Candida maltosa with increased rate of growth

Abstract

The present invention relates to a Candida maltosa microorganism which has been genetically modified so as to increase its growth rate, and use of such a microorganism for the production of dicarboxylic acids from monocarboxylic acids, esters of monocarboxylic acids and mixtures thereof.

Description

[0001] CANDIDA MALTOSA WITH INCREASED RATE OF GROWTH

[0002] Cross-Reference to Related Applications

[0003] This Patent Application claims priority from Italian Patent Application No. 102023000028080 filed on

[0004] December 27, 2023, the entire disclosure of which is incorporated herein by reference.

[0005] Technical Field

[0006] The present invention relates to a microorganism Candida maltosa which has been genetically modified to increase its growth rate, and use of such a microorganism for the production of dicarboxylic acids from monocarboxylic acids, monocarboxylic acid esters and mixtures thereof.

[0007] Background of the Invention

[0008] Yeasts have many applications in the field of biotechnology, as a result for example of advantageous growth characteristics such as the possibility of using different substrates as carbon sources, the versatility of their metabolism and the ability to produce and secrete proteins outside the cell. In fact, many yeasts are already widely and variously used for the production of vaccines, therapeutic proteins, antibiotics or food additives. Furthermore, some can be genetically modified and used in biotechnological processes to produce bio-products with high added value, such as organic acids, fatty acids, dicarboxylic acids, bioethanol, etc.

[0009] Over the last 25 years, for example, research has focused on the production of dicarboxylic acids by means of biotechnological processes mediated by microorganisms, including yeasts belonging to the Candida genus, including C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. maltosa, C. parapsilosis and C. tropicalis, which are capable of oxidising alkanes and / or fatty acids to diacids (Shiio I. and Uchio R„ Agr. Biol. Chem., 1971, 35: 2033-2042).

[0010] Biotechnological processes for the production of dicarboxylic acids by means of yeasts (so-called fermentation processes) typically comprise two phases (referred to as biphasic fermentation): an initial phase of cell growth with an increase in biomass and a subsequent production phase in which the compound of interest is produced. During the initial phase of cell growth, the yeast is inoculated into a culture medium in the presence of nutrients, usually including a sugar as a carbon and energy sources, which allow it to replicate and increase its biomass. After an initial latency phase in which the yeast adapts to the environment (lag phase), the yeast begins to grow exponentially (exponential growth phase) and the sugars are rapidly degraded. When the sugars and / or nitrogen source in the growth medium are exhausted, the yeast growth reaches the end of the exponential growth phase. At this moment the next production phase begins. In this second phase, generally in the same medium used for cell growth, the substrate to be converted is added, and this may include alkanes, fatty acids and / or their derivatives or oils, together with the small quantities of sugar that are necessary to maintain the yeast's basic metabolism. Document WO 2019 / 030652, for example, describes a genetically modified microorganism of the Candida maltosa species that is able to produce long-chain dicarboxylic acids from monocarboxylic acids by means of biphasic fermentation.

[0011] The fermentation processes for the production of dicarboxylic acids described above therefore require an initial phase of cell growth that involves the use of time and resources without production of the compound of interest.

[0012] It is therefore important to reduce the duration of this growth phase, without compromising the performance of the entire fermentation process, in order to decrease the associated costs and increase the number of productive runs. One strategy can involve the use of nutrient-rich culture media, such as Yeast Peptone Dextrose (YPD) medium, in which maximum biomass production can be achieved even 12 hours after inoculation. However, nutrient-rich culture media such as YPD are not typically used in the production phase; in addition to being more expensive and therefore less economically viable, they contain many components that would make the product purification process more complex.

[0013] Alternatively, there is a need to find microorganisms characterised by rapid growth, also in the culture media used in the production phase.

[0014] It has now unexpectedly been found that the genetically modified microorganism Candida maltosa, placed in a minimal culture medium and in the presence of a carbon source, has a significantly faster cell growth phase than the unmodified microorganism.

[0015] Summary of the Invention

[0016] The Candida maltosa microorganism according to the invention has a gene for resistance to an antibiotic in its genome, in particular in close proximity to a gene coding for a ribosomal protein.

[0017] The duration of the cell growth phase may be assessed, for example, by monitoring the Oxygen Uptake Rate (OUR). The OUR, i.e. the amount of oxygen consumed, can for example be measured using the following formula:

[0018] OU R — ( Pair in X O2 in) - ( Pair out X 02 out) where Pair in is the flow rate of the incoming air (measured for example in mmol / h), O2 in is the percentage concentration of oxygen in the incoming air, Pair out is the flow rate of the outgoing air, O2 out is the percentage concentration of oxygen in the outgoing air.

[0019] 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.

[0020] 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.

[0021] During the cell growth phase, the microorganism according to the invention reaches the maximum OUR value in a shorter time than the unmodified microorganism grown under the same conditions. According to one aspect, the present invention thus relates to a Candida maltosa microorganism whose genome comprises a gene for resistance to an antibiotic positioned in close proximity to a gene coding for a ribosomal protein.

[0022] This microorganism has also been found to be particularly suitable for the production of dicarboxylic acids from monocarboxylic acids, esters of monocarboxylic acids and their mixtures.

[0023] A second aspect of the invention therefore relates to a process for producing dicarboxylic acids from a substrate chosen from monocarboxylic acids, esters of monocarboxylic acids and mixtures thereof comprising fermenting said Candida maltosa in a culture medium.

[0024] The invention can be better understood through the following detailed description, sequences and figures attached to this patent application.

[0025] Brief Description of the Sequences

[0026] The Applicant has provided a list of sequences in accordance with World Intellectual Property Organization (WIPO) Standard ST.26 and the PCT and EPO sequence list requirements and Rule 37 C.F.R. §1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and / or Amino Acid Sequence Disclosures - the Sequence Rules"). In particular:

[0027] - SEQ ID NO: 1 represents the nucleotide sequence of the gene for nourseothricin resistance without the promoter and terminator sequences;

[0028] - SEQ ID NO: 2 represents the exogenous nucleotide sequence comprising said gene for nourseothricin resistance, said promoter and terminator sequences and the gene coding for the ribosomal protein RPS1;

[0029] - SEQ ID NO: 3 represents the nucleotide sequence of the gene coding for the ribosomal protein RPS1.

[0030] Brief Description of the Figures

[0031] - FIG. 1: Example of the time course of the OUR parameter during the growth phase of a fermentation process. EFT= elapsed fermentation time. The values refer to Example 2;

[0032] - FIG. 2: Strategy used in Example 1 for the insertion by homologous recombination of an exogenous sequence comprising the gene for nourseothricin resistance in Candida maltosa.

[0033] CmRPSl= ribosomal gene of Candida maltosa, backbone: plasmid skeleton;

[0034] FIG. 3: Calculation of the distance between the start of the exogenous nucleotide sequence SEQ ID NO: 2 (comprising the gene for resistance to an antibiotic) respect to the end of the original gene RPS1 present in the genome of the microorganism Candida maltosa obtained in Example 1.

[0035] Description of Embodiments of the Invention The present invention will be described in more detail below.

[0036] The present invention relates to a Candida maltosa microorganism that has been genetically modified to increase its growth rate. The genome of such a microorganism comprises a gene for resistance to an antibiotic positioned in close proximity to a gene coding for a ribosomal protein.

[0037] In the present invention the term "gene for resistance to an antibiotic" means a gene for resistance to an antibiotic flanked by the sequences of a promoter and a terminator, which enable it to be expressed.

[0038] This antibiotic resistance gene (comprising promoter and terminator) may be inserted into the genome as such or it may be comprised within an exogenous nucleotide sequence that is incorporated into the genome of the microorganism.

[0039] Within the meaning of the present invention the expression "in close proximity" means that the gene for resistance to an antibiotic or the exogenous nucleotide sequence comprising it are positioned at a distance of no more than 50000 base pairs (bp), preferably no more than 45000 bp, more preferably not more than 40000 bp, 35000 bp, 30000 bp, 25000 bp, 20000 bp, 15000 bp, 10000 bp, even more preferably not more than 8600 bp, relative to the end of the original gene coding for the ribosomal protein RP present in the genome of the microorganism. This distance is calculated from the start of the inserted gene or sequence. In other words, the present invention relates to a Candida maltosa whose genome comprises a gene for resistance to an antibiotic positioned at a distance of no more than 50000 base pairs (bp) relative to the end of the original gene coding for a ribosomal protein.

[0040] According to a preferred embodiment, said gene is comprised within an exogenous nucleotide sequence and said exogenous nucleotide sequence is positioned at a distance of no more than 50000 base pairs (bp) relative to the end of the original gene coding for a ribosomal protein.

[0041] Said distance is calculated from the start of the gene for resistance to the antibiotic or from the exogenous nucleotide sequence comprising it to the end of the original gene coding for the ribosomal protein RP present in the genome of the microorganism.

[0042] For example, the antibiotic may be selected from the different classes of antibiotics known as betalactams, aminoglycosides, tetracyclines, macrolides, fluoroquinolones.

[0043] Examples of antibiotics are nourseothricin, hygromycin, kanamycin, neomycin, geneticin, penicillin, ampicillin or amoxicillin. Preferably, the antibiotic is nourseothricin.

[0044] Preferably, said gene (excluding promoter and terminator sequences) has a sequence with at least 40, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence shown in SEQ. ID NO: 1.

[0045] Preferably the gene for nourseothricin resistance (excluding promoter and terminator sequences) is selected from NAT, STAT, SAT1, SAT2, SAT3 and SAT4. SAT1 is preferred. Even more preferably, said gene (excluding promoter and terminator sequences) has the sequence given in SEQ. ID NO:1. The percentage sequence identity can be determined using any sequence comparison algorithm, e.g. NCBI's BLASTn algorithm, while keeping the search settings on the default parameter settings.

[0046] Said gene for resistance to an antibiotic (comprising promoter and terminator) may be inserted as such or may be comprised within an exogenous nucleotide sequence. Preferably said gene for resistance to an antibiotic is comprised within an exogenous nucleotide sequence. The presence of said gene for resistance to an antibiotic is necessary for the modified microorganism to grow faster than the unmodified one. Exogenous nucleotide sequences that do not include the gene for antibiotic resistance do not allow for growth faster than that of the unmodified microorganism.

[0047] In a preferred embodiment of the invention, in the genome of the microorganism Candida maltosa there is an exogenous nucleotide sequence containing a gene for resistance to an antibiotic, positioned in close proximity to a gene coding for a ribosomal protein.

[0048] Such an exogenous nucleotide sequence preferably has a length of at least 600 base pairs (bp), preferably at least 1100 bp, more preferably at least 1800 bp, even more preferably at least 7500 bp.

[0049] Such an exogenous nucleotide sequence has for example a maximum length of 15000 bp, preferably 12500 bp, even more preferably 8000 bp.

[0050] Preferably, this sequence corresponds to SEQ ID NO:2.

[0051] Faster growth than in the unmodified microorganism is obtained when the gene for resistance to an antibiotic or the exogenous nucleotide sequence comprising it are positioned in the genome of the Candida maltosa microorganism in close proximity of one or two alleles, preferably one, of a gene coding for a ribosomal protein RP. Said gene coding for a ribosomal protein preferably encodes for a protein belonging to the small ribosomal subunit 40S (ribosomal protein RPS) and / or has a sequence identity percentage of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the sequence reported in SEQ. ID NO: 3.

[0052] More preferably, the gene coding for a ribosomal protein is the gene coding for the RPS1 protein. Even more preferably, such a gene has the sequence shown in SEQ ID NO: 3.

[0053] The gene for resistance to an antibiotic or the exogenous nucleotide sequence comprising it are positioned in close proximity to said gene coding for a ribosomal RP protein.

[0054] The expression "in close proximity" means that the gene for resistance to an antibiotic or the exogenous nucleotide sequence comprising it are positioned at a distance of no more than 50000 base pairs (bp), preferably no more than 45000 bp, 40000 bp, 35000 bp, 30000 bp, 25000 bp, 20000 bp, 15000 bp, 10000 bp, even more preferably not more than 8600 bp, relative to the end of the original gene coding for the ribosomal protein RP present in the genome of the microorganism. This distance is calculated from the start of the inserted gene or sequence.

[0055] Preferably, said gene or sequence is positioned between the two ends of the original gene coding for the ribosomal protein RP present in the microorganism's genome, as shown in Fig. 2. The term "original gene" refers to the endogenous gene present in the genome of the microorganism. The gene for resistance to an antibiotic or the exogenous nucleotide sequence comprising it can be inserted by any technique known to those skilled in the art.

[0056] When a genetic technique based on homologous recombination, e.g. by single crossing over, is used, said gene or sequence is positioned between the two ends of the original gene coding for a ribosomal protein RP, as shown for example in Fig. 2. Homologous recombination occurs through the use of a construct or plasmid containing a gene coding for a protein RP with a sequence homologous to that of the original gene coding for the ribosomal protein RP present in the genome of the microorganism within which insertion is desired. Single crossing over also involves duplication of the gene coding for that ribosomal protein RP. Therefore, once inserted, the gene for resistance to an antibiotic or the exogenous nucleotide sequence comprising it is positioned between the two ends of the original gene coding for a ribosomal protein RP. However, duplication of the gene coding for ribosomal protein RP is not implicated in the growthenhancing effect.

[0057] If other genetic modification techniques are used, which for example do not involve duplication of the gene coding for the ribosomal protein RP, the gene for resistance to an antibiotic or the exogenous nucleotide sequence comprising it is placed upstream or downstream of the original gene coding for the ribosomal protein RP, preferably upstream.

[0058] Because a gene for resistance to an antibiotic is present in close proximity to a gene encoding for a ribosomal protein the modified microorganism has a significantly faster growth phase than the microorganism that does not contain it (unmodified microorganism), when placed under the same conditions (e.g. O.D. of the inoculum, composition of the medium, fermentation process parameters, etc.). In the cell growth phase, the microorganism can 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.

[0059] 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 comprise 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 pg / L, trace elements from 0.2 to 8 mg / L, weak organic acid from 1.0 to 2 g / L.

[0060] During the cell growth phase, the microorganism according to the invention reaches the maximum OUR value in at least 10 minutes less time, preferably at least 20 minutes less time, even more preferably at least 40 minutes less time than the unmodified microorganism grown under the same conditions.

[0061] The Candida maltosa microorganism according to the invention has in its genome the genes coding for enzymes involved in the co-oxidation of monocarboxylic to dicarboxylic acids and is advantageously capable of co-oxidising monocarboxylic to dicarboxylic acids. Such a microorganism may advantageously be subjected to genetic modification and / or selection techniques to obtaining an increase in the productivity of dicarboxylic acids, such as for example random mutagenesis and / or inactivation of the function of the CAT (Carnitine Acetyl-Transferase) gene. According to a preferred embodiment, the Candida maltosa according to the invention has the function of the CAT gene inactive.

[0062] The inactivation of CAT gene function is achieved by rendering non-functional one or both alleles of the CAT gene and / or the product of its transcription. Preferably one or both - even more preferably both - alleles of the CAT 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.

[0063] The Candida maltosa according to the invention is advantageously characterised by a dicarboxylic acid productivity of more than 1 g / h / hnit, 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.

[0064] The production of dicarboxylic acids can be quantified by gas chromatography (GC) or high-performance liquid chromatography (HPLC). A person skilled in the art will knows that the result remains the same regardless of the method of analysis used.

[0065] 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 pm) and a flame ionisation detector (GC-FID), using N,O-bis-trimethylsilyl trifluoroacetamide (BSTFA) as a derivativising agent.

[0066] In the present invention optical density is measured using a Perkin Elmer Lambda 35 spectrophotometer. Said Candida maltosa can be advantageously subjected to random mutagenesis. Random mutagenesis may be followed by one or more selection operations conducted to identify, from among the mutant strains obtained, one or more strains producing dicarboxylic acids from monocarboxylic acids with a productivity (i.e. ratio between the grams of DCA produced at the end of the process and the time elapsed since the addition of the substrate in relation to one litre of initial medium, g / h / nit) of more than 0.7 g / h / hnit, preferably more than 0.9 g / h / hnit, more preferably more than 1.3 g / h / hnit, even more preferably more than 1.4 g / h / hnit-

[0067] Such selection operations may, for example, be conducted by inoculating the strains in a culture medium containing a sufficient amount of a monocarboxylic acid and verifying that dicarboxylic acids are present in the fermentation broth after incubation by appropriate analytical techniques; for example, selection operations may be conducted 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 with a flame ionisation detector (GC-FID).

[0068] A further aspect relates to a method for obtaining the Candida maltosa microorganism according to the invention which grows more rapidly than a microorganism not subjected to the method according to the invention. Said method comprises a step of inserting a gene for resistance to an antibiotic or an exogenous nucleotide sequence comprising the same in close proximity to a gene encoding for a ribosomal protein in the genome of Candida maltosa.

[0069] According to the method according to the invention, said antibiotic is to be chosen from, for example, nourseothricin, hygromycin, kanamycin, neomycin, geneticin, penicillin, ampicillin and amoxicillin.

[0070] The gene for resistance to an antibiotic is preferably the gene for resistance to nourseothricin, flanked by the promoter and terminator sequences.

[0071] Preferably said gene has a sequence (excluding promoter and terminator sequences) with a percentage identity of at least 40, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% with the sequence reported in SEQ ID NO: 1. More preferably the gene for nourseothricin resistance (excluding promoter and terminator sequences) is selected from NAT, STAT, SAT1, SAT2, SAT3 and SAT4. SAT1 is preferred. Even more preferably, said gene (excluding promoter and terminator sequences) has the sequence given in SEQ. ID NO:1.

[0072] According to the method according to the invention the nucleotide sequence comprising the gene for resistance to an antibiotic has a minimum length of at least 600 base pairs (bp), preferably at least 1100 bp, more preferably at least 1800 bp, even more preferably at least 7500 bp.

[0073] Such an exogenous nucleotide sequence preferably has a maximum length of 15000 bp, preferably 12500 bp, even more preferably 8000 bp.

[0074] Preferably, this sequence corresponds to SEQ. ID NO:2.

[0075] According to the method according to the invention the gene for resistance to an antibiotic or the nucleotide sequence comprising it is positioned in close proximity of one or two alleles, preferably one, of any gene coding for a ribosomal protein RP in the genome of the microorganism Candida maltosa. Said gene coding for a ribosomal protein preferably encodes for a protein belonging to the 40S small ribosomal subunit (RPS ribosomal protein) and / or has a sequence identity percentage of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the sequence reported in SEQ ID NO: 3.

[0076] More preferably the gene coding for a ribosomal protein is the gene coding for the RPS1 protein. Even more preferably, such a gene has the sequence shown in SEQ ID NO: 3.

[0077] The gene for resistance to an antibiotic or the nucleotide sequence comprising it in close proximity to a gene coding for a ribosomal protein may be inserted by any genetic technique known to those skilled in the art. It may, for example, be carried out by homologous recombination by single crossing over, using a construct or plasmid comprising the nucleotide sequence containing the gene for antibiotic resistance and the sequence of the gene coding for the ribosomal protein within which said nucleotide sequence is to be inserted. In this way, once inserted, the gene for resistance to an antibiotic or the exogenous nucleotide sequence comprising it is positioned between the two ends of the original gene coding for a ribosomal protein RP (as shown for example in Fig. 2).

[0078] The Candida maltosa microorganism according to the invention may optionally be subjected to one or more of the following operations: a) random mutagenesis; b) selection of one or more strains producing dicarboxylic acids from monocarboxylic acids; c) inactivation of the CAT gene function.

[0079] Operations a), b) and c) may be performed in any order.

[0080] Preferably, the random mutagenesis operations a) precede CAT gene function inactivation operations c). Operations b) for the selection of one or more strains producing dicarboxylic acids from monocarboxylic acids may precede or follow random mutagenesis a). Preferably, selection operations b) follow random mutagenesis a).

[0081] The operations b) of selecting one or more strains producing dicarboxylic acids from monocarboxylic acids may precede or follow inactivation of the function of the CAT gene c). Preferably, the operations of selection b) precede inactivation of the function of the CAT gene c).

[0082] Selection operations b) are carried out to identify 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, from among the strains of the microorganism Candida maltosa and / or from among the mutants obtained at point a) and / or from among the strains with inactive CAT gene function.

[0083] According to the present invention, "productivity" is defined as the ratio of the 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 / it).

[0084] According to the present invention, "amount of DCA produced" means the amount (grams) of dicarboxylic acids produced in a fermentation process conducted from 1 litre of initial broth (g).

[0085] According to the present invention, "selectivity" means the ratio of moles of DCA produced to moles of substrate consumed (mol / mol).

[0086] According to one aspect, the operations b) of selecting one or more strains that produce dicarboxylic acids from monocarboxylic acids precede random mutagenesis a), and this random mutagenesis precedes inactivation of the CAT gene function.

[0087] According to a preferred aspect, the operations b) of selecting one or more strains producing dicarboxylic acids from monocarboxylic acids follow random mutagenesis a) and such random mutagenesis precedes inactivation of the function of the CAT gene. Advantageously, through selection operations b) one or more strains are identified from among mutants obtained by the random mutagenesis operation a) which produce dicarboxylic acids from monocarboxylic acids with productivity of more than 0.7 g / h / it, preferably more than 0.9 g / h / it, more preferably more than 1.3 g / h / it, even more preferably more than 1.4 g / h / linit-

[0088] Alternatively, or in combination, by means of selection operations b) one or more strains producing dicarboxylic acids from monocarboxylic acids in quantities greater than 15 g, preferably greater than 30 g, more preferably greater than 60 g, more preferably greater than 70 g, will be identified from among mutants obtained by the random mutagenesis operation a).

[0089] Alternatively, or in combination, by means of selection operations b) one or more strains will be identified from among mutants obtained by the random mutagenesis operation a) that produce dicarboxylic acids from monocarboxylic acids with a selectivity of more than 0.15 mol / mol, preferably more than 0.2 mol / mol.

[0090] 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 selected from: X-rays, ultraviolet (UV) radiation, treatment with chemical mutagens (such as, for example, nitrosoguanidine (NTG), 4-nitroquinolone-l- 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.

[0091] Operations b) to select one or more strains producing dicarboxylic acids from monocarboxylic acids may for example be conducted by inoculating the strains in a culture medium comprising a sufficient amount of a monocarboxylic acid and verifying the presence of dicarboxylic acids in the fermentation broth after incubation by appropriate analytical techniques; for example, selection operations may be conducted by inoculating the strains at an optical density (OD625) of 1.5 in culture medium containing 10-20% by weight of oleic acid as the sole carbon source and incubating them at 30°C for 24 hours. The production of dicarboxylic acids may, for example, be verified by GC-FID analysis.

[0092] Operations to inactivate the function of the CAT gene may be conducted 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 the operations for inactivating the function of the CAT gene are conducted by any known technique that renders both alleles of the CAT gene non-functional.

[0093] The genetic technique of inactivation by deletion is particularly preferred. Within the meaning of the present invention, a 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. The CAT gene can for example be deleted as described in WO 2019 / 030652 in the example on pages 4 to 6, which is therefore intended to form part of this description.

[0094] The Applicant has therefore identified a method for accelerating growth of the microorganism Candida maltosa optionally subjected to random mutagenesis a), selection b) and the deletion of both alleles of the CAT gene c) mentioned above, comprising the steps of i) inserting an exogenous nucleotide sequence comprising a gene for resistance to an antibiotic into the genome of said microorganism in or in close proximity to a gene coding for a ribosomal protein, ii) growing the microorganism obtained in step a) in a culture medium in the presence of a carbon and nitrogen source.

[0095] Growth of the microorganism (phase ii) is preferably conducted by inoculating the Candida maltosa microorganism according to the invention in a culture medium at an optical density (OD625) of 1.5 to 4.

[0096] Growth is typically conducted at a temperature of 25 to 35°C, preferably 28 to 32°C.

[0097] The pH of the culture medium is preferably maintained at values of 5 to 8, more preferably 6 to 7. Dissolved oxygen is preferably maintained at values from 5% to 40%, more preferably from 10% to 30%, through an appropriate combination of factors, including agitation, aeration.

[0098] The culture medium in which the Candida maltosa microorganism according to the invention is placed typically comprises at least one carbon source, at least one nitrogen source (inorganic and / or organic) and at least one inorganic salt.

[0099] Of these, the carbon source preferably includes one or more sugars such as glucose and / or sucrose. Glucose is preferred. The carbon source is preferably added in an amount of 40 to 90 g / L, more preferably 60 to 80 g / L.

[0100] The nitrogen source preferably includes one or more sources of organic nitrogen and / or one or more sources of inorganic nitrogen.

[0101] Examples of sources of organic nitrogen are urea, amino acids, oligopeptides or proteins. Urea is preferred. Examples of inorganic nitrogen sources are ammonia and ammonium salts such as ammonium sulfate or ammonium nitrates / nitrites. Ammonium sulfate is preferred.

[0102] The amount of total nitrogen is preferably added in amounts of 2 to 6, more preferably 3 to 4 g / L. The inorganic salt preferably but not limitedly comprises one or more of potassium monobasic phosphate, potassium dibasic phosphate, potassium chloride, magnesium sulfate, calcium chloride, ferric chloride, copper sulfate, sodium molybdate, nickel sulfate, cobalt sulfate, manganese sulfate, zinc sulfate. The inorganic salt is added in varying quantities depending on the specific fermentation conditions and the metabolic needs of the microorganism, as known to those skilled in the art. In a preferred embodiment of the present invention, the culture medium comprises glucose 60 to 80 g / L, phosphate salts 2 to 10 g / L, inorganic nitrogen 1.4 to 1.6 g / L, organic nitrogen 1.5 to 1.8 g / L, magnesium salt 0.5 to 0.7 g / L, vitamins 0.5 to 2 pg / L, trace elements 0.2 to 8 mg / L, weak organic acid 1.0 to 2 g / L.

[0103] The duration of the cell growth phase may, for example, be assessed by monitoring the OUR, as described above. Surprisingly it has been found that, by applying the method for accelerating growth described above, in the cell growth phase the OUR reaches its maximum value in at least 10 minutes less time, preferably at least 20 minutes less time, even more preferably at least 40 minutes less time, than in the growth phase conducted without applying this method.

[0104] Insertion step i) may be preceded or followed by further genetic modifications, such as random mutagenesis and / or inactivation of the CAT gene function as described above.

[0105] Said insertion step i) may therefore precede or follow the operations of random mutagenesis a), selection b) and inactivation of the CAT gene function c). Preferably, said insertion step i) follows the operations of random mutagenesis a), selection b) and inactivation of the CAT gene function c).

[0106] 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 according to the invention in a culture medium.

[0107] In the process according to the invention, fermenting preferably comprises a phase of Candida maltosa cell growth and a subsequent phase of dicarboxylic acid production.

[0108] These monocarboxylic acids are preferably C12-C24 acids, more preferably C16-C22.

[0109] These 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 and arachidic acid.

[0110] Preferably, these monocarboxylic acids are unsaturated, more preferably monounsaturated. Oleic acid is preferred.

[0111] 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 70%, more preferably more than 80% by weight of monounsaturated monocarboxylic acids. Preferably said monounsaturated monocarboxylic acids preferably comprise more than 80%, more preferably more than 90% by weight oleic acid.

[0112] One example of a particularly preferred mixture comprises at least 70% oleic acid (preferably 80-90% by weight), up to 20% linoleic acid (preferably 4-12% by weight), up to 5% stearic acid (preferably 1.5-4% by weight) and up to 6% palmitic acid (preferably 2-5% by weight). Such mixtures are obtained, for example, by the hydrolysis of vegetable oils or a mixture of vegetable oils or waste oils. Vegetable oils are either the unmodified product of pressing or oils that have 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.

[0113] 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.

[0114] 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 Cl- C9 alkyl groups; more preferred are methyl, ethyl, propyl and butyl alcohols. An example of a preferred polyalcohol is glycerol.

[0115] Methyl and ethyl esters of unsaturated carboxylic acids may be obtained by transesterification of methanol and ethanol with the triglycerides present in vegetable oils.

[0116] The production of dicarboxylic acids with the Candida maltosa microorganism according to the invention is preferably carried out by means of biphasic fermentation, in which the previously described cell growth phase (phase (ii)) is followed by a dicarboxylic acid production phase. Advantageously, the growth and production phases are conducted in the same culture medium.

[0117] The production step is preferably a fed-batch process aimed at keeping the metabolism of the cell biomass active and catalytically performing in producing dicarboxylic acids. Advantageously, this phase has a dual feed: a sugar, aimed at keeping the cells active, and a substrate chosen from monocarboxylic acids, monocarboxylic acid esters and their mixtures for biotransformation. The sugar may be at least one of glucose, sucrose, fructose. Preferably, such sugar is glucose. The 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.

[0118] 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 culture medium is preferably maintained at 5 to 8, more preferably 6 to 7. Dissolved oxygen is preferably maintained at 5 to 50%, more preferably 10 to 40%, by adjusting agitation and aeration.

[0119] 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.

[0120] The applicant has also surprisingly found that, in a process for the production of dicarboxylic acids by means of biphasic fermentation with a Candida maltosa microorganism, preferably subjected to the random mutagenesis a), selection b) and inactivation of the CAT gene function c) mentioned above, reducing the amount of phosphate supplied during the preparation of the culture medium can result in improved production performance. Reducing the phosphate content favours better emulsification of the substrate added at the start of the fermentation phase. Moreover, it has the additional 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 is reduced at the end of the fermentation process.

[0121] The process for the production of dicarboxylic acids with a Candida maltosa microorganism according to the invention, preferably subjected to the above-mentioned random mutagenesis operations a), selection b) and inactivation of the CAT gene function c), is preferably conducted in a culture medium comprising an amount of phosphate greater than 1 g / l, preferably greater than or equal to 2 and less than 5 g / l, more preferably less than 4.5, even more preferably less than 4, even more preferably less than 3 g / l. With an amount of phosphate of less than 5 g / l in the growth 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 an amount of phosphate of more than 5 g / l.

[0122] Fermentation performance means productivity and / or grams of dicarboxylic acids produced and / or selectivity.

[0123] In said process for the production of dicarboxylic acids, the amount of phosphate of less than 5 g / L may advantageously be achieved by supplying 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, into the culture medium, 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, preferably less than 1 g / L. According to a preferred embodiment, the process for the production of dicarboxylic acids with said Candida maltosa microorganism is conducted in a culture medium comprising an amount of dibasic potassium phosphate of 3.5 to 5 g / L and in the absence of monobasic phosphate.

[0124] The dicarboxylic acid production process according to the present invention is therefore advantageously carried out in a culture medium containing less than 5g / l phosphate. The Applicant has also surprisingly found that, in a process for the production of dicarboxylic acids by biphasic fermentation with a Candida maltosa microorganism, preferably subjected to the operations of random mutagenesis a), selection b) and inactivation of the CAT gene function c) mentioned above, by adding 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 cellular growth phase of said microorganism, the duration of the cellular growth phase is reduced in comparison with when said compounds were not added.

[0125] During the cell growth phase, this microorganism reaches the maximum OUR value in at least 20 minutes less time, preferably at least 30 minutes less time, even more preferably at least 40 minutes less time, than if no such compounds were added.

[0126] Even more surprisingly, if the process for the production of dicarboxylic acids is carried out with the Candida maltosa microorganism according to the invention, preferably subjected to the random mutagenesis a), selection b) and inactivation of the CAT gene function c) mentioned above, 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 growth phase of said microorganism results in a further reduction in the duration of the cell growth phase. During the cell growth phase said microorganism reaches the maximum OUR value in a time at least 30 minutes less, preferably at least 40 minutes less, even more preferably at least 60 minutes less than if said compounds were not added.

[0127] Preferably, said compounds are selected from fatty acids, methyl or ethyl esters of fatty acids, fatty acid salts 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.

[0128] Said compounds are advantageously added in amounts of 0.01 to 5% by weight, preferably 0.05 to 5% by weight, with respect to the initial volume of the culture medium. Said compounds are preferably added to the culture medium prior to inoculation of the microorganism.

[0129] According to one embodiment, the process for the production of dicarboxylic acids by the biphasic fermentation according to the invention, in which an initial cell growth phase of the microorganism is followed by a subsequent production phase, thus comprises the addition of one or more compounds selected from fatty acids, methyl and ethyl esters of fatty acids, fatty acid salts, alkanes and vegetable oils at the beginning of the cell growth phase, preferably prior to inoculation of the microorganism.

[0130] 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 the slowing down of foam formation and reduction in the amount of foam that typically forms during the production phase.

[0131] 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, in many cases the foam will reform within minutes of addition despite the addition of antifoaming agent. On the other hand, in the process according to the invention, foam formation when the substrate to be converted is added to the culture medium is slowed down by the addition of 0.05 to 5% by weight, relative to the initial volume of the culture medium, of one or more compounds selected from fatty acids, fatty acid salts, methyl or ethyl esters of fatty acids, alkanes and vegetable oils.

[0132] Advantageously, foam formation is also slowed down after addition of the defoaming agent during the production phase. This defoaming agent may be chosen from silicones and non-silicones, preferably the defoaming agent is a silicone.

[0133] 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 the addition of the defoaming agent.

[0134] 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.

[0135] The applicant has therefore disclosed a method for slowing down and decreasing foam formation in a process for the production of dicarboxylic acids by a Candida maltosa microorganism, preferably subjected to the above-mentioned random mutagenesis a), selection b) and inactivation of the CAT gene function c), 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, said compounds are selected from fatty acids and vegetable oils. According to a preferred aspect, said Candida maltosa microorganism has a genome comprising a gene for resistance to an antibiotic positioned in close proximity to a gene coding for a ribosomal protein. 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.

[0136] These compounds are preferably added to the culture medium before inoculation of the microorganism. The following examples illustrate the present invention for non-limiting purposes. EXAMPLES

[0137] EXAMPLE 1: Insertion of the sequence SEQ ID NO:2 containing the nourseothricin resistance gene SEQ ID NO:1 into Candida maltosa in close proximity to the RPS1 gene (SEQ. ID NO:3).

[0138] A Candida maltosa microorganism subjected to random mutagenesis and capable of producing dicarboxylic acids was deleted of the CAT gene using the protocol described in the example on pages 4-6 of WO 2019 / 030652, obtaining strain A.

[0139] Strain A was modified by inserting into its genome the 7891 bp sequence reported in SEQ ID NO:2 comprising the gene for resistance to nourseothricin SEQ IN NO:1. Through homologous recombination by double crossing over, the start of the sequence (SEQ ID NO:2) was inserted at a distance of 8514 bp from the end of the original gene encoding for the ribosomal protein RPS1 present in the genome of the microorganism, having the sequence given in SEQ ID NO:3. This insertion resulted in duplication of the RPS1 gene.

[0140] Homologous recombination was achieved using a plasmid constructed from the pSFS2 plasmid developed for gene deletion in the species Candida albicans (O. ReuR et al., Gene 341 (2004) 119-127).

[0141] The final plasmid used to modify strain A comprised: the gene for nourseothricin resistance (with promoter and terminator), the gene for a recombinase under the control of an inducible promoter, the RPS1 gene, a suitable sequence for correct replication of the entire plasmid in E.coli.

[0142] The linearised plasmid was used for the transformation of strain A: the transformation was successful, and the transformed clones were selected on medium containing the antibiotic and were verified by PCR.

[0143] EXAMPLE 2: Growth with the microorganism from Example 1

[0144] The Candida maltosa microorganism obtained in Example 1 was streaked onto YPD plate (glucose 20 g / L, yeast extract 10 g / L, tryptone 20 g / L) and incubated at 30°C. After 24 hours, a small quantity of colonies was inoculated into liquid YPD medium and incubated at 30°C while shaking at 250 rpm. After approximately another 24 hours, a volume of culture was taken and used to inoculate, at an QD600 of 2.5, a fermenter containing 1 litre of culture medium at pH 6.4 and of composition: phosphate buffer, inorganic nitrogen source (5.63g / L), organic nitrogen source (3.5 g / L), vitamins (0.5 mg / L) 2ml / L, magnesium salts (0.6 g / L), 1.0 ml trace elements (T.E. Titolchimica 1000X), glucose (70g / L), weak organic acid 1.5 g / L.

[0145] The microorganism was grown by setting the temperature at 30°C, aeration at 1 vvm and agitation in cascade with respect to pO2, the latter set at 30%. When the OUR reached its maximum value, the growth phase was considered finished. The duration of the growth phase was found to be approximately 900 minutes. EXAMPLE 3: COMPARATIVE

[0146] The procedure in Example 2 was applied to strain A, in which the nucleotide sequence SEQ ID NO:2 comprising the gene for resistance to nourseothricin SEQ. ID NO:1 was not inserted.

[0147] When the OUR reached its maximum value, the growth phase was considered to have ended. The duration of the growth phase was found to be about 940 minutes, about 40 minutes longer than in Example 2.

[0148] EXAMPLE 4: COMPARATIVE

[0149] The same procedure as described in Example 2 was applied to strain A modified by inserting by homologous recombination by single crossing over a sequence of 7968 base pairs, which did not include the sequence of the nourseothricin gene (SEQ ID NO:1).

[0150] When the OUR reached its maximum value, the growth phase was considered to have ended. The duration of the growth phase was found to be about 960 minutes, about 60 minutes longer than in Example 2.

[0151] EXAMPLE 5

[0152] The same procedure described in Example 2 was applied to the Candida maltosa microorganism of Example 1. Before the microorganism was inoculated into the fermenter, 2 g oleic acid (0.2% by weight) 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 was 836 minutes, 64 minutes less than in Example 2 (conducted with the same microorganism but without the addition of 0.2% oleic acid by weight).

[0153] EXAMPLE 6 COMPARATIVE: Insertion of a sequence containing the nourseothricin resistance gene SEQ ID NO:1 into Candida maltosa genome NOT in close proximity to a RPS gene.

[0154] Strain A of Example 1 was modified by inserting into its genome a sequence of 4186 bp comprising the gene for resistance to nourseothricin SEQ IN NO:1. The inserted sequence had 100% of identity respect to the portion of the sequence reported in SEQ ID NO:2 comprised between nucleotides in position 786 and 4972.

[0155] Therefore, the sequence SEQ ID NO:2 overlapped with for 53% of its length with the inserted sequence and in the overlapping interval the two sequences had 100% identity.

[0156] Through homologous recombination by double crossing over, the start of the inserted sequence was positioned at a distance of more than 50000 base pairs from the end of the original gene encoding for the ribosomal protein RPS1 present in the genome of the microorganism.

[0157] EXAMPLE 7 COMPARATIVE: Growth with the microorganism from Example 6 comparative The procedure in Example 2 was applied to the strain obtained in Example 6 comparative. When the OUR reached its maximum value, the growth phase was considered to have ended. The duration of the growth phase was found to be about 944 minutes, about 44 minutes longer than in Example 2. Therefore, the insertion of a sequence comprising a gene for resistance to an antibiotic at a distance of more than 50000 base pairs from the end of the original gene encoding for the ribosomal protein RPS1 does not allow to obtain an acceleration of the growth phase of the microorganism.

[0158] EXAMPLE 8:

[0159] Strain A was modified by inserting into its genome a sequence of 10113 bp sequence comprising the gene for resistance to nourseothricin SEQ IN NO:1. Said sequence contained the whole sequence SEQ ID NO:2 and a further fragment of 2222 bp. Therefore, the inserted sequence overlapped with the sequence SEQ. ID NO:2 for 77% of its length and in the overlapping interval had 100% identity with SEQ ID NO:2.

[0160] Through homologous recombination by double crossing over, the start of the sequence was inserted at a distance of 10736 bp from the end of the original gene encoding for the ribosomal protein RPS1 present in the genome of the microorganism.

[0161] EXAMPLE 9: fermentation process with the microorganism of Example 8

[0162] The microorganism obtained in Example 8 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.

[0163] The process was conducted by biphasic fermentation, with decoupling of the biomass cell growth phase from the DCA production phase.

[0164] When the OUR reached its maximum value, the growth phase was considered to have ended. The duration of the growth phase was found to be about 900 minutes.

[0165] When the OUR reached its maximum value and the biomass reached a steady state of growth, 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 5.9%, stearic acid 2.6%, palmitic acid 0.4%).

[0166] 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

[0167] 6.4 by the addition of KOH. Dissolved oxygen was kept at 30%.

[0168] Samples of the fermentation broths were extracted and analysed to quantify the DCAs produced.

[0169] The results obtained are shown in Table 1 below. Table 1.

Claims

CLAIMS1. Candida maltosa whose genome comprises a gene for resistance to an antibiotic positioned at a distance of no more than 50000 base pairs (bp) relative to the end of the original gene coding for a ribosomal protein.

2. Candida maltosa according to claim 1, wherein said antibiotic is nourseothricin.

3. Candida maltosa according to claim 2, wherein the gene for nourseothricin resistance has at least 90% sequence identity with the sequence reported in SEQ. ID NO: 1.

4. Candida maltosa according to claim 3, wherein said gene for nourseothricin resistance is SAT1.

5. Candida maltosa according to any of claims 1-4, wherein said gene for resistance to an antibiotic is comprised within an exogenous nucleotide sequence and said exogenous nucleotide sequence is positioned at a distance of no more than 50000 base pairs (bp) relative to the end of the original gene coding for a ribosomal protein.

6. Candida maltosa according to claim 5, wherein said exogenous nucleotide sequence has a length of at least 600 base pairs.

7. Candida maltosa according to any of claims 1-6, wherein said gene coding for a ribosomal protein encodes for a protein belonging to the small ribosomal subunit 40S.

8. Candida maltosa according to any of claims 1-7, having the function of the CAT gene inactive.

9. Candida maltosa according to claim 8, having both alleles of the CAT gene deleted.

10. Candida maltosa according to any of claims 1-9, characterised by a dicarboxylic acid productivity of more than 1 g / h / hnit, 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.

11. 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 1-10 in a culture medium.

12. Process according to claim 11, wherein said monocarboxylic acids are C12-C24 acids.

13. Process according to any of claims 11-12, wherein said monocarboxylic acids are in the form of mixtures of acids of different chain lengths and unsaturation.

14. Process according to claim 13, wherein said mixtures comprise at least 70% by weight of unsaturated monocarboxylic acids.

15. Process according to claim 14, wherein said unsaturated monocarboxylic acids comprise more than 70% by weight of monounsaturated acids.

16. Process according to claim 15, wherein said monounsaturated monocarboxylic acids comprise more than 80% by weight of oleic acid.

17. Process according to any of claims 11-16, wherein the medium contains less than 5 g / l phosphate.

18. Process according to any of claims 11-17 wherein fermenting comprises a phase of Candida maltosa cell growth and a subsequent phase of dicarboxylic acids production.

19. Process according to claim 18, 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 the cellular growth phase.

20. Process according to claim 19, wherein said compounds are selected from fatty acids and alkanes.

21. Process according to any of claims 19 to 20, wherein said compounds are added in an amount of 0.01 to 5% by weight, with respect to the initial volume of culture medium.

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