Patent Description:
Medium-long chain dicarboxylic acids (in the meaning of this invention, from <NUM> carbon atoms upwards) have a vast range of applications: from use as monomers for the synthesis of polymers to use in various industries such as cosmetics, pharmaceuticals, and plant protection products. For example, sebacic acid (C10) is used for the synthesis of polyesters, which have applications in fibres, films, resins, plasticisers, synthetic lubricants and adhesives. Producing them by a chemical route is however technically difficult, not environmentally very sustainable and economically expensive.

In the last <NUM> years research has therefore turned its attention to the production of dicarboxylic acids through biotechnological processes carried out by microorganisms capable of using alkanes, oils or fatty acids as substrates, in order to obtain sustainable low-cost processes.

In particular, fatty acids are metabolised via the β-oxidation pathway so that inhibition or deletion of the genes which take part in this metabolic pathway will result in reduced degradation of fatty acids, whereas in microorganisms provided with metabolic pathways for the functionalisation of fatty acids (e.g. ω-oxidation) it will result in increased conversion of such compounds. For example, <CIT> describes complete blocking of the β-oxidation pathway in Candida tropicalis achieved through deletion of the POX genes, which makes it possible to obtain dicarboxylic acids by a fermentation route.

Alternative strategies to block the β-oxidation pathway, through which the β-oxidation metabolic pathway is not blocked directly but its functionality is inhibited, are also described in the literature via deletion of the CAT gene in Candida tropicalis. This restricts the transport of acetyl-CoA (a derivative from the degradation of fatty acids via β-oxidation) between peroxisomes and mitochondria, consequently inhibiting β-oxidation functionality. For example, <NPL>) describes a homozygote strain of Candida tropicalis with inactivation of the two alleles of the CAT gene by gene disruption. However, the homozygote Δcat strain shows no production of dicarboxylic acids and produces less cellular biomass than the original strain in the presence of glucose as the sole carbon source.

Furthermore,<NPL>) describes strains of Candida tropicalis with deletion of the two alleles of the CAT gene which were previously subjected to random mutagenesis leading to a favourable genetic background and amplifying the effect of the deletions of both the alleles. In this case, the mutated strain from which the mutant originated with deletion of the two alleles is in fact already capable of producing quantities of dodecanedioic (C<NUM>) acid from dodecane.

<CIT> discloses a process for producing dicarboxylic acids using genetically engineered Candida maltosa. The Candida maltosa has gene disruptions in the <NUM>-oxidation pathway by knocking out the POX4 genes encoding for isoenzymes of the acyl CoA oxidase. <NPL>, and <NPL> disclose genetically engineered strains of C. tropicalis, in which the CAT gene encoding for carnitine-acetyl transferase has been deleted.

<NPL>, refers to a method of gene deletion in C. albicans using a deletion cassette.

<NPL> relates to C. tropicalis strains subjected to random mutagenesis and subsequent deletion of one or both alleles of the CAT gene.

This invention instead relates to a process for the production of dicarboxylic acids from monocarboxylic acids through a wild type microorganism Candida maltosa provided with the ω-oxidation metabolic pathway and having the peroxisome and mitochondrial carnitine acetyl-transferase coded by a single gene, characterized by inactivation of the function of the CAT (Carnitine Acetyl-Transferase) gene, in which that inactivation is done by making non-functional both the alleles of the CAT (Carnitine Acetyl-Transferase) gene, and/or the product of its transcription, without the microorganism being subjected to random mutagenesis techniques that are expensive and of insecure outcome.

For the purpose of the invention, the term "Candida maltosa" is used according to its usual meaning as it distinguishes from Candida tropicalis in that they are two distinct species in the field of yeast taxonomy and show significant differences at the molecular and biochemical level. See, for instance, <NPL>), <NPL>), and<NPL>).

In particular, this invention relates to the development of a strain of wild type Candida maltosa through deletion of both the alleles of the CAT (Carnitine Acetyl-Transferase) gene, the said deletion making it possible to restrict intracellular transport of acetyl-coenzyme A deriving from the dissipative metabolism of fatty acids, thus therefore inhibiting the dissipative pathway itself and encouraging the ω-oxidation metabolic pathway, with consequent conversion to dicarboxylic fatty acids. The CAT gene has been inactivated by double crossing over recombination with a deletion cassette.

This invention is also particularly advantageous because of the fact that Candida maltosa is included in biological risk class <NUM>, while other known microorganisms used for the production of dicarboxylic acids and belonging to the Candida genus are included in biological risk class <NUM> (e.g. Candida tropicalis).

The wild type microorganism Candida maltosa used in the process of the invention is advantageously a (CAT/CAT) strain, in particular a CGMCC <NUM> strain.

Carnitine acetyltransferase is an enzyme belonging to the class of transferases and catalyses the following reaction: acetyl-CoA + carnitine ⇄ CoA + acetylcarnitine.

Acetyl-coenzyme A (acetyl-CoA), which is subsequently used in the glyoxylate cycle and in the tricarboxylic acid cycle, is produced during metabolism of the fatty acids. Within eukaryotic cells some of these processes take place in well-defined compartments or organelles, such as in peroxisomes (e.g. the glyoxylate cycle), mitochondria (e.g. the tricarboxylic acids cycle) or in the cytosol, and it is therefore necessary to ensure intracellular transport of acetyl-CoA.

Movement of acetyl-CoA is facilitated by enzymes of the carnitine acetyltransferase (CAT) family which catalyse transport of the acetyl group of the acetyl-CoA to carnitine with the consequent production of acetylcarnitine (and vice versa) that is subsequently transported across the membrane. It is thought that the peroxisome CAT enzyme is involved in exporting acetyl-CoA produced during β-oxidation and that the main function of mitochondrial CAT is associated with the release of acetyl-CoA from acetyl carnitine in the mitochondrion. Peroxisome carnitine acetyl-transferase and the mitochondrial type are generally coded by a single gene. Although some microorganisms are strictly dependent on CAT for the movement of acetyl-CoA, others (such as S. cerevisiae) showed two parallel metabolic pathways dependent on CAT or peroxisome citrate synthase (Cit2).

In the meaning of this invention, by inactivation of the CAT gene is meant any technique which makes non-functional both the alleles of the CAT gene or the product of its transcription or the product of its translation. The genetic technique of inactivation by deletion is particularly preferred. Even more preferred is the genetic technique of deletion performed by a process of double crossing over recombination, in particular using a deletion cassette. For the purposes of this invention, a plasmid containing a gene for resistance to an antibiotic under the control of a constitutive promoter and a gene for recombinase under the control of an inducible promoter is particularly preferred for allowing the deletion of both the alleles of the CAT gene.

A wild type strain (CAT/CAT) of Candida maltosa CGMCC2. <NUM> (BNCC340010=BNCC140359; hereinafter indicated as strain '<NUM>) from the BNBIO microorganisms collection as characterised in the table was used to create the mutant with the deletion of <NUM> alleles of the CAT gene (cat/cat). Candida maltosa '<NUM> grows in the presence of oleic acid as the sole carbon source but does not produce dicarboxylic acids.

A Sigma-Aldrich technical grade oleic acid having the following composition - oleic acid <NUM>%, linoleic acid <NUM>%, stearic acid <NUM>%, palmitic acid <NUM>% - was used as substrate. The strain subsequently underwent genetic modifications through a process of double crossing over recombination using a suitably constructed deletion cassette to allow the deletion of the CAT gene, at first partly (<NUM> deleted allele, the so-called cat/CAT mutant) and then completely (<NUM> deleted alleles, the so-called cat/cat mutant). The deletion cassette was constructed starting from a pSFS2 plasmid developed for deleting genes in the species Candida albicans. The plasmid contains:.

The two genes mentioned are located between two FRT (Flippase Recognition Target) sequences. Recombinase is an enzyme promoting recombination between FRT sites, substantially eliminating the sequences lying between them. Expression of recombinase therefore makes it possible to eliminate the gene for resistance to the antibiotic once the gene has been deleted, making it possible to reuse the same antibiotic/plasmid for a further deletion. The nucleotide sequence of the gene coding for peroxisome and mitochondrial CAT 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 CAT gene in C. maltosa Xu316 was in turn used to design the primers to amplify the CAT gene in C. maltosa '<NUM> and corresponding portions of interest by PCR (Polymerase Chain Reaction). As far as deletion of the first allele of the CAT gene is concerned, a deletion cassette was constructed using the plasmid described above and the regions in <NUM>' (Flanking Region <NUM>) and <NUM>' (Flanking Region <NUM>) in the CAT gene amplified by PCR and subsequently inserted into the plasmid. The deletion cassette constructed in this way was used to transform the C. maltosa '<NUM> strain and the antibiotic resistant clones with one deleted allele were identified by PCR. In order to be able to delete the second allele of the CAT gene, the recombinase was expressed to eliminate resistance to the antibiotic; this step was performed for one clone showing that one CAT allele was deleted. The clones which then were sensitive to the antibiotic were analysed by PCR and one clone from which the gene for resistance to the antibiotic had been excised was then transformed by a new deletion cassette.

This new deletion cassette was constructed using the plasmid described above and a different region in <NUM>' (Flanking Region <NUM>) of the CAT gene, while the region in <NUM>' corresponds to Flanking Region <NUM>; these regions were amplified by PCR and subsequently inserted into the plasmid. The clones transformed with the construct in which the second allele was deleted were then selected as reported above and identified by PCR.

Finally, the fragment containing resistance to the antibiotic was removed as indicated above and the definitive clone was evaluated using PCR allowing the identification of a strain of C. maltosa '<NUM> in which both the alleles of the CAT gene were deleted. Results for the first preliminary comparative tests between the wt and cat/CAT and cat/cat strain are shown below.

It is clear that the strain of Candida maltosa '<NUM> in which both the alleles of the CAT gene were deleted ('<NUM> cat/cat) is unable to grow using oleic acid as the sole source of carbon, thus suggesting that the modification of the metabolic pathway involving the use of acetyl-CoA has an inhibiting effect on the β-oxidation pathway.

Subsequently, the fermentation performance of the Candida maltosa '<NUM> cat/cat strain in comparison with the '<NUM> CAT/CAT (wt) strain was evaluated under different process conditions. The process provides for a two-stage fermentation with uncoupling between the stage of cell growth to biomass (with consumption of glucose initially provided in the substrate) and the stage of producing dicarboxylic acids from mixtures of monocarboxylic acids.

The technical oleic acid used as substrate had the following composition: oleic acid <NUM>%, linoleic acid <NUM>%, stearic acid <NUM>%, palmitic acid <NUM>%.

Low = feeding rate controlled to maintain the substrate concentration at approximately <NUM>/L for the duration of the production stage.

High = feeding rate controlled to maintain the substrate concentration at approximately <NUM>/L for the duration of the production stage. The high feeding rate ensures a supply of fatty acids which is consistent with a wt microorganism capable of mainly consuming fatty acids via the β-oxidation metabolic pathway thus allowing the transport of derived acetyl-CoA through the "carnitine shuttle" process.

Fermentation samples were collected and analysed, first by Gas Chromatography using a Flame Ionisation Detector (GC-FID) and subsequently by Gas Chromatography associated with Mass Spectrometry (GC-MS). On the basis of the GC-FID data the output of dicarboxylic acid was <NUM>/L in the case of the Candida maltosa '<NUM> cat/cat samples. GC-MS analysis subsequently confirmed that the peak quantified in GC-FID actually corresponded to <NUM>,<NUM>-octadecenedioic acid.

Claim 1:
Process for the production of dicarboxylic acids from monocarboxylic acids, through a wild type microorganism Candida maltosa having the ω-oxidation metabolic pathway and having the peroxisome and mitochondrial carnitine acetyl-transferase coded by a single gene, characterized by inactivation of the function of the CAT (Carnitine Acetyl-Transferase) gene, in which that inactivation is done by making non-functional both the alleles of the CAT gene, and/or the product of its transcription.