity of the fermentation broth, measured using Fungilab™ Alpha Series Rotational Viscometerset at 50 rpm, was 359.1 cST. Samples of the fermentation broths were extracted and analysed to quantify the DCAs produced. The results obtained are shown in Table 1. EXAMPLE 3 COMPARATIVE The process in Example 2 was applied using strain A. In this process, as the β-oxidation pathway is still active in the microorganism, the substrate supply was adjusted to maintain its concentration at around 50 g / l. The results obtained are shown in Table 1 where: - DCA titre (g / lfin) = grams of dicarboxylic acids (DCA) produced in relation to one litre of final fermentation medium, - DCA (g) = grams of dicarboxylic acids (DCA) produced in the process from 1 litre of initial broth, - Productivity g / h / linit = ratio between the amount in grams of DCA at the end of the process and the time elapsed since the addition of the substrate in relation to one litre of initial medium (i.e. the duration of the production phase), - Selectivity molDCA / molMCA = the ratio of moles of all DCA produced to moles of all MCA consumed. Table 1. Selectivity Strain DCA titre (g / lfin) DCA (g) Productivity (g / h / linit) (mol / mol ) Strain A 43.58 71.80 1.44 0.25 Strain B 101.34 135.41 2.71 0.95 The results shown in Table 1 show that use of the microorganism according to the invention (strain B) in a process for the production of dicarboxylic acids yields significantly higher results, both in terms of productivity and selectivity, than the use of strain A, which underwent random mutagenesis but had the CAT gene function active. In particular, productivity and the amount of total DCA produced are 46% higher and selectivity as high as 73%. EXAMPLE 4 The strain A of Example 1 was subjected to deletion of both alleles of the MFE gene using the protocol described below, resulting in the strain referred to as strain C. Strain A underwent genetic modifications through a process of double crossing over recombination using a suitably constructed deletion cassette to allow the deletion of the MFE2 gene, at first partly (1 deleted allele, the so-called mfe2 / MFE2 mutant) and then completely (2 deleted alleles, the so-called mfe2 / mfe2 mutant). The strategy used for the deletion of the alleles of the MFE2 gene was the same described in the example on pages 4-6 of WO 2019 / 030652 (mentioned also for the deletion of the alleles of the CAT gene in Example 1 of the Application). The nucleotide sequence of the gene coding for MFE2 gene in C. tropicalis was used to search for the homologous sequence in the genome of C. maltosa Xu316 deposited in the NCBI database by BLAST software. The nucleotide sequence of the MFE2 gene in C. maltosa Xu316 was in turn used to design the primers to amplify the MFE2 gene in strain A through PCR reactions. EXAMPLE 5 The strain B of Example 1 was subjected to deletion of one allele of MFE2 gene, using the strategy described in Example 4, resulting in the strain referred to as strain D. EXAMPLE 6 Strain C obtained in Example 4 was used in a fermentation process using the same protocol described in Example 2. Samples of the fermentation broth were extracted and analysed to quantify the DCAs produced. The results obtained are reported in Table 2. EXAMPLE 7 Strain D obtained in Example 5 was used in a fermentation process using the same protocol described in Example 2. Samples of the fermentation broth were extracted and analysed to quantify the DCAs produced. The results obtained are reported in Table 2. Table 2. Selectivity Strain DCA titre (g / lfin) DCA (g) Productivity (g / h / linit) (mol / mol) Strain C 86.22 116.38 2.33 0.88 Strain D 76.5 111.9 2.03 0.91 The results shown in Table 2 show that use of the microorganism according to the invention, having the function of the MFE gene inactive (strain C) or having the function of the CAT gene inactive and one allele of the MFE gene deleted (strain D) in a process for the production of dicarboxylic acids yields significantly higher results, in terms of quantity of DCA produced, productivity and selectivity, than the use of strain A, which underwent random mutagenesis but had the CAT and MFE gene function active. In particular, compared to strain A, both strains C and D produce a quantity of DCA that is 36% higher and with a selectivity that is 72% higher. Furthermore, the productivity of strain C is 38% higher than that of strain A, while that of strain D is 29% higher. EXAMPLE 8 Strain B obtained in Example 1 was used in a fermentation process using the same protocol described in Example 2. The performances obtained in a fermentation process wherein the culture medium comprised 2,51 g / l of phosphate have been compared to the performances obtained in a fermentation process wherein the medium contained 9.5 g / l of phosphate (the other components remain unchanged). Thanks to the use of a medium with a reduced content of phosphate (2,51 g / l), an increment of: - 6.7% of DCA titre (g / l)fin, - 6.5% of grams of DCA, - 5.3% of Productivity (g / h / l)init, has been obtained, keeping the selectivity almost unchanged. At the end of the fermentation process the quantity of phosphates remained in the medium was lower than 1 g / l. EXAMPLE 9 The same procedure described in Example 2 was applied to the strain B, with the difference that before the microorganism was inoculated into the fermenter or after 5, 6, 7 or 9 hours from inoculation, 0.6% by weight of oleic acid was added to the culture medium. When the OUR reached its maximum value, the growth phase was considered to have ended. The duration of the growth phase is reported in Table 3. Table 3. Time of addition of 0.6 w% of Duration of the Growth acceleration oleic acid growth phase (min) compared to Example 2 (min) Before inoculation 896 64 5 hours after inoculation 941 19 6 hours after inoculation 945 15 7 hours after inoculation 943 17 9 hours after inoculation 960 0 The results shown in Table 3 show that the addition of 0.6 w% of oleic acid before inoculation of the microorganism or during the first 5, 6 o 7 hours after inoculation, the duration of the cell growth phase is shortened of at least 15 minutes (i.e. the growth phase is accelerated) respect to not adding it (Example 2) or to adding it after 9 hours from inoculation of the microorganism.

Claims

CLAIMS 1. Candida maltosa having the function of the CAT gene (carnitine acetyltransferase) and / or of the MFE2 gene (multifunctional enzyme type 2) inactive, characterised by a dicarboxylic acid productivity of more than 1 g / h / linit at an optical density (OD625) of at least 90 and after incubation at 30°C for at least 6 hours in a culture medium fed with an oleic acid, in a quantity to maintain its concentration of at least 13 g / l, and 1.5 g / l / h glucose, wherein the productivity is the ratio of grams of DCA produced at the end of the process to the time elapsed since the addition of the substrate compared to one litre of initial medium (g / h / linit).

2. Candida maltosa according to claim 1, wherein the production of dicarboxylic acids is quantified by gas chromatography analysis using a gas chromatograph equipped with a ZB- 5MS plus column (30 m x 0.25 mm x 0.25 µm) and a flame ionisation detector (GC-FID), using N,O-bis-trimethylsilyl-trifluoroacetamide (BSTFA) as a derivatising agent.

3. Candida maltosa according to any of claims 1-2, having one or both alleles of the CAT gene and / or the product of its transcription non-functional.

4. Candida maltosa according to claim 3, having both alleles of the CAT gene non-functional.

5. Candida maltosa according to claim 4, having both alleles of the CAT gene deleted.

6. Candida maltosa according to any of claims 1-5 subjected to the operations of: a) random mutagenesis; b) selection of one or more strains producing dicarboxylic acids from monocarboxylic acids; c) inhibition of β-oxidation by inactivation of the function of (i) the CAT gene, or (ii) the MFE2 gene, or a combination thereof.

7. Process for producing dicarboxylic acids from a substrate selected from monocarboxylic acids, monocarboxylic acid esters and mixtures thereof comprising fermenting a Candida maltosa according to any of claims 1-6 in a culture medium.

8. Process according to claim 7, wherein said monocarboxylic acids are C12-C24 acids.

9. Process according to any of claims 7-8, wherein said monocarboxylic acids are in the form of mixtures of acids of different chain lengths and unsaturation content.

10. Process according to claim 9, wherein said mixtures comprise at least 70% by weight of unsaturated monocarboxylic acids.

11. Process according to claim 10, wherein said unsaturated monocarboxylic acids comprise more than 70% by weight of monounsaturated acids.

12. Process according to claim 11, wherein said monounsaturated monocarboxylic acids comprise more than 80% by weight of oleic acid.

13. Process according to any of claims 7-12, wherein the culture medium comprises an amount of phosphate of more than 1 g / l and less than 5 g / l.

14. Process according to any of claims 7-13, wherein fermenting comprises a phase of cell growth of Candida maltosa and a subsequent phase of production of dicarboxylic acids.

15. Process according to claim 14, comprising the addition to the culture medium of one or more compounds selected from fatty acids, methyl or ethyl esters of fatty acids, alkanes and vegetable oils before inoculation of the microorganism or during the first 8 hours after inoculation.

16. Process according to claim 15, wherein said compounds are selected from fatty acids and alkanes.

17. Process according to any of claims 15-16, wherein said compounds are added in quantities of 0.01 to 5% by weight respect to the initial volume of culture medium.