EP4127201A1 - Procédé de fabrication d'un micro-organisme oléagineux - Google Patents

Procédé de fabrication d'un micro-organisme oléagineux

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Publication number
EP4127201A1
EP4127201A1 EP21722286.8A EP21722286A EP4127201A1 EP 4127201 A1 EP4127201 A1 EP 4127201A1 EP 21722286 A EP21722286 A EP 21722286A EP 4127201 A1 EP4127201 A1 EP 4127201A1
Authority
EP
European Patent Office
Prior art keywords
oleaginous
oleaginous microorganism
seq
oil
engineering
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21722286.8A
Other languages
German (de)
English (en)
Inventor
Paola Branduardi
Raffaella Desiré DI LORENZO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pirelli and C SpA
Pirelli Tyre SpA
Original Assignee
Pirelli SpA
Pirelli Tyre SpA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pirelli SpA, Pirelli Tyre SpA filed Critical Pirelli SpA
Publication of EP4127201A1 publication Critical patent/EP4127201A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
    • C12Y114/19001—Stearoyl-CoA 9-desaturase (1.14.19.1), i.e. DELTA9-desaturase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • 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.
  • 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.
  • 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).
  • MES mild extraction solvent
  • TDAE treated distillate aromatic extracts
  • NAP naphthenic oil
  • RAE Residual Aromatic Extract
  • 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.
  • biorefinery there are often bioprocesses, or transformations carried out by living organisms or enzymatic activities derived from them, accompanied by sustainable chemical processes.
  • homogeneous and easily transformable substrates such as sugars in monomeric form or starches: in this case the biorefineries are defined as first-generation.
  • 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.
  • 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.
  • microorganisms it is possible to use microorganisms to transform residual biomass into compounds of interest, including oils.
  • 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.
  • 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).
  • 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.
  • 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.
  • a first aspect of the present invention consists in a process of engineering an oleaginous microorganism comprising the following steps:
  • the microorganism is an oleaginous yeast of the group comprising the genera Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon, Yarrowia.
  • 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.
  • 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.
  • 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.
  • the gene encoding the enzyme delta-9 desaturase overexpressed in yeast is OLE1 of Lipomyces starkeyi having the sequence (SEQ ID NO: 1).
  • the gene encoding the enzyme delta-12 desaturase overexpressed in yeast is FAD2 of Lipomyces starkeyi having the sequence (SEQ ID NO: 2).
  • the oleaginous microorganism is Lipomyces starkeyi.
  • 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.
  • 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:
  • 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.
  • 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);
  • HygR hygromycin B
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the coding sequence must be correctly and functionally connected to regulatory elements of the transcription, functioning in the selected expression host.
  • transformation means that DNA, once introduced into the cell, can replicate outside chromosomes or as part of a chromosome.
  • 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.
  • oleaginous microorganism refers to a microorganism capable of accumulating at least 20% of lipids with respect to its dry weight.
  • 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.
  • 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.
  • OIL 1 and OIL 2 The microbial oils with plasticising action (OIL 1 and OIL 2) were produced according to the procedures described below.
  • 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.
  • 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 Na 2 HP04, 2.7 g KH2PO4, 0.2 g MgS0 4 * 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 corresponds to lOOOmL in the presence of about 40 g/L of glycerol.
  • 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.
  • 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.
  • HCI 2M acid lysis
  • 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.
  • 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.
  • 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 corresponds to 1000 mL in the presence of about 60 g/L of glycerol.
  • 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.
  • 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.
  • 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.
  • HCI 2M acid lysis
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ).
  • 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 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 ZnS0 4 * 7H20, 0.076 g MnS0 4 * 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).
  • 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 ) 2 S04, 0.95 Na 2 HP0 4 , 2.7 g KH2PO4, 0.2 g MgS0 4 * 7H 0, per litre enriched with mineral stock 100 times concentrated, containing (4 g CaCl2 * 2H2O, 0.55 g FeS0 4 * 7H 2 0, 0.52 g citric acid, 0.10 g ZnS0 4 * 7H 2 0, 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.
  • 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.
  • 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.
  • HCI 2M acid lysis
  • 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.
  • 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.
  • 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.
  • 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 Na 2 HP04, 2.7 g KH2PO4, 0.2 g MgS0 4 * 7H 0, per litre enriched with mineral stock 100 times concentrated, containing (4 g CaCL * 2H2O, 0.55 g FeS0 4 * 7H 2 0, 0.52 g citric acid, 0.10 g ZnS0 4 * 7H 2 0, 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.
  • 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 corresponds to 5000mL in the presence of about 25g/L of glycerol.
  • the operating volume of medium used in the bioreactor is 5000mL.
  • 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.
  • 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.
  • 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.
  • HCI 2M acid lysis
  • 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.
  • Figure 15 shows the fermentation profile of L. starkeyi-OLE1-FAD2 with respect to the biomass trend over time and the corresponding substrate consumption.
  • 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 Na 2 HP04, 2.7 g KH2PO4, 0.2 g MgS0 4 * 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 ZnS0 4 * 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.
  • 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.
  • HCI 2M acid lysis
  • 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.
  • Figure 16 shows the composition of fatty acids relating to OIL 3 compared to the composition of OIL 2.
  • 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.
  • 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.
  • 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 ZnS0 4 * 7H20, 0.076 g MnS0 4 * 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.
  • HCI 2M acid lysis
  • 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.

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Abstract

La présente invention concerne un procédé de fabrication d'un micro-organisme oléagineux, un micro-organisme oléagineux modifié obtenu à partir d'un tel procédé, ainsi qu'un procédé de production d'une huile plastifiante obtenue à partir d'un tel micro-organisme oléagineux modifié par mise en culture dans un milieu de culture contenant de la biomasse.
EP21722286.8A 2020-03-31 2021-03-30 Procédé de fabrication d'un micro-organisme oléagineux Pending EP4127201A1 (fr)

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