EP4127201A1

EP4127201A1 - Process of engineering an oleaginous microorganism

Info

Publication number: EP4127201A1
Application number: EP21722286.8A
Authority: EP
Inventors: Paola Branduardi; Raffaella Desiré DI LORENZO
Original assignee: Pirelli SpA; Pirelli Tyre SpA
Current assignee: Pirelli and C SpA; Pirelli Tyre SpA
Priority date: 2020-03-31
Filing date: 2021-03-30
Publication date: 2023-02-08
Also published as: WO2021198904A1; CN116034164A

Abstract

The present invention relates to a process of engineering an oleaginous microorganism, to the engineered oleaginous microorganism obtained from such a process, and to the process for the production of a plasticising oil obtained from such an engineered oleaginous microorganism through cultivation in a culture medium containing biomass.

Description

TITLE

“PROCESS OF ENGINEERING AN OLEAGINOUS MICROORGANISM”

DESCRIPTION FIELD OF THE INVENTION

The present invention relates to a process of engineering an oleaginous microorganism, to the engineered oleaginous microorganism obtained from such a process, and to a process for the production of a plasticising oil obtained from such an engineered oleaginous microorganism through cultivation in a culture medium containing biomass.

PRIOR ART

Plasticising or process oils are used in industry, for example in the tyre industry. Plasticising oils are products of petrochemical derivation, like other products present in daily life such as fuels, plastics, synthetic fibres, solvents, fertilizers, fine chemicals, and ingredients for the formulation of drugs.

These oils are further classified according to the content of paraffinic, naphthenic and aromatic hydrocarbons.

Plasticising oils include mild extraction solvent (MES) mineral oils, treated distillate aromatic extracts (TDAE), naphthenic oil (NAP), Residual Aromatic Extract (RAE) (adapted from SUCHIVA, Krisda “Introduction to Process oils.” Research and Development Centre for Thai Rubber Industry, Mahidol University).

The problems associated with mineral oils are linked to the non- renewability of the raw material (in times compatible with its consumption).

The new bioeconomy trends are based on the exploitation and enhancement of freshly synthesised biomass through sustainable processes with reduced environmental impact.

This biomass represents the raw material of biorefinery, where this term means a production system capable of transforming a renewable substrate in times compatible with its use into a spectrum of products that can include bioenergy, biofuels and biomaterials, as is now possible starting from oil. At the heart of biorefinery there are often bioprocesses, or transformations carried out by living organisms or enzymatic activities derived from them, accompanied by sustainable chemical processes. In this technology it is possible to use homogeneous and easily transformable substrates, such as sugars in monomeric form or starches: in this case the biorefineries are defined as first-generation. Alternatively, second-generation biorefineries offer the possibility of using residual biomass as raw material, often of an inhomogeneous nature such as lignocellulose.

The first-generation biorefinery constitutes an alternative source of plasticising oils, as in patents (W02012012133; US 8,969,454 B2; WO201 2085014; WO2013189917; WO2012085012) in which vegetable oils consisting of a mixture of triglycerides are used as extender (and/or plasticising) oils for tyre formulations. As previously introduced, the raw material or starting substrate raises practical and ethical problems: in fact, the use of edible biomass overlaps and therefore competes with the agricultural and food chain, and its availability is subject to seasonality and climatic variability.

It is possible to use microorganisms to transform residual biomass into compounds of interest, including oils. Among microorganisms, yeasts constitute a valid platform for the development of bioprocesses, as many of them are genetically treatable and stable, easy to grow, safe for use (few yeasts are in fact known for their pathogenicity for humans, plants or animals, not subject to phage attack.

In particular, various yeast species are described as oleaginous, i.e. characterized by an oil content higher than 20% of the dry biomass. Furthermore, by suitably varying the growth conditions, their accumulation capacity rises to over 70% (as described in Thevenieau, F. et al., “Microorganisms as sources of oils.” Ocl 20.6 (2013): D603).

By way of non-exhaustive description, oils produced by oil microorganisms can be used for various applications, such as (i) in the biodiesel industry, where microbial oils obtained from Rhodosporidium toruloides yeast and Chlorella spp. microalgae are used (as described in X. Zhao et al., “Effects of some inhibitors on the growth and lipid accumulation of oleaginous yeast Rhodosporidium toruloides and preparation of biodiesel by enzymatic transesterification of the lipid”, Bioprocess Biosyst. Eng., 35 (2012); Li, Yecong, et al. “Characterization of a microalga Chlorella sp. well adapted to highly concentrated municipal wastewater for nutrient removal and biodiesel production.” Bioresource technology 102.8 (2011): 5138-5144) and (ii) in the biofuel industry and in the food industry as an alternative source of polyunsaturated fatty acids, where oils produced by the oleaginous yeast Lipomyces starkeyi are used (as described in Angerbauer, Christoph, et al. “Conversion of sewage sludge into lipids by Lipomyces starkeyi for biodiesel production.” Bioresource technology 99.8 (2008): 3051-3056; and in Blomqvist, Johanna, et al. Oleaginous yeast as a component in fish feed.” Scientific reports 8.1 (2018): 15945).

In second-generation biorefineries, residual biomass deriving from other industrial processes and/or biomass not in competition with the food chain are therefore used (as described in Naik, S. N, et al. “Production of first and second generation biofuels: a comprehensive review.” Renewable and sustainable energy reviews 14.2 (2010): 578-597; and in Stichnothe, H., et al. “Development of Second-Generation Biorefineries.” Developing the Global Bioeconomy. 2016. 11-40).

Furthermore, further examples of applications obtained after engineering of oleaginous microorganisms are reported in the literature: the yeast Criptococcus curvatus, mutated due to the partial block of the delta-9 desaturase enzymatic activity, allows oils with a composition similar to cocoa butter to be obtained (as described in Hassan, Mainul, et al. “Production of cocoa butter equivalents from prickly-pear juice fermentation by an unsaturated fatty acid auxotroph of Cryptococcus curvatus grown in batch culture.” Process Biochemistry 30.7 (1995): 629-634); and wild and mutated strains of R. toruloides have been used as producers of oils for the production of biodiesel and oleochemicals (as described in WO2016/185073 and Koutinas, Apostolis A., et al. “Design and techno-economic evaluation of microbial oil production as a renewable resource for biodiesel and oleochemical production.” Fuel 116 (2014): 566-577).

SUMMARY OF THE INVENTION

In view of what is known in the art, the Applicant has also developed an engineering process that allows an oleaginous yeast to be obtained which overexpresses a combination of endogenous genes encoding for enzymatic activities involved in the biosynthetic process of fatty acids, in particular (i) the enzyme delta-9 desaturase and (ii) the enzyme delta-12 desaturase.

The Applicant has surprisingly observed that the oil obtained from the oleaginous yeast thus engineered had a particular enrichment in monounsatu rated fatty acids, unlike the expected enrichment in polyunsaturated fatty acids.

Therefore, a first aspect of the present invention consists in a process of engineering an oleaginous microorganism comprising the following steps:

(a) providing an oleaginous microorganism;

(b) inserting the gene encoding the enzyme delta-9 desaturase into the oleaginous microorganism;

(c) inserting the gene encoding the enzyme delta-12 desaturase into the oleaginous microorganism;

(d) selecting the resulting engineered microorganism.

According to an embodiment of the first aspect of the present invention, the microorganism is an oleaginous yeast of the group comprising the genera Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon, Yarrowia.

According to a preferred embodiment of the first aspect of the present invention, the microorganism belongs to strains of the species Cryptococcus curvatus, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon fermentans, and Yarrowia lipolytica, more preferably Rhodosporidium toruloides and Lipomyces starkeyi.

According to an alternative embodiment of the first aspect of the present invention, the microorganism is a microalgae of the group consisting of Chlorella ellipsoidea, Chlorella protothecoides, Chlorella vulgaris, Chlorella vulgaris, Dunaliella sp., Haematococcus pluvialis, Neochloris oleoabundans, Neochloris oleabundans, Pseudochlorococcum sp., Scenedesmus obliquus, Tetraselmis chui, Tetraselmis sp., Tetraselmis tetrathele, Chaetoceros calcitrans CS 178, Chaetoceros gracilis, Chaetoceros muelleri, Nitzschia of. pusilla YSR02, Phaeodactylum tricornutum F&M-M40, Skeletonema sp. CS 252, Thalassiosira pseudonana CS 173 Crypthecodinium cohnii, Isochrysis sp., Isochrysis sp., Isochrysis zhangjiangensis, Nannochloropsis oculate, Nannochloropsis oculata NCTU-3, Nannochloropsis sp., Pavlova salina CS 49, Rhodomonas sp., Thalassiosira weiss f log ii.

According to an alternative embodiment of the first aspect of the present invention, the microorganism is selected from the group consisting of fungi and protists, such as for example Aspergullus terreus, Claviceps purpurea, Tolyposporium, Mortierella alpina, Mortierella isabellina, Schizochitrium limacynum.

According to a preferred embodiment of the first aspect of the present invention, the gene encoding the enzyme delta-9 desaturase overexpressed in yeast is OLE1 of Lipomyces starkeyi having the sequence (SEQ ID NO: 1).

According to a preferred embodiment of the first aspect of the present invention, the gene encoding the enzyme delta-12 desaturase overexpressed in yeast is FAD2 of Lipomyces starkeyi having the sequence (SEQ ID NO: 2).

According to a preferred embodiment of the first aspect of the present invention, the oleaginous microorganism is Lipomyces starkeyi.

In a second aspect thereof, the present invention also relates to the engineered oleaginous microorganism obtained with the process of the first aspect of the present invention, characterised by a carbon flow directed towards the synthesis of oil with a modified lipid profile compared to the wild oleaginous microorganism.

The engineered oleaginous microorganism obtained with the process of the first aspect of the present invention is suitable for the production of oil starting from biomass.

Therefore, a third aspect of the present invention relates to a process for the production of a plasticising oil comprising a plasticising oil comprising the following steps of:

(a) cultivation of engineered oleaginous microorganisms obtained by the process of the first aspect of the present invention in a culture medium comprising biomass containing a carbon to nitrogen molar ratio between 5 and 20;

(b) imbalance by variation of the carbon to nitrogen molar ratio at values equal to or greater than 30, preferably between 30 and 100;

(c) separation of engineered oleaginous microorganisms from the culture medium; and

(d) extraction of the oil from oleaginous microorganisms.

According to an embodiment of the third aspect of the present invention, the biomass comprises at least one source of organic carbon selected from the group consisting of crude glycerol, molasses, lignocellulose, sugar beet pulp, whey, starch residues, waste water, waste oils, glucose, xylose, arabinose, fructose, galactose, mannose, acetate, and/or a combination thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described, for non-limiting illustrative purposes, according to preferred embodiments thereof, with particular reference to the accompanying figures, in which:

Figure 1 shows the fermentation profile of R. toruloides in a bioreactor with the main parameters related to obtaining the microbial oil 1 (OIL 1);

Figure 2 shows the fermentation profile of L. starkeyi in a bioreactor with the main parameters relating to obtaining the microbial oil 2 (OIL 2);

Figure 3 shows the map of the recombinant vector pLS01 bearing the expression cassette of the gene for resistance to nurseotricin (NrsR) deriving from plasmid pZs (Branduardi et al., “Biosynthesis of vitamin C by yeast leads to increased stress resistance.” PLoS One, 2, e1092, 2007);

Figure 4 shows the map of the recombinant vector pLS02, derived from pLS01 and bearing a multiple cloning site (MCS);

Figure 5 shows the map of the recombinant vector pLS02-OLE1, deriving from pLS02 and bearing (in the MCS) the expression cassette for the putative endogenous gene encoding for the enzyme delta-9 desaturase of L. starkeyi DSM70295;

Figure 6 shows the fragment derived from pLS02-OLE1 which includes the expression cassette bearing the putative gene encoding for delta-9 desaturase (OLE1) and the gene for resistance to nurseotricin (NrsR): such a cassette is preferably integrative in the homologous ends;

Figure 7 shows the map of the recombinant vector pLS03, bearing the expression cassette of the gene for resistance to hygromycin B (HygR) deriving from plasmid pZ4 (Branduardi et al., “The yeast Zygosaccharomyces bailii: a new host for heterologous protein production, secretion and for metabolic engineering applications.” FEMS yeast research 4.4-5 (2004): 493- 504);

Figure 8 shows the map of the recombinant vector pLS04, deriving from pLS03 and bearing a multiple cloning site (MCS);

Figure 9 shows the map of the recombinant vector pLS04-FAD2, derived from pLS03 and bearing (in the MCS) the expression cassette for the endogenous gene encoding for the enzyme delta-12 desaturase deriving from L. starkeyi DSM70295;

Figure 10 shows the fragment derived from pLS04-FAD2 which includes the expression cassette bearing the gene encoding for delta-12 desaturase (FAD2) and the gene for resistance to hygromycin B (FlygR): such a cassette is preferably integrative in the homologous ends;

Figure 11 shows the image relating to the electrophoretic run carried out to confirm the successful integration of the expression cassette bearing the putative gene encoding for delta9 desaturase ( OLE1 ) and the gene for resistance to nurseotricin ( NrsR ) (Photo A), and to confirm the successful integration of the expression cassette bearing the gene coding for delta12 desaturase ( FAD2 ) and the gene for resistance to hygromycin B ( HygR ) (Photo B), where 1 represents the PCR negative control (water), 2 represents the integration negative control (DNA L. starkeyi), 3 represents the integration positive control pl_S04-OLE1 (Photo A) or pl_S04-FAD2 (Photo B), and 4 represents the engineered strain L. Starkeyi-OLE1 -FAD2;

Figure 12 shows the graph representative of the number of copies of the OLE1 gene (Graph A) and of the FAD2 gene (Graph B) per cell, in the engineered strain, and in the wild control strain, to which unit value has been attributed;

Figure 13 shows a representative graph of the expression levels of the putative gene encoding for the delta-9 desaturase enzyme activity ( OLE1 - Graph A) and of the gene encoding for the delta-12 desaturase enzyme activity ( FAD2 - Graph B) in the engineered strain, and in the wild control strain, to which unit value has been attributed;

Figure 14 shows a graph representative of the trend over time of the growth and production of oily biomass of an engineered strain for the production of OIL 3, compared to the consumption of the supplied substrate (glycerol 100 g/L);

Figure 15 shows the fermentation profile of engineered L. starkeyi in bioreactor, with the main parameters relating to obtaining the microbial oil (OIL 3), where the imbalance phase is shown in the graph with a dashed line;

Figure 16 shows a histogram representative of the fatty acid composition related to OIL 3 compared to the composition of OIL 2. In Figure 16, asterisks indicate statistical significance according to Student's t-test in the difference in lipid composition between OIL 2 and OIL 3 (^* p<0.05, ^** p<0.005 and ^*** p<0.0005);

Figure 17 shows a graph representative of the number of copies of the OLE1 gene (Graph A) and of the FAD2 gene (Graph B) in the respective engineered strains compared to the wild control strain to which unit value has been attributed;

Figure 18 shows a representative graph of the expression levels of the putative gene encoding for the delta-9 desaturase enzyme activity (OLE1 - Graph A) and of the gene encoding for the delta-12 desaturase enzyme activity (FAD2 - Graph B) in the respective engineered strains with respect to the wild control strain to which unit value has been attributed;

Figure 19 shows a histogram representative of the fatty acid composition related to OIL 8 compared to the composition of OIL 3. In Figure 19, asterisks indicate statistical significance according to Student's t-test in the difference in lipid composition between OIL 8 and OIL 3 (^* p<0.05, ^** p<0.005 and ^*** p<0.0005);

Figure 20 shows a histogram representative of the fatty acid composition related to OIL 9 compared to the composition of OIL 3. In Figure 20, asterisks indicate statistical significance according to Student's t-test in the difference in lipid composition between OIL 9 and OIL 3 (^* p<0.05, ^** p<0.005 and ^*** p<0.0005).

For the purposes of the present description and of the following claims, unless indicated otherwise, all the numbers expressing quantities, values, percentages and so on must be interpreted as modified in all cases by the term "about". Furthermore, all ranges include any combination of the maximum and minimum points described and include any intermediate range, which may or may not have been specifically listed herein.

The term “biomass” defines any substance of an organic nature that can regenerate in times compatible with its consumption, which can be used for the production of bioenergy, biofuels and biomaterials. This is in contrast to fossil biomass, whose regeneration times exceed those of consumption by many orders of magnitude.

The term “expression vector” defines a DNA construct comprising a DNA sequence linked to a control sequence capable of leading to the expression of said DNA in a suitable host. In the present invention, the typical plasmid expression vector used has: a) an origin of replication which allows the actual replication of the plasmid so that in each cell of the selected host there are 1- 2 or tens of copies of the plasmid vector, or a DNA sequence that allows the integration of the plasmid vector into a chromosome of each cell of the selected host; b) a selection marker such that a cell correctly transformed with the plasmid vector can be selected; c) a DNA sequence comprising cleavage sites for restriction enzymes in order to be able to introduce exogenous DNA into the plasmid vector by a process called ligation. As generally reported in the prior art, in order to reach high levels of expression of the gene inserted in the host cell, the coding sequence must be correctly and functionally connected to regulatory elements of the transcription, functioning in the selected expression host.

The term “transformation” used herein means that DNA, once introduced into the cell, can replicate outside chromosomes or as part of a chromosome.

The term “lipid bodies” refers to the intracellular compartments present in animals, plants, fungi and even bacteria specialised for the accumulation of energy in the form of neutral lipids such as triglycerides and sterol esters.

The term “oleaginous microorganism” refers to a microorganism capable of accumulating at least 20% of lipids with respect to its dry weight.

The term “delta-9 desaturase” refers to a polypeptide belonging to the family of enzymes EC 1.14.19.1 which catalyses the introduction of a double bond in the delta-9 position of the fatty acid chain. Such a reaction has palmitic and/or stearic acid as its predominant substrate, giving rise to palmitoleic and/or oleic acid, respectively.

The term “delta-12 desaturase” refers to a polypeptide belonging to the family of enzymes EC 1.14.19.6 which catalyses the introduction of a double bond in the delta-12 position of the fatty acid chain. Such a reaction has oleic acid as its predominant substrate, giving rise to linoleic acid.

By way of non-limiting example of the present invention, the following examples are given to support and demonstrate the metabolic path developed by the inventors of the present invention for the production of modified oil.

EXAMPLE 1

The microbial oils with plasticising action (OIL 1 and OIL 2) were produced according to the procedures described below.

The following Table 1 shows the fatty acid composition relating to OIL 1 and OIL 2, expressed as a percentage weight by weight (% w/w) at the final time.

TABLE 1 A) Production of microbial oil with plasticising action in the oleaginous yeast Rhodosporidium toruloides DSM4444 by fermentation process

(OIL 1)

The cells of the oleaginous yeast strain R. toruloides DSM4444 were pre- inoculated into the medium from the following composition: 1 g of yeast extract, 1.31 g (NH bO^ 0.95 Na₂HP04, 2.7 g KH2PO4, 0.2 g MgS0₄ ^* 7H2O, per litre enriched with mineral stock 100 times concentrated, containing (4 g CaCl2 ^* 2H2O, 0.55 g FeSC>4 ^* 7H2O, 0.52 g citric acid, 0.10 g ZnSC>4 ^* 7H2O, 0.076 g MnSC>4 ^* H2O, 100 microlitres of H2SO4 18 M, per litre of solution), in the presence of glycerol 15 g/L as a source of energy and carbon. This concentration allows a C:N ratio of 10:1 to be obtained, which is defined as balanced to support the growth and division of yeasts. The pre inoculation was carried out in 200 ml_ of medium in 1 L flasks placed at 25 °C on an orbital shaker at 220 rpm. After 72 hours of growth, the cells were inoculated in a 2L bioreactor at an initial DO660 of approximately 1. The operating volume of medium, the same used for the pre-culture, corresponds to lOOOmL in the presence of about 40 g/L of glycerol.

After a fermentation time suitable for the development of an exponential growth phase, the imbalancing phase began, where crude glycerol was added to the medium to reach a final concentration of about 50 g/L. The C:N molar ratio therefore switches to the value of about 30:1 , causing a metabolic variation towards the accumulation of microbial oils in the so-called lipid bodies at the expense of cell divisions, which cannot be performed due to the scarcity of the nitrogen source.

The fermentation parameters require the bioreactor to maintain a constant temperature of 25 °C; an amount of dissolved oxygen greater than 25% with an air flow of 1 vvm (volume of air per volume of culture medium); the pH is maintained at 5.5 with the addition, if necessary, of NaOH 4M and H3PO4 at 25% (v/v); stirring is dependent on the percentage of oxygen dissolved in the medium.

After 264 hours from inoculation, the cells were recovered by centrifugation and subjected to acid lysis (HCI 2M), in order to break the cells themselves, and treatment with 2:1 chloroforr methanol solution and chloroform 100% for the separation of the lipids. The chemical extraction of the oil was carried out according to the following protocol: the cells were dissolved in a hydrochloric acid solution 2 M; the preparation was heated in a thermostated bath for 60 minutes at 95 °C with constant stirring. An equal volume (1 :1 v/v) of a 2:1 chlorofornrmethanol solution was added to the suspension and then it was subjected to centrifugation for 10 minutes at 10000 rpm until separation in phases was obtained; the lower phase was recovered and 10 mL of 100% chloroform were added to the suspension in order to recover the remaining lipids. The microbial oil obtained from chemical extraction was subjected to a transesterification reaction and subsequent analysis with gas chromatography [SAVI LABORATORI & SERVICE S.r.l., Roncoferraro (MN), Italy]

Figure 1 shows the fermentation profile of R. toruloides with respect to the biomass trend over time (symbol ·) and the corresponding substrate consumption (symbol A), where the imbalance phase is shown in the graph with a dashed line, the line with the symbol A represents the trend of the glycerol concentration, and the line with the symbol · represents the trend of the biomass.

B) Production of microbial oil with plasticising action in the oleaginous yeast Lipomyces starkeyi DSM70295 by fermentation process (OIL 2).

The cells of the oleaginous yeast strain Lipomyces starkeyi DSM70295 were pre-inoculated into the medium from the following composition: 1 g of yeast extract, 1.31 g (NH bO^ 0.95 Na2HP04, 2.7 g KH2PO4, 0.2 g MgSC>4 ^* 7H2O, per litre enriched with mineral stock 100 times concentrated, containing (4 g CaCl2 ^* 2H2O, 0.55 g FeSC>4 ^* 7FI2O, 0.52 g citric acid, 0.10 g ZnSC>4 ^* 7FI2O, 0.076 g MnSC>4 ^* FI2O, 100 microlitres of FI2SO4 18 M, per litre of solution), in the presence of glycerol 15 g/L as a source of energy and carbon. This concentration allows a C:N molar ratio of 10:1 to be obtained, which is defined as balanced to support the growth and division of yeasts. The pre-inoculation was carried out in 200 mL of medium in 1 L flasks placed at 25 °C on an orbital shaker at 220 rpm. The cells were inoculated in a 2L bioreactor at an initial DO660 of 3. The operating volume of medium, the same used for the pre-culture, corresponds to 1000 mL in the presence of about 60 g/L of glycerol.

After a fermentation time suitable for the development of an exponential growth phase, the imbalancing phase began, where crude glycerol was added to the medium to reach a final concentration of 60 g/L. The C:N molar ratio therefore switches to the value of about 40:1 , causing a metabolic variation towards the accumulation of microbial oils in the so-called lipid bodies at the expense of cell divisions, which cannot be performed due to the scarcity of the nitrogen source.

After about 160 hours from inoculation, the cells were recovered by centrifugation and subjected to acid lysis (HCI 2M), in order to break the cells themselves, and treatment with 2:1 chloroforr methanol solution and chloroform 100% for the separation of the lipids. The chemical extraction of the oil was carried out according to the following protocol: the cells were dissolved in a hydrochloric acid solution 2 M; the preparation was heated in a thermostated bath for 60 minutes at 95 °C with constant stirring. An equal volume (1 :1 v/v) of a 2:1 chloroforrmmethanol solution was added to the suspension and then it was subjected to centrifugation for 10 minutes at 10000 rpm until separation in phases was obtained; the lower chloroform phase was recovered and 10 mL of 100% chloroform were added to the suspension in order to recover the remaining lipids. The microbial oil obtained from chemical extraction was subjected to a transesterification reaction and subsequent analysis with gas chromatography [SAVI LABORATORI & SERVICE S.r.l., Roncoferraro (MN), Italy]

Figure 2 shows the fermentation profile of L. starkeyi with respect to the biomass trend over time (symbol ·) and the corresponding substrate consumption (symbol A), where the imbalance phase is shown in the graph with a dashed line, the line with the symbol A represents the trend of the glycerol concentration, and the line with the symbol · represents the trend of the biomass.

EXAMPLE 2

This example describes the procedure for preparing the expression cassette containing the putative sequence encoding for the delta9 desaturase activity under the control of the pTDH3 promoter and tPGK1 terminator together with the resistance cassette to nurseotricin NsrR.

A) Construction of the recombinant expression vector pLS01 bearing the sequence that encodes for the enzyme nurseotricin N-acetyl transferase (NrsR) capable of giving resistance to the antibiotic nurseotricin.

The sequences for the pURA3 promoter and tGAL1 terminator of L. starkeyi were amplified by PCR using as a template the genomic DNA of L. starkeyi DSM70295 and specific oligonucleotides (SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO:5; SEQ ID NO:6). The NAT gene encoding for the resistance to nurseotricin was amplified by PCR using as a template the plasmid pZs and specific oligonucleotides (SEQ ID NO: 7; SEQ ID NO: 8). The program used for both amplifications is as follows: after 30 seconds of denaturation at 98 °C, 35 cycles (10 second denaturation at 98 °C, 30 second pairing at 64 °C and 30 seconds elongation at 72 °C ), followed by a final elongation of 2 minutes at 72 °C. The PCR products were loaded onto 0.8% agarose gel and the fragments of interest were recovered by excision and purified with the NucleoSpin Gel and PCR clean-up kit (MACHEREY- NAGEL GmbH & Co. KG). The NrsR gene, amplified by plasmid pZs, and pURA3 and t GAL1, amplified by the genomic DNA of L. starkeyi, were inserted into the pStblue-1 vector by cloning using the Gibson assembly cloning kit (New England Biolab, NEB) and the product amplified by transformation of Escherichia coli. Once the plasmid extraction from E. coli was performed, visualized by electrophoretic run on 0.8% agarose gel, the correct insertion of the pURA3, NAT, t GAL1 fragments in the pSTBIue plasmid was verified through analytical digestions tests carried out with the restriction enzymes Hhel and Sphl. The vector named pl_S01 -L/rsft was purified on a column using the NucleoSpin Gel and PCR clean-up kit (MACHEREY-NAGEL GmbH & Co. KG) and sequenced using the specific oligonucleotides (SEQ ID NO: 9; SEQ ID NO: 10).

Figure 3 shows the recombinant vector pl_S01 -L/rsft.

B) Construction of the recombinant vector pLS02 bearing the multiple cloning site (MCS).

The pLS01 vector was subjected to preparative digestion with the restriction enzyme EcoRV for linearization. The vector was recovered by removal from agarose gel and then purified with NucleoSpin Gel and PCR clean-up and quantified with Nanodrop [Euroclone (Spa)]. The constitutive and strong promoter of the endogenous gene of L. starkeyi TDH3 and the terminator of the endogenous gene of L. starkeyi PGK1 were inserted in the linearized pl_S01 plasmid: these sequences were amplified by PCR using as model the genomic DNA of L. starkeyi DSM70295 and specific oligonucleotides (SEQ ID NO: 11 ; SEQ ID NO: 12; SEQ ID NO:13; SEQ ID NO:14), interspersed with specific sites for five different restriction enzymes (Bglll, Spel, EcoRV, Blpl, Fsel) in order to create an MCS between promoter and terminator. This was done again using the Gibson assembly cloning kit (New England Biolab, NEB). Once the extraction of the pl_S02 plasmid from E. coli was carried out, it was verified through an analytical gel of 0.8% agarose. The correct insertion of the p TDH3, tPGK1 fragments inside the pl_S01 -L/rsft plasmid was verified through the analytical digestions carried out with Pstl and Pmll. The vector pLS02 was purified on a column using NucleoSpin Gel and PCR clean-up kit (MACHEREY-NAGEL GmbH & Co. KG) and sequenced using the specific oligonucleotides (SEQ ID NO: 9; SEQ ID NO: 15).

Figure 4 shows the vector pLS02.

C) Construction of the recombinant vector pLS02 -OLE1 bearing the putative gene encoding for the delta-9 desaturase enzyme activity.

The sequences encoding for the enzyme delta-9 desaturase were obtained by aligning the sequence of the yeast protein Saccharomyces cerevisiae against the putative amino acid sequences of the coding sequences present in the whole genome of Lipomyces starkeyi (www.genome.jgi.doe.gov/Lipst1_1/Lipst1_1.home.html), using the BlastP program (https://blast.ncbi. nlm.nih.gov/Blast.cgi?PAGE=Protein).

The sequence encoding for the enzyme delta-9 desaturase was amplified by PCR using the genomic DNA of L. starkeyi DSM70295 as a template and specially designed oligonucleotides (SEQ ID NO: 16; SEQ ID NO:17). The program used for amplification is as follows: after 30 seconds of denaturation at 98 °C, 30 cycles (10 second denaturation at 98 °C, 30 second pairing at 72 °C and 60 second elongation at 72 °C ), followed by a final elongation of 2 minutes at 72 °C. The PCR product and the target vector pLS02-MCS were digested with the restriction enzyme Spel and their ligation led to the obtainment of the recombinant expression vector pLS02 -OLE1. The vector pLS02 -OLE1 was removed from agarose gel and purified on a column using NucleoSpin Gel and PCR clean-up kit (MACHEREY-NAGEL GmbH & Co. KG), quantified and sequenced using the specific oligonucleotides (SEQ ID NO: 15; SEQ ID NO: 14).

Figure 5 shows the vector pl_S02 -OLE1.

D) Isolation of the expression cassette for the putative gene encoding for the delta9 desaturase activity and for resistance to nurseotricin.

The pl_S02 vector was digested with the EcoRI-HF restriction enzyme. The fragment corresponding to the expression cassette (4556 bp) was recovered by removal from 0.8% agarose gel and purified with NucleoSpin Gel and PCR clean-up.

Figure 6 shows the expression cassette for the sequence encoding for the delta9 desaturase activity ( OLE1 ).

EXAMPLE 3

This example describes the procedure for preparing the expression cassette containing the sequence encoding for the delta12 desaturase activity under the control of the pTDH3 promoter and tPGK1 terminator together with the resistance cassette to hygromycin HygR.

A) Construction of the recombinant vector pLS03 bearing the sequence encoding for the enzyme hygromvcin-B 4-O-kinase (HygR) capable of giving resistance to the antibiotic hygromycin.

The HygR gene for resistance to hygromycin B (4-O-kinase) was amplified by PCR using as a template the plasmid pZ4 and specific oligonucleotides (SEQ ID NO: 18; SEQ ID NO: 19). The program used for the amplification is as follows: after 30 seconds of denaturation at 98 °C, 35 cycles (10 second denaturation at 98 °C, 30 second pairing at 68°C and 30 second elongation at 72 °C ), followed by a final elongation of 2 minutes at 72 °C. The PCR product was loaded onto 0.8% agarose gel and the fragments of interest were recovered by excision and purified with the NucleoSpin Gel and PCR clean-up kit (MACHEREY-NAGEL GmbH & Co. KG). The hph gene, amplified by the plasmid pZ4, was inserted into the pStblue-1 vector by cloning using the Gibson assembly cloning kit (New England Biolab, NEB). Once the plasmid extraction from E. coli was performed, visualized by electrophoretic run on 0.8% agarose gel, the correct insertion of the hph fragment in the pSTBIue plasmid was verified through analytical digestions tests carried out with the restriction enzymes Hhel and Hindi. The vector named pLS03 -HygR was purified on the column using the kit described above and sequenced using the specific oligonucleotides (SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:18).

Figure 7 shows the recombinant vector pLS03.

B) Construction of the recombinant vector pLS04 bearing the multiple cloning site (MCS).

The target vector pLS02 was digested with the restriction enzyme Nhel to obtain the linearized pl_S02 vector and remove the nurseotricin resistance gene ( NrsR ). Similarly, using the same restriction enzyme Nhel, the pl_S03 vector was digested to obtain the HygR gene, which confers resistance to the hygromycin-B antibiotic.

The HygR gene and the pl_S02 vector were recovered by removal from agarose gel and then purified with NucleoSpin Gel and PCR clean-up and quantified with Nanodrop [Euroclone (Spa)]. The ligation was then carried out which led to the obtainment of the recombinant expression vector pl_S04 bearing resistance to the antibiotic hygromycin B. The correct insertion of the HygR fragment inside the pl_S02 plasmid was verified through the analytical digestion carried out with Sacll.

Figure 8 shows the recombinant vector pl_S04.

C) Construction of the recombinant vector pLS04 -FAD2 bearing the gene encoding for the delta-12 desaturase enzyme activity.

The sequences encoding for the enzyme delta-12 desaturase were obtained from the identification and characterisation work described in Matsuzawa, Tomohiko, et al. “Identification and characterization of D12 and D12/D15 bifunctional fatty acid desaturases in the oleaginous yeast Lipomyces starkeyi.” Applied microbiology and biotechnology 102.20 (2018): 8817-8826.

The sequence encoding for the enzyme delta-12 desaturase was amplified by PCR using the genomic DNA of L. starkeyi DSM70295 as a template and specific oligonucleotides (SEQ ID NO: 20; SEQ ID NO:21). The program used for the amplification is as follows: after 30 seconds of denaturation at 98 °C, 10 cycles (10 second denaturation at 98 °C, 30 second pairing at 59 °C and 40 second elongation at 72 °C ), 25 cycles (10 second denaturation at 98 °C, 30 second pairing at 64 °C and 40 second elongation at 72 °C), followed by a final 2 minute elongation at 72 °C. The target vector pLS04- MCS was digested with the restriction enzyme EcoRV-HF, and ligated to the PCR product leading to the obtainment of the recombinant expression vector pLS04-F4D2. The vector pLS04-F4D2 was purified on a column using NucleoSpin Gel and PCR clean-up kit (MACHEREY-NAGEL GmbH & Co. KG) and sequenced using the specific oligonucleotides (SEQ ID NO: 15; SEQ ID NO: 14).

Figure 9 shows the vector pl_S04-F4D2.

D) Isolation of the expression cassette for the gene encoding for the desaturase delta-12 activity and for resistance to hygromvcin B.

The pl_S04-F4D2 vector was digested with the restriction enzyme Xhol. The fragment corresponding to the expression cassette (4765 bp) was recovered by removal from 0.8% agarose gel and purified with NucleoSpin Gel and PCR clean-up.

Figure 10 shows the expression cassette for the sequence encoding for the delta12 desaturase activity ( FAD2 ).

EXAMPLE 4

Construction of the recombinant strain L. sfar/cey/-OLE1-FAD2 from L. starkeyi (DSM70295) for the modification of the lipid profile and for obtaining the microbial oil (OIL 3).

The laboratory strain of L. starkeyi DSM70295 was transformed using two expression cassettes of which (i) one containing the putative sequence encoding for the delta9 desaturase activity under the control of the pTDH3 promoter and t PGK1 terminator together with the resistance cassette to nurseotricin NsrR, described in example 2, and (ii) one containing the sequence encoding for the delta12 desaturase activity under the control of the p TDH3 promoter and the t PGK1 terminator together with the hygromycin resistance cassette HygR, described in example 3. The integration of both expression cassettes was verified by PCR with the oligonucleotides (SEQ ID NO: 15; SEQ ID NO:22; SEQ ID NO:23) (Calvey et al„ “An optimized transformation protocol for Lipomyces starkeyi .’’ Current genetics 60.3 (2014): 223-230) (Figure 11).

EXAMPLE 5

Evaluation of the number of gene copies inserted in the genome of L. starke -OLE1-FAD2 through relative quantitative Real Time PCR.

The evaluation of the number of gene copies inserted in the genome of L. starkeyi-OLE1 -FAD2 was carried out through a relative quantitative Real Time PCR. Starting from the genomic DNA, extracted from both the parental strain and the engineered strain, the number of copies of OLE1 and FAD2 per cell was quantified. Real-time PCR was performed using specific oligonucleotides (SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27) and actin as internal control (SEQ ID NO: 28; SEQ ID NO: 29) (Figure 12).

EXAMPLE 6

Evaluation of the expression levels of the sequences encoding for delta-9 desaturase and delta-12 desaturase over-expressed in L. starkeyi - OLE1- FAD2 by relative quantitative Real Time PCR.

The evaluation of the expression levels of the putative sequence encoding for the enzyme delta-9 desaturase and delta-12 desaturase was performed by relative quantitative Real Time PCR.

The messengers for delta-9 desaturase and delta-12 desaturase in the recombinant strain L. starkeyi-OLE1-FAD2 and in the wild strain are quantified from the cDNA obtained by retro-transcription of the total RNA (Figure 13).

The cells were pre-inoculated in 5 ml of the medium containing: Glucose 25%, xylose 25%, 1 g of yeast extract, 1.31 g (NH bO^ 0.95 Na2HPC>4, 2.7 g KH2PO4, 0.2 g MgSC ^* 7H2O, per litre enriched with mineral stock 100 times concentrated, containing (4 g CaCl2 ^* 2H2O, 0.55 g FeSC>4 ^* 7FI2O, 0.52 g citric acid, 0.10 g ZnS0₄ ^* 7H20, 0.076 g MnS0₄ ^* H2O, 100 microlitres of FI2SO4 18 M, per litre of solution) for 24 h. RNA extraction was performed on a sample of cells in the exponential phase, using the ZR Fungal/Bacterial RNA Miniprep kit (Zymoresearch/The epigenitics company). The extraction was then controlled with electrophoretic run on 1.5% agarose gel. The cDNA was obtained using the iScript cDNA Synthesis (BIORAD) kit. Real-time PCR was performed using specific oligonucleotides (SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27) and actin as internal control (SEQ ID NO: 28; SEQ ID NO: 29).

EXAMPLE 7

Flask production kinetics of the oleaginous yeast L. starkevi-OLE1 -FAD2 engineered for the modification of the lipid profile of the microbial oil obtained from the wild strain.

The cells of the oleaginous yeast strain L. starkeyi-OLE1-FAD2, engineered for the production of modified lipid oil (OIL 3) were pre-inoculated into the medium with the following composition: 1 g yeast extract, 1.31 g (NH )₂S04, 0.95 Na₂HP0₄, 2.7 g KH2PO4, 0.2 g MgS0₄ ^* 7H 0, per litre enriched with mineral stock 100 times concentrated, containing (4 g CaCl2 ^* 2H2O, 0.55 g FeS0₄ ^* 7H₂0, 0.52 g citric acid, 0.10 g ZnS0₄ ^* 7H₂0, 0.076 g MnS04 ^* FI2O, 100 microlitres of FI2SO4 18 M, per litre of solution), in the presence of glycerol 15 g/L as a source of energy and carbon. This concentration allows a C:N ratio of 10:1 to be obtained, which is defined as balanced to support the growth and division of yeasts. The pre-inoculation was carried out in 100 mL of medium in 500L flasks placed at 25 °C on an orbital shaker at 220 rpm. After 72 hours of growth, the cells were inoculated at an optical density of 3 (OD 660 nm) in 50 mL of medium, the same used for pre-inoculation in the presence of about 100 g/L of glycerol, in 250 imL flasks placed at 25 °C on an orbital shaker at 220 rpm. Cell growth was monitored by measuring OD at 660 nm at regular time intervals. The extracellular concentration of glycerol was determined by HPLC using H2SO4 0.01 M as mobile phase and a Rezex ROA-Organic (Phenomenex) column.

After 240 hours from inoculation, the cells were recovered by centrifugation and subjected to acid lysis (HCI 2M), in order to break the cells themselves, and treatment with 2:1 chloroforr methanol solution and chloroform 100% for the separation of the lipids. The chemical extraction of the oil was carried out according to the following protocol: the cells were dissolved in a hydrochloric acid solution 2 M; the preparation was heated in a thermostated bath for 60 minutes at 95 °C with constant stirring. An equal volume (1 :1 v/v) of a 2:1 chloroforrmmethanol solution was added to the suspension and then it was subjected to centrifugation for 10 minutes at 10000 rpm until separation in phases was obtained; the lower phase was recovered and 10 mL of 100% chloroform were added to the suspension in order to recover the remaining lipids. The microbial oil obtained from chemical extraction was subjected to a transesterification reaction and subsequent analysis with gas chromatography [SAVI LABORATORI & SERVICE S.r.l., Roncoferraro (MN), Italy].

Figure 14 shows the fermentation profile of the L. starkeyi-OLE1-FAD2 strain with respect to the biomass trend over time and the corresponding substrate consumption. The following Table 2 shows the fatty acid composition relating to OIL 3 compared with the composition of OIL 1 and 2 and of some vegetable oils, in particular castor oil (OIL 4), sunflower oil AP- 75^® (Cargill) (OIL 5), sunflower oil AP-88^® (Cargill) (OIL 6). TABLE 2

The following table 3 summarises the characterisation of the oils of Table 2 and of a mineral oil MES (TUDALEN 4226, H&R Group) (OIL 7) as a functional reference carried out using Differential Scanning Calorimetry (DSC), starting from a temperature of -140 °C to +60 °C to establish the melting temperature and the glass transition temperature. The iodine number was determined using the ISO 3961 method, which involves treating the oil with an excess Wijs solution. Wijs solution contains iodine monochloride dissolved in acetic acid. The iodine monochloride reacts with the unsaturated part of the oil and the unreacted iodine is released as iodine by adding potassium iodide. The released iodine is determined by titration with sodium thiosulfate. TABLE 3

EXAMPLE 8

Production of microbial oil (OIL 3) with plasticising action in the oleaginous yeast L. starkevi-OLE1 -FAD2 by a fermentation process in a bioreactor with a volume of 10 litres.

The cells of the oleaginous yeast strain L. starkeyi-OLE1-FAD2, engineered for the production of modified lipid oil (OIL 3) were pre-inoculated into the medium with the following composition: 1 g of yeast extract, 1.31 g (NH )2S04, 0.95 Na₂HP04, 2.7 g KH2PO4, 0.2 g MgS0₄ ^* 7H 0, per litre enriched with mineral stock 100 times concentrated, containing (4 g CaCL ^* 2H2O, 0.55 g FeS0₄ ^* 7H₂0, 0.52 g citric acid, 0.10 g ZnS0₄ ^* 7H₂0, 0.076 g MnS04 ^* H2O, 100 microlitres of H2SO4 18 M, per litre of solution), in the presence of glycerol 15 g/L as a source of energy and carbon. This concentration allows a C:N ratio of 10:1 to be obtained, which is defined as balanced to support the growth and division of yeasts. The pre-inoculation was carried out in 200 mL of medium in 1000L flasks placed at 25 °C on an orbital shaker at 220 rpm. After about 48 hours of growth, the cells were inoculated in a 10L bioreactor at an initial DO660 of 0.2. The operating volume of medium, the same used for the pre-culture, corresponds to 5000mL in the presence of about 25g/L of glycerol. The operating volume of medium used in the bioreactor is 5000mL.

After a fermentation time suitable for the development of an exponential growth phase, the imbalancing phase began, where crude glycerol was added to the medium to reach a final concentration of about 80 g/L. The C:N molar ratio therefore switches to the value of about 50:1 , causing a metabolic variation towards the accumulation of microbial oils in the so-called lipid bodies at the expense of cell divisions, which cannot be performed due to the scarcity of the nitrogen source.

After 150 hours from inoculation, the cells were recovered by centrifugation and subjected to acid lysis (HCI 2M), in order to break the cells themselves, and treatment with 2:1 chloroforr methanol solution and chloroform 100% for the separation of the lipids. The chemical extraction of the oil was carried out according to the following protocol: the cells were dissolved in a hydrochloric acid solution 2 M; the preparation was heated in a thermostated bath for 60 minutes at 95 °C with constant stirring. An equal volume (1 :1 v/v) of a 2:1 chloroforrmmethanol solution was added to the suspension and then it was subjected to centrifugation for 10 minutes at 10000 rpm until separation in phases was obtained; the lower chloroform phase was recovered and 10 mL of 100% chloroform were added to the suspension in order to recover the remaining lipids. The microbial oil obtained from chemical extraction was subjected to a transesterification reaction and subsequent analysis with gas chromatography [SAVI LABORATORI & SERVICE S.r.l., Roncoferraro (MN), Italy]

Figure 15 shows the fermentation profile of L. starkeyi-OLE1-FAD2 with respect to the biomass trend over time and the corresponding substrate consumption.

EXAMPLE 9 Production kinetics of oils in flasks with the oleaginous yeast L. starkevi- OLE1-FAD2 engineered for the modification of the lipid profile of the oil obtained with respect to the wild strain.

The cells of the oleaginous yeast strain L. starkeyi-OLE1-FAD2, engineered for the production of modified lipid oils (OIL 3) were pre inoculated into the medium with the following composition: 1 g yeast extract, 1.31 g (NH )2S04, 0.95 Na₂HP04, 2.7 g KH2PO4, 0.2 g MgS0₄ ^* 7H 0, per litre enriched with mineral stock 100 times concentrated, containing (4 g CaCL ^* 2H2O, 0.55 g FeS04 ^* 7H2O, 0.52 g citric acid, 0.10 g ZnS0₄ ^* 7H2O, 0.076 g MnS04 ^* H2O, 100 microlitres of H2SO4 18 M, per litre of solution), in the presence of glycerol 15 g/L as a source of energy and carbon. This concentration allows a C:N ratio of 10:1 to be obtained, which is defined as balanced to support the growth and division of yeasts. The pre-inoculation was carried out in 100 mL of medium in 500L flasks placed at 25 °C on an orbital shaker at 220 rpm. After 72 hours of growth, the cells were inoculated at an optical density of 3 (OD 660 nm) in 50 mL 20 of medium, the same used for pre-inoculation in the presence of about 100 g/L of glycerol, in 250 mL flasks placed at 25 °C on an orbital shaker at 220 rpm. Cell growth was monitored by measuring OD at 660 nm at regular time intervals. The extracellular concentration of glycerol was determined by HPLC using H2SO4 0.01 M as mobile phase and a Rezex ROA-Organic (Phenomenex) column. After 240 hours from inoculation, the cells were recovered by centrifugation and subjected to acid lysis (HCI 2M), in order to break the cells themselves, and treatment with 2:1 chloroforr methanol solution and chloroform 100% for the separation of the lipids. The chemical extraction of the oil was carried out according to the following protocol: the cells were dissolved in a hydrochloric acid solution 2 M; the preparation was heated in a thermostated bath for 60 minutes at 95 °C with constant stirring. An equal volume (1 :1 v/v) of a 2:1 chloroforrmmethanol solution was added to the suspension and then it was subjected to centrifugation for 10 minutes at 10000 rpm until separation in phases was obtained; the lower phase was recovered and 10 mL of 100% chloroform were added to the suspension in order to recover the remaining lipids. The microbial oil obtained from chemical extraction was subjected to a transesterification reaction and subsequent analysis with gas chromatography [SAVI LABORATORI & SERVICE S.r.l., Roncoferraro (MN), Italy].

Figure 16 shows the composition of fatty acids relating to OIL 3 compared to the composition of OIL 2.

EXAMPLE 10

Construction of the recombinant strain L. starkevi- OLE1 from L. starkevi (DSM70295) for the modification of the lipid profile and for obtaining the microbial oil (OIL 8).

The laboratory strain of L. starkeyi DSM70295 was transformed using the expression cassette containing the putative sequence encoding for the delta9 desaturase activity under the control of the pTDH3 promoter and tPGK1 terminator together with the nurseotricin NsrR resistance cassette, described in Example 2.

EXAMPLE 11

Construction of the recombinant strain L. starkevi- FAD2 from L. starkevi (DSM70295) for the modification of the lipid profile and for obtaining the microbial oil (OIL 9).

The laboratory strain of L. starkeyi DSM70295 was transformed using the expression cassette containing the sequence encoding for the delta12 desaturase activity under the control of the pTDH3 promoter and tPGK1 terminator together with the hygromycin HygR resistance cassette, described in Example 3.

EXAMPLE 12

Evaluation of the number of gene copies inserted in the genome of L. starkevi- OLE1 and L. starkevi- FAD2 through relative quantitative Real Time PCR.

The evaluation of the number of gene copies inserted in the genomes of L. starkeyi- OLE1 and L. starkeyi- FAD2 was carried out through a relative quantitative Real Time PCR, as described in Example 5. Starting from the genomic DNA, extracted from both the parental strains and the engineered strains, the number of copies of OLE1 and FAD2 per cell was quantified. Real-time PCR was performed using specific oligonucleotides (SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27) and actin as internal control (SEQ ID NO: 28; SEQ ID NO: 29) (Figure 17).

EXAMPLE 13

Evaluation of the expression levels of the sequences encoding for delta- 9 desaturase and delta-12 desaturase over-expressed in the L. starkevi- OLE1 and L. starkevi- FAD2 strains by relative quantitative Real Time PCR.

The evaluation of the expression levels of the putative sequence encoding for the enzyme delta-9 desaturase and delta-12 desaturase was performed by relative quantitative Real Time PCR, as described in Example 5.

The messengers for delta-9 desaturase and delta-12 desaturase in the recombinant strains L. starkeyi- OLE1 , L. starkeyi- FAD2 and in the wild strain are quantified from the cDNA obtained by retro-transcription of the total RNA (Figure 18).

The cells were pre-inoculated in 5 ml of the medium containing: Glucose 25%, xylose 25%, 1 g of yeast extract, 1.31 g (NFl4)2S04, 0.95 Na2FIP04, 2.7 g KFI2PO4, 0.2 g MgS04 ^* 7FI2O, per litre enriched with mineral stock 100 times concentrated, containing (4 g CaCl2 ^* 2FI2O, 0.55 g FeS04 ^* 7H2O, 0.52 g citric acid, 0.10 g ZnS0₄ ^* 7H20, 0.076 g MnS0₄ ^* H2O, 100 microlitres of FI2SO4 18 M, per litre of solution) for 24 h. RNA extraction was performed on a sample of cells in the exponential phase, using the ZR Fungal/Bacterial RNA Miniprep kit (Zymoresearch/The epigenitics company). The extraction was then controlled with electrophoretic run on 1.5% agarose gel. The cDNA was obtained using the iScript cDNA Synthesis (BIORAD) kit. Real-time PCR was performed using specific oligonucleotides (SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27) and actin as internal control (SEQ ID NO: 28; SEQ ID NO: 29).

EXAMPLE 14 Production kinetics of oil in flasks of the oleaginous yeasts L. starkevi - OLE1 and L. starkevi -FAD2, engineered for the modification of the lipid profile of the oil with respect to the wild strain.

The cells of the oleaginous yeast strains L. starkeyi-O El and L. starkeyi- FAD2, engineered for the production of oils with a modified lipid profile (OIL 8 and OIL 9) were pre-inoculated in the medium with the following composition: 1 g yeast extract, 1.31 g (NH bO^ 0.95 Na2HP04, 2.7 g KH2PO4, 0.2 g MgS04 ^* 7H2O, per litre enriched with mineral stock 100 times concentrated, containing (4 g CaCL ^* 2H2O, 0.55 g FeS04 ^* 7H2O, 0.52 g citric acid, 0.10 g ZnS04 ^* 7H2O, 0.076 g MnS04 ^* H2O, 100 microlitres of H2SO4 18 M, per litre of solution), in the presence of glycerol 15 g/L as a source of energy and carbon. This concentration allows a C:N ratio of 10:1 to be obtained, which is defined as balanced to support the growth and division of yeasts. The pre-inoculation was carried out in 100 mL of medium in 500L flasks placed at 25 °C on an orbital shaker at 220 rpm. After 72 hours of growth, the cells were inoculated at an optical density of 3 (OD 660 nm) in 50 mL 20 of medium, the same used for pre-inoculation in the presence of about 100 g/L of glycerol, in 250 mL flasks placed at 25 °C on an orbital shaker at 220 rpm. Cell growth was monitored by measuring OD at 660 nm at regular time intervals. The extracellular concentration of glycerol was determined by HPLC using H2SO40.01 M as mobile phase and a Rezex ROA-Organic (Phenomenex) column. After 240 hours from inoculation, the cells were recovered by centrifugation and subjected to acid lysis (HCI 2M), in order to break the cells themselves, and treatment with 2:1 chloroforr methanol solution and chloroform 100% for the separation of the lipids. The chemical extraction of the oil was carried out according to the following protocol: the cells were dissolved in a hydrochloric acid solution 2 M; the preparation was heated in a thermostated bath for 60 minutes at 95 °C with constant stirring. An equal volume (1 :1 v/v) of a 2:1 chloroforrmmethanol solution was added to the suspension and then it was subjected to centrifugation for 10 minutes at 10000 rpm until separation in phases was obtained; the lower phase was recovered and 10 mL of 100% chloroform were added to the suspension in order to recover the remaining lipids. The microbial oil obtained from chemical extraction was subjected to a transesterification reaction and subsequent analysis with gas chromatography [SAVI LABORATORI & SERVICE S.r.l., Roncoferraro (MN), Italy]. Figures 19 and 20 show the compositions of fatty acids relating to OIL 8 and OIL 9 compared with the composition of OIL 3.

LIST OF OLIGONUCLEOTIDE SEQUENCES

SEQ ID NO: 1 OLE1

AT GACTGCCAGTGCT G AG ACAACGT CCGCGCAGCCT GT CGT CG AGT CGGCT CGCGCAAGGCCACCCAG AT CAAGCT CAACGT CGCCTT CT CG TT CGGTTGGT AGT GCTGCGT CG ACTGCG AAGCAAGCGT CT CCT ACAT T CGT CCACAT CT CCG AGCAACCGTT CACT CT CCAG AACTGGT ACAAG CACAT CAGCTGGCT CAAT GT CACGCT GAT CAT CTT CAT CCCT GT CATT GGCTGCACT ACCGCGGTTTT CACT CCT CTGCAAT CT AAG ACTGCCAT CCTTGCCTTT GT CT ACT ACGCCCT GACGGGCCT CGGT AT CACTGCGG GTT AT CACCGCCT CTGGT CGCACCGTGCTT ACAGTGCCCGT CTT CCT CT CCGT ATT CT ACT CGCT GCTTT CGGCGGCGGTGCT GTT GAGGGTT C CATT CGCTGGTGGT CCGCTGGT CACCGT GT CCAT CACAGATT CACCG AT ACT GAG AAGG ACCCTT ACT CT GT CCGCAAGGGT CTGCT CT ATT CT CACATGGGCTGG ATGGT GTTT CT CCACAACCCCAAG AAGT CCGGCC GGGT CG AT AT CACCG ACTT G AACGCT G ACCCT GT CGT CAG ATGGCAG CACAAG AACT ACATT CT CGT CCTT CT CTTT AT GGGTTT CAT CTT CCCC AT GGT AGTTGCCGGCCT CGG ATGGGGT G ACTGG AAGGGTGGT CT CA T CTGGGCTGGCATT GT CCGTTT G ACAGTT GT CCACCATGCCACTTTTT GT GT CAACT CGCT CGCT CACTGGCT CGGT G ACCAGCCTTT CG ACG AC CGCCGCT CT CCGCGT G ACCACTT CTT G ACTGCCAT CGTT ACGTT CGG CG AGGGCT AT CACAACTT CCACCAT G AGTT CCCCT CT G ACT ACCGT A ACGCCAT AAG ATGGT AT CAGT AT GAT CCCACT AAGTGGCT CAT CTGG TT CCT CAAG AAG AT CGGCTTTGCTT AT G ACCTT AAG ACCTT CT CT CAC AATGCCAT CCAGCAAGGCCT CGT CCAGCAG AGGCAG AAAAAGCT CG ACAAGTGGCGCGCACGT CTT AACTGGGGT GTT CCT CT CG AGCAGCT C CCGGT CATGG AATTT G AAGAGT ACCAGGAGCAGGCCAAG ACGCGT G CGCT CGT CCT CATT GCTGGT GTT GT CCACG AT GT CACCAACTTT ATT G AGCAGCAT CCTGGT GG AAAGGCT CT GAT CCAGT CAGGT ATTGGCAAG G ATGCCACCGCT GT CTT CAATGGCGGT GT CT ACG ACCACT CCAATGC TGCCCACAACCTGCT CGGT ACCATGCGT GTTGGT GT CATT CGCGGCG GCAT GG AAGT CGAGGT CT GG AAG AT GGCT CAGCG AG AG AAT AAGG A GT CAACG AT CAAGT CCG ATT CG AAT AATGCCAAT AT CGT CCGTGCAG GTT CT CAGGCAACCCGG AT ACAAGCT CCCAT CCAGGGCGCT GGTGC CGCTTAG

SEQ ID NO: 2 FAD2

AT GT CCACAAT AACAT ACACACAGCGCAGGCCGT CAGT GT CGCT G AC TT CG AAGCCCGT CT ACAAGG ATGCCTT CGGCCACG ACTT CG AACCGC CGG AGT ACACAAT CAAAG AT AT CCTT G ATGCCAT CCCCAAGCACTGC TT CG ACCGCT CT CTT AGCCACT CT CT CGCCT AT GT CGCCCGCG ACCT CTT CT ACGCCT CCTGCTT GTT CGGCCT AGCG ACACAG AT CCAT AGCA T CCCCT AT CT ACCT GCCCGCGT CGT CGCCTGGGTT CT CT ACGGCTTT TGCCAAGGCCTT GT CGGCACAGGCTT GTGGGT CCT CGCCCACG AGT GCGGCCAT GG AGCCTT CT CCCCCT ACAAGCT CGCCAACG ACGT CGT CGGGTGGCT CCT CCACT CCGCCCT CTTT GTGCCGT ACCACT CATGGC GG AT CACT CACT CCAAGCACCACAAAGCCACCGGCCACCT CACCCG CG AT ATGGT CTT CGT CCCGCGCG ACGT G ACCCGTT ACAAGCT CT CCC G AAACCT G ACT G AGCT CACCG AGGAGGCGCCG AT CGCG ACCCT CT A TTT CCT ATTT AT CCAGCAGGT CTTT GGTTGGCCCGCGT ACCT CGCCT A CAAT GT CACCGGCCAG AAAT ACCCTGGT GT GT CCAGCTT CAG ACGGT CACATTTTGCGCCGT CCGCGCCCAT GTT CG AT GT AAAGG ACTT CTGG GAT ATT AT AAT CT CCG AT GT CGGT AT ACTT GT CGCCGGCACT CTT ACC T ATCTCG G CAT CC AG AAAT GGGGTTGGGCT A ACTT CG CTCTCTACT A CTTT AT CCCTT AT CT CTGGGT CAACAACTGGTT GGTTTGCAT CACGT A T CT ACAGCACACT G ACCCAACT CT ACCCCATT ACG ACGCGAGCG AGT GG AACTTTGCCCGCGGCGCCGCGGCAACAGT AG AT CGCG ACTTTGG CTT CAT CGGCCGCCACCT CTT CCACG AGAT CAT CG AG ACGCAT GT CG CGCACCACT ACT CGT CGCG AATT CCATT CT ACCACGCCG AGG AGGCC ACGCAAGCCAT CCGCAAGGT CATGGGCAAGCATT ACCGT CAGG ACA AG ACAAACCT CATT CT CGCACT CTGG AAGACCGCGCGG ACATGCCAT TTT GTGGAGGGCG ACGGCGT CAAG AT GT ACAG AAATGCCAACGGAAT CGGT ATT CCGCCAAAGG AGGGAAGAAGGGCT CAGT AA

SEQ ID NO: 3; pURA3 fw

TCG ATC CAG AAT TCG TGA TTC TAG ACT CGA GAT ATC ATG ATC ACT GAG CGA TAG TTC

SEQ ID NO: 4; pURA3 rev

TCG TCA AGA GTG GTA CCC ATG TTG AAT TTA GGG ATA TAC TGT AGA AGA C

SEQ ID NO: 5;tGAL1 fw

TGA GCA TGC CCT GCC CCT AAA GTT TAG AGA TGT ACA AGG GGT SEQ ID NO: 6; tGAL1 rev

GCT TGT CGA CGA ATT CAG ATT GCC ACG ATA ACT TTG TGC

SEQ ID NO: 7; NsrR fw ATG GGT ACC ACT CTT GAC GAC

SEQ ID NO: 8; NsrR rev

TTA GGG GCA GGG CAT GCT CA

SEQ ID NO: 9; T7 ATT TAG GTG ACA CTA TAG

SEQ ID NO: 10; SP6

TAA TAC GAC TCA CTA TAG GG SEQ ID NO: 11 ; pTDH3 fw

CGT GAT TCT AGA CTC GAG ATT TAA TTT GCT GAA GCG GTT TGC SEQ ID NO: 12; pTDH3 rev

CGC TTA GCG ATA TCA CTA GTA GAT CTT GCG AAT GTG GAT TAG AGT AAG A

SEQ ID NO: 13; p PGK1 fw

ACT AGT GAT ATC GCT AAG CGG CCG GCC CTC CCG TTA ATG TTG GGA TTC

SEQ ID NO: 14; t PGK1 rev

TAT CGC TCA GTG ATC ATG ATC CTG TCA ATT ATG CTA CCA CTT G

SEQ ID NO: 15; LsTDH3 fw ATA ATA ATC CGA ACT GCC GC SEQ ID NO: 16; OLE1 fw

TCA GAT ACT AGT ATG ACT GCC AGT GCT GAG ACA ACG TCC

SEQ ID NO: 17; OLE1 rev

AGA TAC ACT AGT CTA AGC GGC ACC AGC GCC CT

SEQ ID NO: 18; HygR fw

ATG GGT AAAAAG CCT GAA CTC ACC

SEQ ID NO: 19; HygR rev TTA TTC CTT TGC CCT CGG ACG

SEQ ID NO: 20; FAD2 fw

CTA GTA AAC TAG TAT GTC CAC AAT AAC ATA CAC AC SEQ ID NO: 21 ; FAD2 rev

TAT TCT ACT AGT TTA CTG AGC CCT TCT TCC

SEQ ID NO: 22; OLE1 CNT rev CAA CTA CCA TGG GGA AGA TG

SEQ ID NO: 23 FAD2 CNT rev

CAA GTA TAC CGA CAT CGG AGA TTA SEQ ID NO: 24 OLE1 RT fw CCA CTT CTT GAC TGC CAT GC

SEQ ID NO: 25 OLE1 RT rev GAG GAA CCA GAT GAG CCA CT

SEQ ID NO: 26 FAD2 RT fw GAT CGC GAC TTT GGC TTC AT SEQ ID NO: 27 FAD2 RT rev

GAA TTC GCG ACG AGT AGT GG

SEQ ID NO: 28 ACT fw CAT TGC CGA CAG AAT GCA GA

SEQ ID NO: 29 ACT rev

ACG GAG TAC TTA CGC TCA GG

Claims

1. A process of engineering an oleaginous microorganism comprising the following steps of:

(a) providing an oleaginous microorganism;

(d) selecting the resulting engineered microorganism.

2. Process of engineering an oleaginous microorganism according to claim 1 , wherein said oleaginous microorganism is an oleaginous yeast selected from the group consisting of the genera Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon, and Yarrowia.

3. Process of engineering an oleaginous microorganism according to claim 1 , wherein said oleaginous microorganism belongs to strains of the species Cryptococcus curvatus, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon fermentans, and Yarrowia lipolytica.

4. Process of engineering an oleaginous microorganism according to claim 1 , wherein said oleaginous microorganism is Lipomyces starkeyi.

5. Process of engineering an oleaginous microorganism according to claim 1 , wherein said oleaginous microorganism is a microalgae selected from the group consisting of Chlorella ellipsoidea, Chlorella protothecoides, Chlorella vulgaris, Dunaliella sp., Haematococcus pluvialis, Neochloris oleoabundans, Neochloris oleabundans, Pseudochlorococcum sp., Scenedesmus obliquus, Tetraselmis chui, Tetraselmis sp., Tetraselmis tetrathele, Chaetoceros calcitrans CS 178, Chaetoceros gracilis, Chaetoceros muelleri, Nitzschia of. pusilla YSR02, Phaeodactylum tricornutum F&M-M40, Skeletonema sp. CS 252, Thalassiosira pseudonana CS 173 Crypthecodinium cohnii, Isochrysis sp.., Isochrysis sp., Isochrysis zhangjiangensis, Nannochloropsis oculate, Nannochloropsis oculata NCTU-3, Nannochloropsis sp., Pavlova salina CS 49, Rhodomonas sp., and Thalassiosira weissflogii.

6. Process of engineering an oleaginous microorganism according to claim 1 , wherein said oleaginous microorganism is selected from the group consisting of fungi and protists, preferably Aspergullus terreus, Claviceps purpurea, Tolyposporium, Mortierella alpina, Mortierella isabellina, and Schizochitrium limacynum.

7. Process of engineering an oleaginous microorganism according to claim

1 , wherein said gene encoding the enzyme delta-9 desaturase overexpressed in yeast is OLE1 of Lipomyces starkeyi having the sequence (SEQ ID N: 1).

8. Process of engineering an oleaginous microorganism according to claim

1 , wherein said gene encoding the enzyme delta-12 desaturase, overexpressed in yeast is FAD2 of Lipomyces starkeyi having the sequence (SEQ ID N:2).

9. The engineered oleaginous microorganism obtained by the process according to any one of claims 1 to 8, wherein the engineered oleaginous microorganism comprises a carbon flow directed towards the synthesis of oil with a modified lipid profile compared to the wild oleaginous microorganism.

10. A process for the production of a plasticising oil which comprises the following steps:

(a) cultivation of engineered oleaginous microorganisms obtained by the process according to any one of claims 1 to 8 in a culture medium comprising biomass containing a carbon to nitrogen molar ratio between 5 and 20;

(b) imbalance by variation of the carbon to nitrogen molar ratio at values equal to or greater than 30, preferably between 30 and 100; (c) separation of engineered oleaginous microorganisms from the culture medium; and

(d) extraction of the oil from oleaginous microorganisms.

11. Process according to claim 10, wherein said biomass comprises at least one source of organic carbon selected from the group consisting of crude glycerol, molasses, lignocellulose, sugar beet pulp, whey, starch residues, waste water, waste oils, glucose, xylose, arabinose, fructose, galactose, mannose, acetate, and/or a combination thereof.