WO2021198895A1 - Fabrication de composés élastomères comprenant des huiles à action plastifiante obtenue à partir de cellules microbiennes oléagineuses - Google Patents

Fabrication de composés élastomères comprenant des huiles à action plastifiante obtenue à partir de cellules microbiennes oléagineuses Download PDF

Info

Publication number
WO2021198895A1
WO2021198895A1 PCT/IB2021/052611 IB2021052611W WO2021198895A1 WO 2021198895 A1 WO2021198895 A1 WO 2021198895A1 IB 2021052611 W IB2021052611 W IB 2021052611W WO 2021198895 A1 WO2021198895 A1 WO 2021198895A1
Authority
WO
WIPO (PCT)
Prior art keywords
oil
oleaginous
seq
desaturase
microorganism
Prior art date
Application number
PCT/IB2021/052611
Other languages
English (en)
Inventor
Luca Castellani
Luca Giannini
Silvia GUERRA
Paola Branduardi
Raffaella Desiré DI LORENZO
Original Assignee
Pirelli Tyre S.P.A.
Universita' Degli Studi Di Milano Bicocca
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 Tyre S.P.A., Universita' Degli Studi Di Milano Bicocca filed Critical Pirelli Tyre S.P.A.
Priority to EP21722284.3A priority Critical patent/EP4127200A1/fr
Priority to CN202180024254.5A priority patent/CN115698311A/zh
Publication of WO2021198895A1 publication Critical patent/WO2021198895A1/fr

Links

Classifications

    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B60—VEHICLES IN GENERAL
    • B60C—VEHICLE TYRES; TYRE INFLATION; TYRE CHANGING; CONNECTING VALVES TO INFLATABLE ELASTIC BODIES IN GENERAL; DEVICES OR ARRANGEMENTS RELATED TO TYRES
    • B60C1/00—Tyres characterised by the chemical composition or the physical arrangement or mixture of the composition
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B60—VEHICLES IN GENERAL
    • B60C—VEHICLE TYRES; TYRE INFLATION; TYRE CHANGING; CONNECTING VALVES TO INFLATABLE ELASTIC BODIES IN GENERAL; DEVICES OR ARRANGEMENTS RELATED TO TYRES
    • B60C1/00—Tyres characterised by the chemical composition or the physical arrangement or mixture of the composition
    • B60C1/0016—Compositions of the tread
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B60—VEHICLES IN GENERAL
    • B60C—VEHICLE TYRES; TYRE INFLATION; TYRE CHANGING; CONNECTING VALVES TO INFLATABLE ELASTIC BODIES IN GENERAL; DEVICES OR ARRANGEMENTS RELATED TO TYRES
    • B60C1/00—Tyres characterised by the chemical composition or the physical arrangement or mixture of the composition
    • B60C1/0025—Compositions of the sidewalls
    • 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
    • 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
    • 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)
    • C12Y114/19006—DELTA12-fatty-acid desaturase (1.14.19.6), i.e. oleoyl-CoA DELTA12 desaturase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • 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 manufacturing process of elastomeric compounds comprising oils with a plasticising action obtained by means of native or engineered oleaginous microorganisms, cultivated in a culture medium containing biomass, the use of the elastomeric compounds thus obtained for the production of tyres, and tyres containing such compounds.
  • Plasticising or process oils are used in the tyre industry to promote the workability of rubber, reduce the viscosity thereof, ensure good distribution of fillers as well as reduce fuel consumption.
  • 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.
  • properties required to have a good plasticising product are: a) good miscibility/compatibility with the elastomer, depending on the aromaticity and molecular weight as well as on the solubility of the oil; b) stable colour, depending on the chemical composition of the oil; c) resistance to ageing; d) low toxicity, currently regulated by the European directive 2005/69/EC which has prohibited, since 2010, the use of oils containing a quantity of polycyclic aromatic hydrocarbons (PAH) greater than or equal to 10 mg/kg.
  • PAH polycyclic aromatic hydrocarbons
  • Plasticising oils currently used in tyres 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.
  • oils which can be used as plasticisers in the tyre industry can be produced by fermentation of biomass by oleaginous microorganisms.
  • the Applicant has also found a manufacturing process which allows elastomeric compounds to be obtained comprising plasticising oils obtained through the cultivation of oleaginous microorganisms in a culture medium containing biomass with an imbalanced carbo nitrogen molar ratio in favour of carbon, the subsequent separation of the oil thus obtained from the culture medium, and finally the mixing of the oil with the elastomeric compound.
  • 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 cross-linked elastomeric material obtained by cross-linking a cross-linkable elastomeric compound comprising at least one oil obtained from oleaginous yeasts had comparable or even better static mechanical properties than the use of conventional plasticising oils or vegetable oils, further showing improving dynamic mechanics, in particular hysteresis and tan5, predicting a lower rolling resistance of the tyre made with such elastomeric material, and consequently lower fuel consumption and carbon dioxide emissions.
  • a first aspect of the present invention consists in a manufacturing process of an elastomeric compound comprising a plasticising oil comprising the following steps of:
  • the elastomeric compound obtained in the process according to the first aspect of the present invention is used for the manufacture of tyres.
  • the microorganism is an oleaginous yeast of the group comprising the genera Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon, Yarrowia.
  • 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.
  • 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 oleaginous microorganism is an engineered oleaginous microorganism obtained by means of an engineering process of an oleaginous microorganism which comprises the following steps of:
  • 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 relates to an oil for use as a plasticiser in a cross-linkable elastomeric compound, characterised by the following composition expressed as a percentage by weight with respect to the total weight of the fatty acids in the oil (w/w):
  • the total saturated fatty acids represent 30-45% w/w, preferably 30-40% w/w.
  • palmitic acid represents 25-35% w/w, preferably 27-32% w/w.
  • stearic acid represents 4-11% w/w, preferably 4-9% w/w.
  • the total monounsaturated fatty acids represent 35-65% w/w, preferably 45-65% w/w, and more preferably 55-65% w/w.
  • palmitoleic acid represents 2-9% w/w, preferably 3-8% w/w, and more preferably 4-7% w/w.
  • oleic acid represents 35-60% w/w, preferably 45-60% w/w, and more preferably 50-60% w/w.
  • the total polyunsaturated fatty acids represent 2-20% w/w, preferably 3-15% w/w, and more preferably 3-10% w/w.
  • linoleic acid represents 2-15% w/w, preferably 2-10% w/w, and more preferably 2-5% w/w.
  • the present invention relates to a tyre for vehicle wheel comprising at least one component of said tyre comprising a cross-linked elastomeric material obtained by cross-linking a cross-linkable elastomeric compound comprising at least one oil obtained from oleaginous microorganisms starting from biomass.
  • 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.
  • the cross-linkable elastomeric compound comprises the elastomeric compound obtained by the process according to the first aspect of the present invention.
  • the cross-linkable elastomeric compound comprises at least one oil obtained from an engineered oleaginous microorganism.
  • the cross-linkable elastomeric compound comprises an oil having the composition defined in the second aspect of the present invention.
  • 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 pl_S03, bearing the expression cassette of the gene for resistance to hygromycin B (FlygR) 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 coding 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 delta-9 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 delta-12 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 pLS04-OLE1 (Photo A) or pLS04-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);
  • Figure 21 shows a cross half-section showing a tyre for motor vehicle wheels according to an embodiment of the fifth aspect of the present invention.
  • Tyre 100 for four-wheeled vehicles comprises at least one carcass structure, comprising at least one carcass layer 101 made of an elastomeric compound having respectively opposite end flaps engaged with respective annular anchoring structures 102, referred to as bead cores, possibly associated to a bead filler 104.
  • the tyre area comprising the bead core 102 and the filler 104 forms a bead structure 103 intended for anchoring the tyre onto a corresponding mounting rim, not shown.
  • An anti-abrasive strip 105 made with an elastomeric compound is arranged in an outer position of each bead structure 103.
  • a protective layer 121 consisting of a plurality of cords incorporated within an elastomeric compound rubber layer, generally known as “chafer”, can be added between the at least one carcass layer 101 and the anti-abrasive strip 105.
  • the carcass structure is associated to a belt structure 106 comprising one or more belt layers 106a, 106b placed in radial superposition with respect to one another and with respect to the carcass layer, having typically textile and/or metallic reinforcement cords incorporated within a layer of elastomeric compound.
  • At least one zero degree reinforcement layer 106c can be applied, which incorporates typically textile and/or metal reinforcement cords incorporated within a layer of elastomeric compound.
  • a tread band 109 of elastomeric compound is applied in a position radially outer to the belt structure 106.
  • respective sidewalls 108 of elastomeric compound are applied in an axially outer position on the lateral surfaces of the carcass structure, each extending from one of the lateral edges of the tread 109 at the respective bead structure 103.
  • An under-layer 111 of elastomeric compound can be arranged between the belt structure 106 and the tread band 109.
  • the elastomeric compound comprising at least one oil obtained from oleaginous microorganisms starting from biomass according to the first aspect of the present invention can be advantageously incorporated into one or more of the components of the tyre selected from belt structure, carcass structure, tread band, underlayer, sidewall, mini-sidewall, sidewall insert, bead, flipper, chafer, sheet and anti-abrasive strip, it is preferably incorporated at least in the tread band 109, in the sidewall 108, in the mini sidewall 110, and/or in the under-layer 111.
  • the elastomeric polymer which may be used in the present invention can be selected from those commonly used in sulphur cross-linkable elastomeric materials, which are particularly suitable for producing tyres, i.e. from elastomeric polymers or copolymers with an unsaturated chain having a glass transition temperature (Tg) generally less than 20°C, preferably within the range of 0°C to -110°C.
  • Tg glass transition temperature
  • These polymers or copolymers may be of natural origin or may be obtained by polymerization in solution, emulsion polymerization or polymerization in gaseous phase of one or more conjugated diolefins, optionally mixed with at least one comonomer selected from monovinylarenes and/or polar comonomers in an amount not higher than 60% by weight.
  • the diene elastomeric polymer which can be used in the present invention can be selected, for example, from: cis-1 ,4-polyisoprene (natural or synthetic, preferably natural rubber), 3,4-polyisoprene, polybutadiene (in particular polybutadiene with a high content of 1 ,4-cis), optionally halogenated isoprene/isobutene copolymers, 1 ,3-butadiene/acrylonitrile copolymers, styrene/1 ,3-butadiene copolymers, styrene/isoprene/1 ,3- butadiene copolymers, styrene/1 ,3-butadiene/acrylonitrile copolymers, and mixtures thereof.
  • cis-1 ,4-polyisoprene natural or synthetic, preferably natural rubber
  • 3,4-polyisoprene polybutadiene (in particular poly
  • elastomeric compound refers to the product obtained by mixing and, optionally, heating at least one elastomeric polymer with at least one of the additives commonly used in the preparation of tyre compounds.
  • additives commonly used in the preparation of tyre compounds are represented by (a) reinforcing fillers, such as carbon black and/or silica, (b) coupling agents, typically comprising a silane group, (c) vulcanising agents, such as sulphur or sulphur derivatives, (d) accelerating agents, such as dithiocarbamates, guanidine, thiourea, thiazoles, sulfenamides, thiurams, amines, xanthates and mixtures thereof, (e) activators, typically zinc and/or zinc compounds, (f) retardant agents, (g) antioxidants, (h) anti-ageing agents, (i) adhesives, (I) anti-ozone agents, (m) modifying resins, or mixtures thereof.
  • reinforcing fillers such as carbon black and/or silica
  • coupling agents typically comprising a silane group
  • vulcanising agents such as sulphur or sulphur derivatives
  • 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.
  • EXAMPLE 1 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 (2M HCI), in order to break the cells themselves, and treatment with 2:1 chloroforr methanol solution and chloroform 100% for the separation of the lipids.
  • acid lysis 2M HCI
  • 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 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 FeS0 4 * 7FI2O, 0.52 g citric acid, 0.10 g ZnS0 4 * 7FI2O, 0.076 g MnS0 4 * FI2O, 100 microlitres of Fl2S0 4 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 delta-9 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.
  • the vector named pLS01 -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 pLS01 -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 vector pLS02 was purified on a column using NucleoSpin Gel and PCR clean-up kit (MACFIEREY-NAGEL GmbFI & 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 delta-9 desaturase activity ( OLE1 ).
  • This example describes the procedure for preparing the expression cassette containing the sequence encoding for the delta-12 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.
  • 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 (2016): 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.
  • 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 delta-12 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 delta-9 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 delta-12 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 * 7H2O, 0.52 g citric acid, 0.10 g ZnS0 4 * 7H20, 0.076 g MnS0 4 * H2O, 100 microlitres of H2SO4 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 (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 * 7FI2O, 0.52 g citric acid, 0.10 g ZnS04 * 7FI2O, 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 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 FIPLC using FI2SO40.01 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. 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.
  • 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).
  • 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 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 * 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 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 FeS04 * 7FI2O, 0.52 g citric acid, 0.10 g ZnS04 * 7FI2O, 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 imL 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.
  • 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 delta- 9 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 delta-12 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 (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 strains L. starkeyi- OLE1 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 (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 * 7FI2O, 0.52 g citric acid, 0.10 g ZnS04 * 7FI2O, 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
  • 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.
  • Figures 19 and 20 show the compositions of fatty acids relating to OIL 8 and OIL 9 compared with the composition of OIL 3.
  • Cross-linkable elastomeric compounds (1-2 and 4-7) were prepared in the laboratory in a 0.05 litre Brabender using OIL 1 (Compound 1) and OIL 2 (Compound 2).
  • OIL 1 Compound 1
  • OIL 2 Compound 2
  • the characterisation results were compared with compound variants using castor oil (Compound 4), sunflower oil AP-75 (Cargill®) (Compound 5), sunflower oil AP-88 (Cargill®) (Compound 6) and MES mineral oil (Compound 7).
  • BR 60 Mooney 63 Polybutadiene manufactured by Versalis Silica Ultrasil 7000: Silica manufactured by Evonik TESPT+N-234: Bis(triethoxysilylpropyl)tetrasulfide (TESPT 50%) supported on carbon black (N-23450%)
  • OIL 1 oil obtained from R. toruloides DSM4444 (Example 1 A)
  • OIL 2 oil obtained from L. starkeyi DSM70295 (Example 1 B)
  • OIL 4 castor oil
  • OIL 5 sunflower oil AP-75 ® (Cargill)
  • OIL 6 sunflower oil AP-88 ® (Cargill)
  • OIL 7 MES mineral oil
  • 6PPD N-(1 ,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine
  • TBZTD Tetrabenzylthiuram disulfide
  • TBBS N-tert-butyl-benzothiazol sulfonamide
  • Table 5 shows a comparison of the results of the static and dynamic properties of the compounds in Table 4.
  • Cross-linkable elastomeric compounds 13 and 6bis-7bis were prepared in the laboratory in a 0.05 litre Brabender using OIL 3 (Compound 3). The results of the characterisation were compared with compound variants that use OIL 6 [sunflower oil AP-88 ® (Cargill) - Compound 6bis] and OIL 7 (mineral oil MES - Compound 7bis).
  • SLR3402 styrene butadiene rubber (15% styrene, 30% vinyl)
  • Silica Ultrasil 7000 Silica manufactured by Evonik Silane JH75S: Mixture of Bis(triethoxysilylpropyl)tetrasulfide (TESPT 50%) supported on carbon black (50%)
  • OIL 7 MES mineral oil
  • OIL 6 sunflower oil AP-88 (Cargill®)
  • OIL 3 oil obtained from L. starkeyi-OLE1 -FAD2 (Example 7)
  • TMQ 2,2,4-Trimethyl-1 ,2-Dihydroquinoline;
  • 6PPD N-(1 ,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine
  • TBZTD Tetrabenzylthiuram disulfide
  • TBBS N-tert-butyl-benzothiazol sulfonamide
  • Compound 3 also has E’ values greater than the E' values of compound 6bis with AP88 vegetable oil, both at 23°C and 70°C, while at the same time exhibiting lower Tanb values at 70°C.
  • OIL 3 is therefore able to give low hysteresis at high T, maintaining high modules at high temperatures, optimal conditions for obtaining a low rolling resistance of the tyre which therefore leads to lower fuel consumption and consequently a reduction in CO2 emission.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Mechanical Engineering (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de fabrication d'un composé élastomère comprenant une huile plastifiante obtenue au moyen de microorganismes oléagineux natifs ou modifiés, cultivé dans un milieu de culture contenant de la biomasse, à faire varier le rapport molaire carbone/azote égal ou supérieur à 30 l'invention concerne également l'utilisation des composés élastomères ainsi obtenus pour la production de pneus, et des pneus contenant de tels composés.
PCT/IB2021/052611 2020-03-31 2021-03-30 Fabrication de composés élastomères comprenant des huiles à action plastifiante obtenue à partir de cellules microbiennes oléagineuses WO2021198895A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP21722284.3A EP4127200A1 (fr) 2020-03-31 2021-03-30 Fabrication de composés élastomères comprenant des huiles à action plastifiante obtenue à partir de cellules microbiennes oléagineuses
CN202180024254.5A CN115698311A (zh) 2020-03-31 2021-03-30 包含从含油微生物细胞获得的具有增塑作用的油的弹性体化合物的制备

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT102020000006688 2020-03-31
IT202000006688 2020-03-31

Publications (1)

Publication Number Publication Date
WO2021198895A1 true WO2021198895A1 (fr) 2021-10-07

Family

ID=71452548

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2021/052611 WO2021198895A1 (fr) 2020-03-31 2021-03-30 Fabrication de composés élastomères comprenant des huiles à action plastifiante obtenue à partir de cellules microbiennes oléagineuses

Country Status (3)

Country Link
EP (1) EP4127200A1 (fr)
CN (1) CN115698311A (fr)
WO (1) WO2021198895A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010017610A1 (fr) * 2008-08-13 2010-02-18 Universidade Estadual De Campinas - Unicamp Procédé de production microbienne de lipides et composition comprenant ces lipides
US20110059204A1 (en) * 2003-05-07 2011-03-10 E.I. Du Pont De Nemours And Company Production of polyunsaturated fatty acids in oleaginous yeasts
US20160265009A1 (en) * 2015-03-11 2016-09-15 The United States Of America, As Represented By The Secretary Of Agriculture Methods and yeast strains for conversion of lignocellulosic biomass to lipids and carotenoids
US20180223082A1 (en) * 2015-07-31 2018-08-09 Compagnie Generale Des Etablissements Michelin Rubber composition including a hydrocarbon resin with a low glass transition temperature
US20190352490A1 (en) * 2014-12-23 2019-11-21 Bridgestone Americas Tire Operations, Llc Oil-containing rubber compositions and related methods

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110059204A1 (en) * 2003-05-07 2011-03-10 E.I. Du Pont De Nemours And Company Production of polyunsaturated fatty acids in oleaginous yeasts
WO2010017610A1 (fr) * 2008-08-13 2010-02-18 Universidade Estadual De Campinas - Unicamp Procédé de production microbienne de lipides et composition comprenant ces lipides
US20190352490A1 (en) * 2014-12-23 2019-11-21 Bridgestone Americas Tire Operations, Llc Oil-containing rubber compositions and related methods
US20160265009A1 (en) * 2015-03-11 2016-09-15 The United States Of America, As Represented By The Secretary Of Agriculture Methods and yeast strains for conversion of lignocellulosic biomass to lipids and carotenoids
US20180223082A1 (en) * 2015-07-31 2018-08-09 Compagnie Generale Des Etablissements Michelin Rubber composition including a hydrocarbon resin with a low glass transition temperature

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ANGERBAUER ET AL: "Conversion of sewage sludge into lipids by Lipomyces starkeyi for biodiesel production", BIORESOURCE TECHNOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 99, no. 8, 7 February 2008 (2008-02-07), pages 3051 - 3056, XP022460644, ISSN: 0960-8524, DOI: 10.1016/J.BIORTECH.2007.06.045 *

Also Published As

Publication number Publication date
EP4127200A1 (fr) 2023-02-08
CN115698311A (zh) 2023-02-03

Similar Documents

Publication Publication Date Title
CN101679990B (zh) △8去饱和酶及其在制备多不饱和脂肪酸中的用途
JP7443656B2 (ja) レチニルエステルの生産
EP2328400B1 (fr) Événement transgénique de soja t mon87705 et procédés pour la détection de celui-ci
CN101627118B (zh) 由靶向诱变工程化的突变型△8去饱和酶基因及其在制备多不饱和脂肪酸中的用途
JP7472427B2 (ja) レチノイドの生合成
CA2870364A1 (fr) Microbes recombinants renfermant des enzymes de passage synthetiques d'acides gras modifies
WO2014176515A2 (fr) Huiles à teneur faible en acides gras polyinsaturés et leurs utilisations
WO2020141168A1 (fr) Nouvelles acétyl-transférases
AU2014236763B2 (en) Thioesterases and cells for production of tailored oils
EP4085132A1 (fr) Souche modifiée par une lipase
CN107636155A (zh) 红冬孢酵母属和红酵母属的d‑氨基酸诱导型基因表达系统
US10081821B2 (en) Methods for efficient production of polyunsaturated fatty acids (PUFA) in Rhodosporidium and Rhodotorula species
CA3177968A1 (fr) Production a grande echelle d'olivetol, d'acide olivetolique et d'autres alkylresorcinols par fermentation
Prasad et al. Stable nuclear transformation of rhodophyte species Porphyridium purpureum: advanced molecular tools and an optimized method
WO2023220060A1 (fr) Lyse enzymatique pour l'extraction de bioproduits à partir de levure
US10308691B2 (en) Methods for tuning carotenoid production levels and compositions in rhodosporidium and rhodotorula genera
WO2013034648A1 (fr) Augmentation de la teneur en lipides de micro-algues par manipulation génétique d'une triglycéride (tag) lipase
WO2021198895A1 (fr) Fabrication de composés élastomères comprenant des huiles à action plastifiante obtenue à partir de cellules microbiennes oléagineuses
WO2021198904A1 (fr) Procédé de fabrication d'un micro-organisme oléagineux
WO2021100419A1 (fr) Protéine de fusion, procédé de production de substance, vecteur, cellule transformée, procédé de fabrication de pneumatique et procédé de fabrication de produit en caoutchouc
DI LORENZO Tailored Bioplasticizer for elastomeric compounds from sustainable biomass
JP2020036560A (ja) ポリイソプレノイドの製造方法、ベクター、形質転換植物、空気入りタイヤの製造方法及びゴム製品の製造方法
WO2014157736A1 (fr) Promoteur manifestant une activité d'expression élevée chez les micro-organismes du genre mortierella
JP2020534806A (ja) レチニルエステルの生産
US20230167465A1 (en) Method for producing polyisoprenoid, vector, transformed plant, method for producing pneumatic tire, and method for producing rubber product

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21722284

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021722284

Country of ref document: EP

Effective date: 20221031