WO2017042413A1 - Production d'huiles microbiennes à teneur élevée en acides gras à chaîne très longue - Google Patents

Production d'huiles microbiennes à teneur élevée en acides gras à chaîne très longue Download PDF

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WO2017042413A1
WO2017042413A1 PCT/ES2016/070634 ES2016070634W WO2017042413A1 WO 2017042413 A1 WO2017042413 A1 WO 2017042413A1 ES 2016070634 W ES2016070634 W ES 2016070634W WO 2017042413 A1 WO2017042413 A1 WO 2017042413A1
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acid
fatty acids
microorganism
gene
long chain
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PCT/ES2016/070634
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English (en)
Spanish (es)
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Sandy Christiane FILLET
Mª del Carmen RONCHEL BARRENO
Beatriz SUÁREZ GONZÁLEZ
Javier VELASCO ÁLVAREZ
José Luis Adrio Fondevila
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Neol Biosolutions, S.A.
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Publication of WO2017042413A1 publication Critical patent/WO2017042413A1/fr

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    • 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

Definitions

  • the present invention relates to the production of microbial oils with a high content of very long chain fatty acids that have at least 20 carbon atoms, by culturing a microorganism in the presence of different carbon sources.
  • Oleochemicals constitute a class of aliphatic molecules derived from lipids. Oleochemicals are used in a wide range of applications including transportation fuels, consumer products (eg cosmetics, shampoos, gels, etc.), and industrial products (eg paints, lubricants, bioplastics, coatings, etc. ) (Biermann et al. 2011. Angew Chem Int Ed, 50 3854-3871). The most common oleochemicals are surfactants, lubricants and biodiesel.
  • oleochemicals such as used oils, fatty fatty residues, or lipids of microbial origin (algae or other microorganisms)
  • lipids of microbial origin algae or other microorganisms
  • microorganisms represent enormous potential since they are capable of producing lipids from a wide variety of raw materials such as starchy sugars, lignocellulosic sugars, carbon dioxide or natural gas, among others (Keasling. 2010. Science, 330: 1355-1358; Lennen and Vietnameser. 2013. Curr Opin Biotechnol, 24: 1044-1053.).
  • Vegetable oils are the most used raw materials for the production of oleochemicals.
  • AGCML Very long chain fatty acids
  • Erucic acid is of great interest since it is a valuable raw material with more than 1,000 patented industrial applications.
  • the main derivative of this acid is erucamide, which is used as a surface activation additive in coatings and in the production of plastic films.
  • behenic acid such as, for example, high temperature lubricants, high molecular weight anionic surfactants, viscoelastic surfactants, EOR surfactants, detergents, plastic and adhesive coatings, gels and epoxy resins, cosmetic formulations, photography and pharmacy.
  • Nerve acid (cis-tetracosa-15-enoic acid; 24: 1 ⁇ 15) is another strategic AGCML that exists in nature as a product of elongation of oleic acid (18: 1 ⁇ 9), with its immediate precursor being erucic acid.
  • Nerve acid has been identified in the seed oils of very few plants such as Lunaria annua (silver plant), Borago officinalis (borage), Cannabis sativa (hemp), Acer truncatum (maple), Tropaeolum speciosum (flame flower) and Cardamine graeca (bitter cress). This acid is also used in industrial applications already mentioned for erucic acid.
  • Two potential solutions include the production of lipids in photoautotrophic algae or the conversion of plant biomass using genetically modified microorganisms.
  • the technologies developed for the production of cellulosic ethanol can be used for the production of biodiesel and oleochemicals with the help of new microbial catalysts.
  • Oleochemicals are synthesized via enzymatic reactions that use free fatty acids or acyl thioesters as substrates. Therefore, the strategies for the production of oleochemicals in microorganisms begin by redirecting the carbon flux of fatty acid metabolism towards the desired product.
  • Oleaginous microorganisms that include bacteria, yeasts, cyanobacteria, microalgae and filamentous fungi are defined by their ability to accumulate at least 20% of their dry weight in the form of lipids (Ratledge and Wynn. 2002. Avd Appl Microbiol, 51: 1-51; Ratledge 2004. Biochemie, 86: 807-815). This characteristic makes these microorganisms considered as very attractive candidates to be used as host strains for the production of oils or compounds derived from them as fatty acids or alcohols.
  • Oleaginous bacteria have been the least studied to date because their lipid content is relatively lower than in yeasts, cyanobacteria, microalgae and filamentous fungi because they are limited by their low growth rates.
  • the cyanobacteria and oleaginous microalgae are attractive hosts for the production of oils and fatty acid derivatives mainly because of their photosynthetic capacity that allows them to convert solar energy and recycle C0 2 .
  • both types are technically difficult to manipulate and their culture and growth processes are more complicated than those of bacteria, yeasts and fungi. These difficulties are those that have prevented its use in the production of fatty acid derived compounds through metabolic engineering.
  • the exploitation of oleaginous filamentous fungi as production organisms has also been impeded by the lack of efficient transformation techniques.
  • the authors of the present invention have genetically modified a microorganism of the Rhodosporidium toruloides strain, by inserting heterologous genes encoding one or more enzymes with Ci 8 3-ketoacyl-CoA synthase activity that allows to produce oils with a high fatty acid content Very long chain (equal to or greater than 20 carbon atoms).
  • the production of oil by using said microorganism is above 50 g / L and the content of very long chain fatty acids is greater than 35% of the total fatty acids present.
  • the present invention relates to a microorganism of the genetically modified Rhodosporidium toruloides species with at least one gene encoding an enzyme with Ci 8 3-ketoacetyl-CoA synthase activity.
  • the present invention relates to a process for obtaining a microbial biomass enriched in oil rich in very long chain fatty acids comprising: i) culturing the microorganism of the invention in a culture medium comprising at least one source of carbon and at least one source of nitrogen under conditions suitable for the growth of said microorganism; Y
  • the invention relates to a microbial biomass enriched in oil rich in very long chain fatty acids obtained by the process to obtain a microbial biomass enriched in oil rich in very long chain fatty acids of the invention.
  • the present invention relates to a process for obtaining a preparation enriched in oil rich in very long chain fatty acids comprising:
  • the present invention relates to a process for obtaining biolubricants comprising: i) obtaining a microbial biomass enriched in oil rich in very long chain fatty acids wherein said obtaining is carried out according to the procedure to obtain a preparation enriched in very long chain fatty acid rich oil of the present invention;
  • step (iii) convert the oil rich in very long chain fatty acids obtained in step (ii) into biolubricants.
  • the invention relates to the use of the microorganism of the invention to obtain a microbial biomass enriched in oil rich in very long chain fatty acids.
  • the present invention also relates to the use of the microorganism of the invention to obtain a preparation enriched in oil rich in very long chain fatty acids.
  • the present invention also relates to the use of the microorganism of the invention to obtain biolubricants.
  • FIG. 1 Integrative cassettes in Rhodosporidium toruloides
  • the present invention relates to a microorganism, hereinafter "microorganism of the invention", of the genetically modified Rhodosporidium toruloides species with at least one gene encoding an enzyme with Cie 3-ketoacyl-CoA synthase activity.
  • gene refers to a deoxyribonucleotide chain that encodes a protein.
  • the term refers to a chain of deoxyribonucleotides that encodes an enzyme with 3-ketoacyl d-8 CoA synthase.
  • enzyme in the context of the present invention, refers to a protein that functions as a highly selective catalyst, accelerating both the speed and the specificity of the metabolic reaction for which it is specific.
  • the microorganism of the invention is a genetically modified microorganism.
  • the enzyme having Ci 8 3-ketoacyl CoA synthase activity may be encoded by a gene encoding said enzyme in another organism.
  • the gene encoding an enzyme having Ci 8 3-ketoacyl-CoA synthase activity can be introduced into the microorganism in any suitable form, for example, comprised in a vector, a plasmid or as a naked nucleic acid.
  • the gene can be expressed exogenously if it is expressed in a vector / plasmid in the microorganism [ie, outside the microbial chromosome (s)], or it can be incorporated in the microbial chromosome (s) by random (ectopic) or homologous recombination or any other suitable method known in the state of the art.
  • Appropriate techniques that allow genetic transformation in yeasts include but are not limited to:
  • Gene bombardment that involves bombarding cells with microprojectiles coated with exogenous DNA.
  • Electroporation which involves administering electrical pulses to yeasts that produces the opening of pores in the spheroplast membrane and intact yeast cells.
  • Agrobacterium tumefaciens-mediated transformation is based on the use of the gene transfer capacity that A. tumefaciens naturally possesses.
  • Transformants are grown in a suitable nutrient medium and under selection conditions to ensure retention of endogenous DNA.
  • the insertion of the gene encoding a Ci 8 3-ketoacyl-CoA synthase in said transformants can determined by any appropriate molecular biology technique, for example, by Southern blot or PCR. Conventional methods of detection and quantification of the expression of a gene can be found, for example, in Sambrook et al, 2001. "Molecular cloning: a Laboratory Manual.”, 3rd ed, Cold Spring Harbor Laboratory Press, NY, Vol.. 1-3.
  • acyl-Coa elongasa acyl-Coa elongasa
  • beta-ketoacyl CoA synthase 3-oxoacil CoA synthase very long chain ", or” fatty acid elongase "as used herein, refers to a polypeptide belonging to the family of EC enzymes 2.3.1.199 and which catalyzes the addition of two carbons to saturated or unsaturated fatty acids of 18-26 carbons at their carboxyl end.
  • the enzyme with Ci 8 3-ketoacyl-CoA synthase activity of the invention is capable of elongating saturated or unsaturated fatty acids of at least 18 carbons into two carbon atoms to give rise to the corresponding fatty acids.
  • said enzyme elongates saturated or unsaturated fatty acids in two carbon atoms whose hydrocarbon chain comprises 18 to 26 carbons.
  • the enzyme with Ci 8 3-ketoacyl-CoA synthase activity catalyzes the aforementioned elongation reaction using as a single substrate monounsaturated fatty acids of at least 18 carbons.
  • said enzyme uses oleic acid as the sole substrate, that is, it has high specificity for said substrate, gondoic acid being obtained as a product of the elongation reaction.
  • said enzyme employs gondoic acid as the sole substrate, that is, it has high specificity for said substrate, obtaining erucic acid as a product of the elongation reaction.
  • said enzyme employs erucic acid as the sole substrate, that is, it has high specificity for said substrate, obtaining nerve acid as a product of the elongation reaction.
  • enzymes with Ci 8 3-ketoacyl-CoA synthase activity suitable for use in the present invention also include those enzymes that have specificity for more than one substrate, in which case they have a substantially higher specificity over saturated and unsaturated fatty acids, preferably monounsaturated, of 18 carbon atoms or more, than those of smaller length (for example, fatty acids of 14-16 carbon atoms).
  • enzymes with Ci 8 3-ketoacyl-CoA synthase activity suitable for use in the present invention include those that show a specificity against saturated fatty acids of at least 18 carbons which is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, or more with respect to acid specificity fatty acids of less than 18 carbons, for example, against fatty acids of 14-16 carbon atoms.
  • This definition also includes enzymes that catalyze the addition of two carbon atoms to a monounsaturated or polyunsaturated fatty acid of at least 18 carbons with a specificity greater than the specificity they present for a monounsaturated or polyunsaturated fatty acid of 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16 or 17 carbons.
  • specificity refers to the efficiency with which the enzyme transforms a particular substrate into the reaction product.
  • greater specificity refers to the efficiency with which the enzyme of the invention transforms a fatty acid of at least 18 carbons, preferably a fatty acid of 18 to 26 atoms.
  • carbon and in particular, oleic acid, gondoic acid and / or erucic acid, in the corresponding reaction products, that is, in gondoic acid, erucic acid and nerve acid, respectively, is at least 1.5 times at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times , at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times or more, greater than the specificity of said enzyme for a fatty acid whose length is less than 18 carbons, in particular for a 16-carbon fatty acid, such as p or, for example, a fatty acid of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 or 15 carbons.
  • an enzyme with Ci 8 3-ketoacyl-CoA synthase activity is specific against oleic acid and its substrate specificity is higher than that observed against a substrate with a different number of carbon atoms and present the same number of unsaturations as oleic acid.
  • an enzyme with Ci 8 3- ketoacyl-CoA synthase activity is specific against gondoic acid and its substrate specificity is higher than that observed against a substrate with a different number of carbon atoms and present the same number of unsaturations as gondoic acid.
  • an enzyme with Ci 8 3- ketoacyl-CoA synthase activity is specific against erucic acid and its substrate specificity is higher than that observed against a substrate with a different number of carbon atoms and present the same number of unsaturations as erucic acid.
  • an enzyme with Ci 8 3- ketoacyl-CoA synthase activity is specific against arachidic acid and its substrate specificity is higher than that observed against a substrate with a different number of carbon atoms and present the same number of unsaturations as arachidic acid.
  • the specificity of an enzyme can be determined by measuring the specificity constant (Kcat / Km).
  • Kcat specificity constant
  • the determination of the specificity of the enzyme with Ci 8 3-ketoacyl-CoA synthase activity of the invention can be determined, for example, by quantifying the number of substrate molecules transformed into product per unit time (Kcat) in the presence of different substrates and dividing said value by the concentration of each of said substrates at which the reaction rate is half of the maximum speed (Km).
  • the specificity of the enzyme with Ci 8 3-ketoacyl-CoA synthase activity of the invention for an 18-carbon fatty acid, for example oleic acid, versus the specificity of said enzyme for a 16-carbon fatty acid, for example palmitoleic acid can be determined by comparing the ratio of the concentration of the 16-carbon fatty acid / 18-carbon fatty acid (palmitoleic acid / oleic acid) at which the reaction rate is half the maximum speed versus the ratio of the concentration of the 20-carbon fatty acid (elongation product of the 18-carbon fatty acid) / the concentration of the 18-carbon fatty acid (elongation product of the 16-carbon fatty acid) at which The reaction rate is half the maximum speed.
  • the concentration of a Fatty acid can be determined by any technique known in the state of the art appropriate for this as, for example, spectrophotomatic or chromatographic techniques.
  • fatty acids refers to biomolecules of lipid nature formed by a long linear hydrocarbon chain, of different length or number of carbon atoms having an alkyl group at one end and an acid group at the other end. Said term includes saturated fatty acids and unsaturated fatty acids. The former do not have double bonds in the hydrocarbon chain that make them up and are flexible and solid at room temperature, while the latter have double or triple bonds, are rigid at the level of these bonds and have a liquid or viscous temperature at room temperature.
  • oleic acid or "cis-9-actadienoic acid”, as used herein, refers to a monounsaturated fatty acid of the omega 9 series typical of vegetable oils such as olive oil, avocado oil etc. and whose empirical formula is Ci 8 H 34 02.
  • arachidic acid or "eicosanoic acid”, as used herein, refers to a saturated fatty acid typical of vegetable oils such as peanut or cocoa butter and whose empirical formula is C 2 or H 40 0 2.
  • glycolic acid or "cis-11-eicosenoic acid”, as used herein, refers to a monounsaturated fatty acid of the omega 9 series typical of vegetable oils such as jojoba and whose formula Empirical is C20H38O2.
  • erucic acid or "cis-13-docosaenoic acid”, as used herein, refers to a monounsaturated fatty acid of the omega 9 series typical of vegetable oils such as mustard and whose formula empirical is
  • cognid acid or "cis-15-tetracosenoic acid”, as used herein, refers to a monounsaturated fatty acid of the omega 9 series typical of vegetable oils such as that of the flower of the silver or coin of the Pope and whose empirical formula is C 24 H 46 0 2.
  • the gene encoding the enzyme having Ci 8 3-ketoacyl-CoA synthase activity has codons optimized for expression in the recombinant microorganism, that is in R. toruloides.
  • codons refers to the alteration of codons in nucleic acid molecules to reflect the use of codons typical of the host organism without altering the DNA encoded polypeptide, to improve expression.
  • Software methods and tools for codon optimization are well known in the state of the art.
  • Codons are known in the art that can be used for gene expression in R. toruloides.
  • Illustrative non-limiting examples of optimized codons that can be employed for gene expression in R. toruloides include: UUU, UUC, UUA, UUG, CUU, CUC, CUA, AUU, AUC, AUG, GUU, GUC, GUA or GUG ( Codon usage data, data source: NCBI-GenBank).
  • the genetically modified microorganism is grown under suitable conditions that allow the expression of the gene having Ci 8 3-ketoacyl-CoA synthase activity and thus produce very long chain fatty acids.
  • Suitable culture media for the appropriate growth of different microorganisms are well known in the art. Normally said culture media comprise carbon sources such as glucose, xylose, sucrose, glycerin, and appropriate nitrogen sources such as, for example, yeast extract, peptone, ammonium salts, macerated corn liquid, urea or glutamate sodium
  • said gene is introduced into a replicative DNA expression or construct vector (cassette) that allows the expression of the gene encoding an enzyme with Ci 8 3-ketoacyl-CoA synthase activity according to the present invention and that includes a transcriptional unit that comprises the assembly of (1) genetic element (s) that plays a regulatory role in gene expression, for example, promoters, operators or enhancers, operably linked to (2) the sequence of the gene encoding an enzyme with Ci 8 3-ketoacyl-CoA synthase activity according to the invention and which is transcribed into messenger RNA and translated into protein and (3) sequences suitable for initiating and terminating transcription and translation.
  • s genetic element
  • s that plays a regulatory role in gene expression
  • promoters for example, promoters, operators or enhancers
  • Vectors that can be used in the context of the present invention typically include an origin of replication in bacteria or yeasts, multiple cloning sites and a genetic marker.
  • the genetic marker is usually a gene that confers resistance to an antibiotic, for example ampicillin or geneticin, or alternatively, an auxotrophic marker in the case of yeasts.
  • Such regulatory elements may include an operator sequence to control transcription.
  • the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can be further incorporated.
  • DNA regions are operably linked when they are functionally related to each other.
  • the DNA for a signal peptide is operably linked to the DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to allow translation.
  • the regulatory sequences useful for the present invention may be nuclear promoter sequences or, alternatively, enhancer sequences and / or regulatory sequences that increase the expression of the nucleotide sequence, suppressor sequences, transcriptional start sites, transcriptional stop sites, sites of polyadenylation and the like. A large number of expression control sequences are known in the art and can be used in the present invention.
  • promoters that ensure the initiation of transcription and optionally poly-A signals that ensure termination of transcription and stabilization of the transcript.
  • Commonly used promoters are the scrofular mosaic virus promoter, the polyubiquitin promoter and the actin promoter for ubiquitous expression.
  • the promoter can be constitutive or inducible. If desired, the constant expression of the gene, then a constitutive promoter is used.
  • An "inducible" promoter is used when a regulated expression of the gene is desired depending on the physiological or developmental conditions.
  • Typical promoters for expression in yeast cells include, but are not limited to:
  • Constitutive promoters such as, for example, the alcohol dehydrogenase (ADH1) promoter, the elongation factor 1-a promoter (TEF) and the gene promoter encoding the triose phosphate isomerase (TPI), the glyceraldehyde promoter 3-phosphate dehydrogenase (GPD) and the 3-phosphoglycerate kinase (GPK) promoter, the MRP7 promoter and the alcohol oxidase promoter (AOX1).
  • ADH1 alcohol dehydrogenase
  • TEZ1 elongation factor 1-a promoter
  • TPI elongation factor 1-a promoter
  • GPD glyceraldehyde promoter 3-phosphate dehydrogenase
  • GPK 3-phosphoglycerate kinase
  • MRP7 promoter
  • AOX1 alcohol oxidase promoter
  • Inducible promoters such as, for example, the metallothionein promoter (CUP1), whose expression is regulated by the addition of copper to the culture medium, the promoter of the gene encoding the FUS1 gene or the FUS2 gene, whose expression is active in the presence of pheromones (to factor a) as described in US5063154, the TET promoter, whose expression is regulated in the presence of tetracyclines, the GAL1 -10, GALL, GALS promoters that are activated in the presence of galactose, the promoter Estrogen-inducible VP16-ER, and the phosphatase promoter (PH05) whose expression is activated in the presence of phosphate and the HSP150 heat shock protein promoter, whose expression is activated at high temperature.
  • CUP1 metallothionein promoter
  • PH05 phosphatase promoter
  • Repressible promoters such as, for example, the promoter of the enolase gene (ENO-1) of S. cerevisiae, whose expression can be repressed when the microorganism is grown in a non-fermentable carbon source, as well as promoters whose expression is subjected under glucose repression so that expression will be repressed when part of the lactose has been hydrolyzed and the concentration of glucose in the medium begins to increase, the glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP) promoter from R. toruloides , and the galactokinase promoter (GAL1).
  • ENO-1 enolase gene
  • GAP galactokinase promoter
  • the promoter used to regulate its expression is preferably an inducible promoter so that the expression of the protein of interest can be delayed until they have been delayed. reached sufficient levels of biomass.
  • the gene encoding enzymes with Ci 8 3-ketoacyl-CoA synthase activity with the ability to increase the concentration of very long chain fatty acids according to the present invention is expressed under the control of a constitutive promoter.
  • the termination sequences associated with these genes are also ligated into the 3 'expression vector of the desired sequence to be expressed to provide mRNA polyadenylation and termination.
  • promoters which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase-2, isocytochrome C, acid phosphatase, degrading enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase , and enzymes responsible for the use of maltose and galactose.
  • Any plasmid vector that Containing a promoter, origin of replication and termination compatible with yeast, is suitable.
  • Yeast vectors suitable for the present invention can be based on the following types of plasmids:
  • Multicopy autonomous plasmids these plasmids contain sequences that allow multiple copies of these vectors to be generated. These sequences may be called 2 ⁇ such as that which appears in episomal plasmids (YEp or yeast episomal plasmids) or ARS-like sequences such as those that appear in replication plasmids (YRps or yeast replication plasmids), Examples of Plasmid-based vectors of this type are p426GPD, p416GPD, p426TEF, p423GPD, P425GPD, p424GPD or p426GAL, YEp24 and YEplac.
  • Plasmids of this type include centromeric plasmids (YCps or yeast centromeric plasmids).
  • Integration plasmids plasmids that can be integrated into the genome of the host cell. Plasmids of this type include integration plasmids (YI Ps or yeast integration plasmids). Examples of plasmid-based vectors of this type are pRS303, pRS304, pRS305 or pRS306 and the like.
  • said genes encoding Ci 8 3-ketoacyl-CoA synthase enzymes are expressed under the control of the R. toruloides glycerol-3-phosphate dehydrogenase promoter.
  • terminator sequence refers to a DNA sequence located at the end of a transcriptional unit that signals the termination of transcription. Terminators are untranslated DNA sequences that contain a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3 ' end of a primary transcript.
  • the terminator sequences are known to those skilled in the art. Illustrative and non-limiting examples of terminator sequences that may be employed in accordance with the present invention include the gene terminator of the nopaline synthase from Agrobacterium tumefaciens (t-nos) or the terminator of the 35S gene from cauliflower mosaic virus (CaMV). In an even more preferred embodiment of the invention the terminator is the terminator of the A. tumefaciens nos gene.
  • the invention contemplates embodiments in which the vectors used for the expression of the gene encoding an enzyme with Ci 8 3-ketoacyl-CoA synthase activity in R. toruloides comprise a genetic marker, such as a gene that confers resistance to an antibiotic.
  • the expression of said gene can be optimized for expression in R. toruloides by the use of promoter, terminator and codon sequences optimized for yeast expression. Examples of such sequences have been mentioned above herein.
  • said promoter and terminator sequences are different from the promoters and terminators used for the correct expression of the gene encoding the enzyme with Ci 8 3-ketoacyl-CoA synthase activity in the microorganism of the invention.
  • the gene encoding an enzyme with Ci 8 3- ketoacyl-CoA synthase activity comprises at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and functionally equivalent variants thereof.
  • the gene encoding an enzyme with Ci 8 3- ketoacyl-CoA synthase activity is the AtFAERt gene of Arabidopsis thaliana whose enzyme comprises the sequence shown in SEQ ID NO: 1 or a functionally equivalent variant thereof.
  • the gene encoding an enzyme with Ci 8 3- ketoacyl-CoA synthase activity is the LaKCS gene of Lunaria annua whose enzyme comprises the sequence shown in SEQ ID NO: 2 or a functionally equivalent variant thereof.
  • the gene encoding an enzyme with Ci 8 3- ketoacyl-CoA synthase activity is the CgKCS gene of Cardamine graeca whose enzyme comprises the sequence shown in SEQ ID NO: 3 or a functionally equivalent variant thereof.
  • the gene encoding an enzyme with Ci 8 3- ketoacyl-CoA synthase activity is the CraFAERt gene of Crambe abyssinica whose enzyme it comprises the sequence shown in SEQ ID NO: 4 or a functionally equivalent variant thereof.
  • the term “is from” or “isolated from” means that the gene or enzyme encoded by said gene is substantially separated or purified from a gene or enzyme encoded in the cell of the organism in which said gene or encoded polypeptide is produced. of natural form.
  • isolated encompasses genes purified by standard purification methods for nucleic acids or encoded proteins.
  • the term also comprises genes or enzymes encoded by said genes prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids or the encoded polypeptide thereof.
  • the term "functionally equivalent variant” as used herein refers to all those polypeptides derived from the sequences of enzymes with Ci 8 3-ketoacyl-CoA synthase activity shown in SEQ ID NO: 1-4 by modification, insertion and / or deletion of one or more amino acids as long as the function of said enzyme is substantially maintained. Specifically, the functionally equivalent variants will maintain the ability to increase the concentration of very long chain fatty acids of at least 18 carbon atoms, particularly oleic, gondoic and erucic acid. Methods for determining the production of these acids from their precursors are known in the state of the art.
  • the determination of the production of long chain fatty acids can be carried out by any method that allows the detection of organic components in a sample such as, for example, chromatographic methods including gas chromatography and mass chromatography.
  • Techniques that allow the extraction of oil from the cellular interior are known in the state of the art and include mechanical extraction methods such as pressing and solid-liquid chemical extraction methods.
  • Ci 8 3-ketoacyl-CoA synthase of the invention include those showing at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity of amino acids with respect to the sequences SEQ ID NO: 1-4 of the Ci 8 3-ketoacyl-CoA synthases indicated above.
  • the degree of identity between two amino acid sequences can be determined by conventional methods mentioned in the context of the first method of the invention such as, for example, BLAST (AltschuI SF et al. Basic Local Alignment Search Tool. J Mol Biol. 1990 Oct 5; 215 (3): 403-10).
  • BLAST AltschuI SF et al. Basic Local Alignment Search Tool. J Mol Biol. 1990 Oct 5; 215 (3): 403-10.
  • the person skilled in the art will understand that the amino acid sequences referred to in this description can be chemically modified, for example, by chemical modifications that are physiologically relevant, such as phosphorylations, acetylations, etc.
  • said gene encoding an enzyme with Ci 8 3-ketoacyl-CoA synthase activity consists of the sequence shown in SEQ ID NO: 1.
  • said gene encoding an enzyme with Ci 8 3-ketoacyl-CoA synthase activity consists of the sequence shown in SEQ ID NO: 2.
  • said gene encoding an enzyme with Ci 8 3-ketoacyl-CoA synthase activity consists of the sequence shown in SEQ ID NO: 3.
  • said gene encoding an enzyme with Ci 8 3-ketoacyl-CoA synthase activity consists of the sequence shown in SEQ ID NO: 4.
  • the microorganism of the invention refers to a mutant of the Rhodosporidium toruloides strain CECT 13085.
  • strain refers to a genetic variant or subtype of an organism. determined.
  • the strain Rhodosporidium toruloides CECT 13085 refers to a microorganism of the Rhodosporidium toruloides species that has the ability to grow in the presence of Undetoxified biomass hydrolysates and / or has the ability to metabolize xylose. Said strain is deposited in the Spanish Type Culture Collection (CECT) dated May 7, 2013. The characteristics of said strain are described in patent application ES2526617A1.
  • the FAD2 gene of the microorganism of the invention is not functional.
  • the term "FAD2”, as used herein, refers to the gene encoding the enzyme with delta-12 desaturase activity that catalyzes the introduction of a double bond in the delta-12 position of a very fatty chain fatty acid. long
  • the FAD2 protein sequence of R. toruloides is deposited in the GenBank database (March 20, 2015 version) under number EMS18237.1.
  • said gene encodes an enzyme with delta-12 desaturase activity whose sequence is shown in the sequence SEQ ID NO: 5.
  • the FAD2 gene is not functional as used herein, refers to said gene encoding an FAD2 protein whose ability to introduce a double bond at the delta-12 position of the carbon chain of an acid Very long chain fat is diminished with respect to the ability to carry out said synthesis by a protein encoded by said functional FAD2 gene.
  • the microorganism of the invention has a non-functional FAD2 gene if the ability to introduce a double bond in the delta-12 position of the carbon chain of a very long chain fatty acid by the FAD2 protein encoded by said non-functional FAD2 gene is reduced by at least 50%, at least 60%, at least 70%, at least 80% at least 90%, at least 95% or more with respect to said synthesis performed by a functional FAD2 gene.
  • the FAD2 gene of R. toruloides, preferably of R. toruloides CECT 13085 may be non-functional due to a mutation in the sequence of said gene.
  • FAD2 gene is not functional also means that said gene is absent in the genome of the microorganism of the invention, a consequence of a total deletion of the sequence of said gene.
  • the microorganism of the invention has the FAD2 gene completely deleted.
  • the determination of the functionality of the FAD2 gene in R. toruloides, preferably in R. toruloides CECT 13085, can be carried out by Any method known in the state of the art to detect the enzymatic activity of FAD2.
  • the determination of the production of long chain fatty acids in which unsaturation has been introduced as a consequence of the enzymatic activity of FAD2 can be carried out by any method that allows the detection of organic components in a sample such as methods Chromatographs that include gas chromatography and mass chromatography.
  • mutation refers to substitutions, insertions or deletions that occur at the level of the nucleotide sequence.
  • insertion is meant the gain of one or more nucleotides.
  • duplications consist of the repetition of a segment of DNA inside a sequence, which can occur three (triplication) or more times.
  • deletion as used herein, the loss of one or more nucleotides is understood. Deletions can be total or partial.
  • total deletion of the sequence of a gene, as used in the present invention, refers to the loss of 100% of the nucleotides that constitute the nucleotide sequence of said gene.
  • partial deletion of the sequence of a gene refers to the loss of at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40 %, 50%, 60%, 70%, 80%, 90% or 99% of the nucleotides that make up the nucleotide sequence of said gene.
  • mutations in the sequence of a gene such as, for example, site-directed mutagenesis by PCR or site-directed mutagenesis by PCR in a plasmid.
  • the KU70 gene and / or the KU80 gene of the microorganism of the invention preferably of the strain R. toruloides CECT 13085, is not functional.
  • KU70 refers to the R. toruloides gene that encodes a protein that participates in non-homologous recombination during the DNA repair process.
  • the KU70 sequence of R. toruloides is deposited in the GenBank database (version of May 27, 2014) under number KF850470.
  • the microorganism of the invention has a non-functional KU70 gene if the aforementioned capacity is reduced by at least 50%, at least 60%, at least 70%, at least 80% at least 90%, at least 95% or more with respect to said synthesis performed by a functional KU70 gene.
  • the KU70 gene of R. toruloides, preferably of R. toruloides CECT 13085 may be non-functional due to a mutation in the sequence of said gene.
  • the expression "KU70 gene is not functional" also means that said gene is absent in the genome of the microorganism of the invention, resulting from a total deletion of the sequence of said gene.
  • the microorganism of the invention has the KU70 gene completely deleted.
  • KU80 refers to the R. toruloides gene encoding a protein that participates in non-homologous recombination during the DNA repair process.
  • the KU80 sequence of R. toruloides is deposited in the GenBank database (May 27, 2014 version) under number KF850471.
  • KU80 gene is not functional
  • the microorganism of the invention has a non-KU80 gene. functional if the capacity mentioned above is reduced by at least 50%, at least 60%, at least 70%, at least 80% at least 90%, at least 95% or more with respect to said synthesis performed for a functional KU80 gene.
  • the KU80 gene of R. toruloides, preferably of R. toruloides CECT 13085, may be non-functional due to a mutation in the sequence of said gene.
  • the expression "KU80 gene is not functional" also means that said gene is absent in the genome of the microorganism of the invention, a consequence of a total deletion of the sequence of said gene.
  • the microorganism of the invention has the KU80 gene completely deleted.
  • the determination of the functionality of the KU80 gene in R. toruloides, preferably in R. toruloides CECT 13085, can be carried out by any method known in the state of the art that allows to detect the enzymatic activity of KU80 as for example, by techniques that allow to detect the sensitivity to agents that cause DNA damage.
  • the microorganism of the invention comprises a gene encoding an enzyme with Ci 6 3-ketoacyl CoA synthase activity.
  • C IB 3-ketoacyl-coA synthase refers to a polypeptide belonging to the family of EC enzymes 2.3.1.199 and which catalyzes the addition of two carbons to an acid 16 carbon fatty at its carboxyl end.
  • the enzyme with Ci 6 3-ketoacyl-CoA synthase activity of the invention is capable of elongating a 16-carbon fatty acid in two carbon atoms, to give rise to an 18-carbon fatty acid.
  • the enzyme with Ci 6 3-ketoacyl-CoA synthase activity catalyzes the aforementioned elongation reaction using 16 carbon fatty acids as the sole substrate.
  • said enzyme employs palmitic or palmitoleic acid as substrates, that is, it has preferential specificity for said substrates, obtaining stearic and, preferably, oleic acid as products of the elongation reaction.
  • enzymes with Ci 6 3-ketoacyl-CoA synthase activity suitable for use in the present invention also include those enzymes that have specificity for more than one substrate, in which case they have a substantially specificity.
  • enzymes with Ci 6 activity 3-Ketoacyl-CoA synthase suitable for use in the present invention include those that show a specificity against fatty acids of 16 carbon atoms that is at least 1.5 times, at least 3 times, at least 3 times, at at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times or more with respect to the specificity against fatty acids of 18 carbon atoms.
  • This definition also includes enzymes that catalyze the addition of two carbons to a fatty acid d e 16 carbons at their carboxyl end with a specificity greater than the specificity they present for a fatty acid of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 17, 18, 19, 20 carbons or more.
  • said gene encoding an enzyme with Ci 6 3-ketoacyl-CoA synthase activity is the R. toruloides EloR1 gene whose enzyme comprises the sequence shown in SEQ ID NO: 6 or a variant functionally equivalent of it.
  • the term "functionally equivalent variant” has been defined earlier in this document. Specifically, the functionally equivalent variants of said Ci 6 3-ketoacyl-CoA synthase will maintain the ability to increase the concentration of oleic acid from 16-carbon acids, particularly palmitic and palmitoleic acid. Methods for determining the production of oleic acid from said precursors are known in the state of the art.
  • the determination of oleic acid production can be carried out by any method that allows the detection of organic components in a sample such as methods Chromatographs that include gas chromatography and mass chromatography. Techniques that allow the extraction of oil from the cellular interior are known in the state of the art and include mechanical extraction methods such as pressing and solid-liquid chemical extraction methods.
  • Ci 6 3-ketoacyl-CoA synthase include those showing at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least one 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with respect to the sequence SEQ ID NO: 6 of said Ci 6 3-Ketoacyl-CoA synthase indicated above.
  • said gene encoding an enzyme with Ci 6 3-ketoacyl-CoA synthase activity consists of SEQ ID NO: 6
  • the present invention relates to a process for obtaining a microbial biomass enriched in oil rich in very long chain fatty acids, hereinafter "first process of the invention", which comprises
  • microbial biomass refers to the biological material of living or recently living organisms, in particular of the microorganism of the invention, and to organic matter originated in a biological, spontaneous or provoked biological process. , usable as a source of energy.
  • biomass can be used directly or indirectly, after conversion into another type of product such as biofuel.
  • microbial biomass is enriched in oil rich in very long chain fatty acids.
  • oil refers to a fatty composition formed by acylglycerides, ie esters in which one, two or three fatty acid molecules bind to a glycerin molecule, forming monoglycerides, diglycerides and triglycerides, respectively.
  • said oil may comprise free fatty acids, and other fat-soluble substances such as phospholipids, sphingolipids or sterols.
  • the oil is composed of triacylglycerols in more than 90% of the total composition.
  • microbial biomass enriched in oil rich in very long chain fatty acids refers to a microbial biomass with an oil content rich in very long chain fatty acids from, to less, 40%, at least, 50%, at least, 60% or at least 70% of its total dry weight.
  • This term also refers to a microbial biomass whose content in very long chain fatty acids represents, at least 5% (w / w), at least 10% (w / w), at least 20%, at least 25% (w / w), at least 30% (w / w), at least 35% (w / w), at least 40% (w / w), or at least 50% (w / w) with respect to the total fatty acids of said microorganism.
  • said very long chain fatty acids represent at least 15% (w / w) with respect to the total fatty acids of said microorganism. In another particular embodiment of the invention, said very long chain fatty acids represent at least 20% (w / w), at least 25% (w / w), at least 30% (w / w), at least 35% (w / w), at least 40% (w / w), at least 50% (w / w) or more with respect to the total fatty acids of said microorganism.
  • said very long chain fatty acids are selected from a group consisting of arachidic acid, gondoic acid, erucic acid, nerve acid, behenic acid, lignoceric acid, eicosadienoic acid, docosadienoic acid, or combinations thereof.
  • gondoic acid represents at least 20% (w / w) with respect to the total fatty acids of said microorganism.
  • erucic acid represents at least 20% (w / w) with respect to the total fatty acids of said microorganism.
  • the nervous acid represents at least 5% (w / w) with respect to the total fatty acids of said microorganism.
  • microorganism of the invention as well as particular and preferred embodiments thereof, have been detailed in the context of the first aspect of the invention and apply equally to the first process of the invention.
  • the first process of the invention comprises culturing the microorganism of the invention in a culture medium comprising at least one carbon source and at least one nitrogen source, under conditions suitable for the growth of said microorganism.
  • cultivate refers to the process of planting, maintaining and causing microorganisms to develop on suitable culture media.
  • culture medium refers to a liquid, semi-solid or solid medium that has the necessary nutrients to allow, under favorable conditions of pH, temperature and oxygenation, the growth of microorganisms
  • the culture medium is a liquid medium.
  • Culture media suitable for growing microorganisms are widely known in the art.
  • the culture medium comprises a carbon source and a nitrogen source.
  • Non-limiting examples of liquid culture media suitable for carrying out the first process of the invention include MB03-2 medium (NH 4 N0 3 composition 0.7 g / L, CaCl 2 .2H 2 0 0.4 g / L, MgS0 4 .7H 2 0 0.4 g / L, KH 2 P0 4 0.75 g / L, macerated corn liquid 9.6 g / L pH 6.0, glycerin 110 g / L), medium MB03-11 (macerated liquid corn 9.6 g / L, sugars Wheat straw hydrolyzate 1 10 g / L, pH 6.0) and half MB03-12 (macerated corn liquid 9.6 g / L, beet molasses sugars 40 g / L, pH 5.0).
  • MB03-2 medium NH 4 N0 3 composition 0.7 g / L, CaCl 2 .2H 2 0 0.4 g / L, MgS0 4 .7H 2 0 0.4
  • the culture medium is a solid medium.
  • solid culture media suitable for carrying out the first process of the invention include MB03-2 medium (NH4NO 3 composition 0.7 g / L, CaCl 2 .2H 2 0 0.4 g / L, MgS0 4 .7H 2 0 0.4 g / L, KH 2 P0 4 0.75 g / L, corn macerated liquid 9.6 g / L pH 6.0, glycerin 110 g / L, 2% agar).
  • the carbon source is a lignocellulosic biomass hydrolyzate.
  • biomass hydrolyzate refers to any saccharification product, which contains the sugars produced in the saccharification process, the remains of non-hydrolyzed biomass and the enzymes used for the hydrolysis of said biomass
  • sacharification or “hydrolysis of biomass”, as used herein, refers to the production of fermentable sugars from polysaccharides.
  • transfermentable sugar refers to oligosaccharides and monosaccharides that can be used as a carbon source by a microorganism in the fermentation process to obtain products such as ethanol.
  • biomass and “biomass substrate”, as used herein, refer to any material suitable for use in saccharification reactions. Such terms include but are not limited to materials comprising cellulose (eg, cellulosic biomass, cellulosic feedstock and cellulosic substrate), lignin or the combination of cellulose and lignin. Biomass can be derived from plants, animals or microorganisms and may include, but is not limited to agricultural, industrial and forestry wastes, agricultural and municipal wastes, and land and aquatic crops for energy purposes.
  • cellulose eg, cellulosic biomass, cellulosic feedstock and cellulosic substrate
  • lignin or the combination of cellulose and lignin.
  • Biomass can be derived from plants, animals or microorganisms and may include, but is not limited to agricultural, industrial and forestry wastes, agricultural and municipal wastes, and land and aquatic crops for energy purposes.
  • biomass substrates include but are not limited to wood, wood pulp, paper pulp, corn fiber, corn grain, corn cobs, crop residues such as corn husks, corn stubble, grasses, wheat, straw wheat, barley, barley straw, hay, rice, rice straw, millet, paper waste, paper, waste pulp, woody or herbaceous processing, fruit or vegetable pulp, grain distillate products, herbs, rice husks, cotton, hemp, flax, sisal, cane bagasse, sorghum, soy, millet, components obtained from the grinding of grains, trees, branches, roots, leaves, wood shavings, sawdust, shrubs and bushes, vegetables, fruits and flowers and any combination thereof.
  • crop residues such as corn husks, corn stubble, grasses, wheat, straw wheat, barley, barley straw, hay, rice, rice straw, millet, paper waste, paper, waste pulp, woody or herbaceous processing, fruit or vegetable pulp, grain distillate products, herbs, rice husks, cotton,
  • the biomass comprises, but is not limited to, cultivated plants (for example, herbs, including C4 grasses, such as rod grass, spinal grass, rye grass, Miscanthus, ribbon grass or combinations thereof), residues of sugar processing, for example but not limited to, bagasse [for example, sugarcane bagasse, beet pulp (for example, sugar beet), or a combination thereof], agricultural residues (for example, soybean stubble, corn stubble, corn fiber, rice straw, straw cane sugar, rice, rice husks, barley straw, corn cobs, wheat straw, cane straw, oat straw, oat shells, fiber corn, hemp, flax, sisal, cotton or any combination thereof), fruit pulp, vegetable pulp, grain distillate products, forest biomass (e.g. wood, wood pulp, fiber, recycled wood pulp fibers sawdust hard, such as poplar wood, softwood or a combination thereof).
  • bagasse for example, sugarcane bagasse, beet pulp (for example, sugar beet), or a combination thereof
  • the biomass comprises cellulosic waste material and / or forest residues including but not limited to paper and paper pulp processing waste, municipal paper waste, newspaper, cardboard and the like.
  • the biomass comprises a kind of fiber while in other alternative embodiments, the biomass comprises a mixture of fibers that originate from different biomass.
  • the biomass may also comprise transgenic plants that express ligninase and / or cellulases.
  • biomass includes any living or dead biological material that contains polysaccharides as substrates including but not limited to cellulose, starch, other forms of long-chain carbohydrate polymers and combinations thereof. It may or may not be completely formed from glucose or xylose, and optionally, it may contain other pentose or hexose monomers.
  • Xylose is an aldopentose that contains five carbon atoms and an aldehyde group. It is the precursor sugar of hemicellulose and is often the main component of the biomass
  • the substrate is suspended before pretreatment. In some embodiments, the consistency of the suspension is between about 2% and about 30% and more typically between about 4% and about 15%.
  • the suspension is washed or treated with acid before pretreatment.
  • the suspension is dehydrated by any suitable method to reduce the consumption of water and chemicals before pretreatment. Examples of dehydration devices include, but are not limited to pressurized screw presses, pressurized filters and extruders.
  • a biomass substrate is "p portrait" when it has been subjected to physical and / or chemical procedures to facilitate saccharification.
  • the biomass substrate is "pretreated” or “treated” to increase the susceptibility of said biomass to cellulose hydrolysis by employing methods known in the state of the art, such as physical-chemical pretreatment methods. (for example, treatment with ammonium, pretreatment with dilute acid, pretreatment with diluted alkali, solvent exposure, steam explosion, milling, extrusion), biological pretreatment methods (for example, the application of lignin-solubilizing microorganisms) and combinations of the same.
  • physical-chemical pretreatment methods for example, treatment with ammonium, pretreatment with dilute acid, pretreatment with diluted alkali, solvent exposure, steam explosion, milling, extrusion
  • biological pretreatment methods for example, the application of lignin-solubilizing microorganisms
  • Grinding consists of a process of crushing plant matter until it is reduced to particles of different sizes that can be separated by mechanical procedures.
  • Extrusion is a process whereby plant material is forced to flow under one or more of a variety of mixing, heating and shearing conditions, through a nozzle designed to shape or expand the ingredients. It can be performed cold where the material is extruded without expansion or hot, where the macromolecules of the components lose their discontinuous native structure and a continuous and viscous mass is formed that dextrinizes and gelatinizes the starch, the proteins are denatured, the enzymes are inactivated responsible for possible deterioration, some non-nutritional compounds are destroyed and the microbial load is destroyed.
  • Acid hydrolysis consists in treating the plant material with acids such as sulfuric acid or hydrochloric acid using high temperatures. Through this process, cellulose hydrolysis is favored but requires pH neutralization at the end of hydrolysis to allow subsequent growth of microorganisms.
  • the alkali treatment consists of the addition of diluted bases to the plant biomass.
  • the efficiency of this procedure depends on the lignin content of the materials. Diluted sodium hydroxide produces a swelling, allowing an increase in the internal surface area reducing the degree of polymerization and crystallinity of the cellulose, causing the separation of the structural junctions between lignin and carbohydrates.
  • the treatment with organic solvents consists of using solvents such as methanol, ethanol or acetone to break the bonds of lignin and cellulose.
  • solvents such as methanol, ethanol or acetone to break the bonds of lignin and cellulose.
  • the removal of solvents from the system is necessary, since they inhibit the growth of organisms.
  • ionic liquids for example, with a solution of sodium chloride
  • ionic liquids favors the degradation of cellulose because the hydrogen and oxygen atoms that are part of it interact separately with the solvent so that rupture occurs of hydrogen bonding links between cellulose chains.
  • the steam explosion treatment consists of treating the biomass with saturated steam at a temperature of 160-260 ° C (0.69-4.83 MPa) for a certain time that will depend on the type of plant material of origin.
  • Treatment with lignin-solubilizing microorganisms consists in treating biomass with microorganisms that produce enzymes capable of degrading lignocellulosic material such as, for example, Trichoderma reesei, Fusarium oxysporium, Piptopus betulinus, Penicillum echinalatum, Penicillum purpurogenum, Aspergillus fus, Aspergillus nius Anaeromyces sp., Caecomices sp., Cyllamcyces sp., Neocallimastix sp., Orpinomyces sp., Piromyces sp., Sporotrichum thermophile, Scytalidium thermophillu, Thermonospora cubata, Rhodosporillum rubrum, Cellulomonas fimi, Clostridium stercocarium, Bacillus polymyxa, Pyrococcus furiosus, Acidothermus cellu
  • lignocellulosic biomass hydrolyzate can be obtained from different plant origins or by-products thereof.
  • the culture medium comprises as a carbon source a hydrolyzate of lignocellulosic biomass that is obtained from wheat straw, sugarcane bagasse, empty bunches of oil palm, pruning palm oil, fiber palm oil , vine pruning, olive pruning and combinations thereof.
  • said hydrolyzate comes from wheat straw.
  • said hydrolyzate comes from cane bagasse.
  • said hydrolyzate comes from empty bunches of oil palm.
  • said combinations of hydrolysates mentioned above have at least 5%, at least 10%, at least 20%, at least 30%, at least 40% hydrolyzed lignocellulosic biomass.
  • the carbon source used for the cultivation of the microorganism of the invention is derived from a mixture of a lignocellulosic biomass hydrolyzate and glycerol.
  • the proportion of hydrolyzate and glycerol may vary so that the growth and lipid production conditions of the microorganism of the invention are optimal.
  • the ratio of biomass hydrolyzate: glycerin is 60:40; more preferably, the ratio of biomass hydrolyzate: glycerin is 70:30; and even more preferably the ratio of biomass hydrolyzate: glycerin is 75:25.
  • the carbon source is selected from the group consisting of glucose, glycerol, glycerin, molasses, xylose, arabinose, mannose, fructose, acetate, starches and combinations thereof.
  • the carbon source is glucose.
  • the carbon source is xylose.
  • the concentration of xylose in the culture medium is 20 g / l. In another more preferred embodiment, the concentration of xylose in the medium is 40 g / l.
  • the source of the nitrogen source is selected from the group consisting of yeast extract, peptone, macerated corn liquid, urea, sodium glutamate, different inorganic nitrogen sources, such as ammonium salts and combinations thereof.
  • the nitrogen source is an ammonium salt, preferably ammonium chloride.
  • the culture medium comprises solid inhibitors.
  • solid inhibitors refers to compounds that inhibit microbial metabolism and negatively affect the growth of the organism.
  • said solid inhibitors come from the degradation of the biomass without detoxifying (for example, they come from the degradation of lignocellulose) and are selected from the group consisting of acetic acid, formic acid, levulinic acid, cumaric acid , ferulic acid, succinic acid, 4-hydroxybenzaldehyde, vanillin, vanillic acid, syringaldehyde, 4-hydroxybenzoic acid, catechol, guaiacol, syringic acid, furfural, 5-hydroxymethylfurfural and combinations thereof.
  • Suitable methods for determining the ability of a strain of R. toruloides to grow in the presence of solid inhibitors from undetoxified biomass hydrolysates include, for example, methods that allow the microorganism to adapt to a culture medium in which it is increased. progressively the concentration of inhibitors.
  • the culture medium comprises other specific means to achieve production of the desired metabolite.
  • Said culture media are widely known in the state of the art and preparing them constitutes a routine practice for the person skilled in the art.
  • the culture is subjected to metabolic stress, so as to produce and accumulate large amounts of fatty acids intracellularly. Metabolic stress can be induced by an excess of carbon source in relation to the source of nitrogen in the culture medium. Triglyceride accumulation occurs when a carbon source is in excess and the nitrogen source limits growth. Under these growth conditions, the cells use the carbon source for the synthesis of neutral lipids and their intermediates (acyl-CoA).
  • the microorganism of the invention is genetically engineered to favor acid accumulation.
  • very long chain fatty acids with at least one gene encoding an enzyme with Ci 8 3-ketoacyl-CoA synthase activity and, with the deletion of the FAD2 gene and, additionally with a gene encoding an enzyme with Ci 6 3-ketoacyl activity CoA synthase.
  • Cultivation can be carried out in flasks or bioreactors until maximum oleic acid production is achieved.
  • the duration of the crop is variable, although typically the culture is carried out for 5 days.
  • condition suitable for the growth of the microorganism of the invention refers to conditions that support the growth of the microorganism of the invention.
  • Such conditions may include pH, nutrients, temperature, humidity, oxygenation, environment and other factors.
  • the conditions suitable for the growth of said microorganism of step i) comprise
  • the conditions under which the culture of the microorganism of the invention is carried out can be adjusted to increase the percentage of oil per unit of dry weight in the resulting microbial biomass. For example, it is possible to grow the microorganism in the presence of limiting concentrations of some nutrient, such as nitrogen, phosphorus or sulfur while maintaining an excess of the carbon source. The limitation of the nitrogen source makes it possible to increase the biomass oil yield per unit of dry weight.
  • the microorganism can be grown under conditions limiting some of the nutrients during the entire culture time or it can be grown by alternating culture cycles at limiting concentrations and culture cycles without limiting concentrations.
  • the cultivation according to the first process of the invention is carried out until the desired amount of biomass has been reached and / or until the biomass contains the Intracellular amount of oil rich in desired long chain fatty acids.
  • a crop monitoring to determine the amount of biomass reached over time (for example, by determining the optical density at 600 nm or by determining the solid weight by unit volume of culture).
  • a crop monitoring to determine the percentage of a certain long-chain fatty acid that accumulates in the biomass over time (for example, by determining the amount of gondoic, erucic or nervous acid per unit mass in the culture using any method appropriate for this known in the state of the art).
  • the first process of the invention comprises separating the microbial biomass from the culture broth.
  • the cells are collected by any of the procedures commonly used for this purpose, such as centrifugation, filtration, decantation, flotation or sedimentation, additionally aided by flocculation or evaporation to remove part or all of the water or the medium from the aqueous fraction of the culture medium.
  • the second stage of the first process of the invention is carried out by a method selected from the group consisting of filtration, microfiltration, centrifugation, pressure, settling and combinations thereof.
  • the first process of the invention further comprises drying the microbial biomass obtained in the second stage.
  • the present invention also relates to oil-rich microbial biomass rich in very long chain fatty acids obtainable according to the first process of the invention, hereinafter referred to as "microbial biomass of the invention".
  • microbial biomass has been described in the second aspect of the invention and is applied equally in the present aspect.
  • the biomass enriched in oil rich in very long chain fatty acids has an oil content of at least 40% of the dry weight, at least 50% of the dry weight, at least 60% of the dry weight , or at least 70% of the dry weight.
  • very long chain fatty acids represent at least 15% (w / w), at least 20% (w / w), at least 25% (w / w), at least 30% (w / w), at least 35% (w / w), at least 40% (w / w), at least 50% (w / w) or more with with respect to the total fatty acids of the microorganism of the invention.
  • said very long chain fatty acids are selected from a group consisting of arachidic acid, gondoic acid, erucic acid, nerve acid, behenic acid, lignoceric acid, eicosadienoic acid, docosadienoic acid, or combinations thereof.
  • gondoic acid represents at least 20% (w / w) with respect to the total fatty acids of said microorganism.
  • erucic acid represents at least 20% (w / w) with respect to the total fatty acids of said microorganism.
  • the nervous acid represents at least 5% (w / w) with respect to the total fatty acids of said microorganism.
  • the present invention relates to a process for obtaining a preparation enriched in oil rich in very long chain fatty acids, hereinafter "second process of the invention", comprising:
  • the first stage of the second process of the invention comprises culturing the microorganism of the invention in a culture medium comprising at least one carbon source and at least one nitrogen source, under conditions suitable for the growth of said microorganism.
  • a culture medium comprising at least one carbon source and at least one nitrogen source.
  • the second stage of the second process of the invention comprises separating the microbial biomass from the culture broth.
  • Microbial biomass can be separated from the culture broth by any appropriate method known in the state of the art. Methods that allow microbial biomass to be separated from the culture broth have been detailed in the context of the first process of the invention. Such methods include but are not limited to filtration, microfiltration, centrifugation, pressure, decantation and combinations thereof.
  • culture broth refers to the culture medium obtained after cultivation of the microorganism of the invention and comprising nutrients from the culture medium and compounds produced by the microorganism of the invention as a consequence of its metabolism.
  • the third stage of the second process of the invention comprises extracting the oil rich in very long chain fatty acids from the microbial biomass obtained in the second stage of said process.
  • Suitable methods for extracting said oil include any method of mechanical extraction and within these, any method of solid-liquid or chemical mechanical extraction known in the state of the art.
  • the mechanical extraction method is performed using screw press, French press or ball mill.
  • the solid-liquid extraction method is performed using a water immiscible organic solvent.
  • organic solvent refers to a substance that dissolves a solute whose molecules contain carbon atoms.
  • water immiscible organic solvent refers to an organic solvent with little or no ability to mix with water.
  • water-immiscible organic solvents include n-hexane, ethyl acetate, petroleum ether, ethyl ether, tert-butyl methyl ether, ethyl acetate, acetone, ethyl methyl ketone, benzene, toluene, xylene .
  • said water immiscible organic solvent is selected from the group consisting of petroleum ether, ethyl ether, tert-butyl methyl ether, ethyl acetate, acetone, ethyl methyl ketone, benzene, toluene, xylene and combinations thereof.
  • the second process of the invention further comprises drying the microbial biomass obtained in the second stage.
  • the preparation enriched with very long chain fatty acids obtained from the microbial biomass according to the present invention can be chemically processed to produce products of interest in the industry.
  • the present invention relates to a process for obtaining biolubricants from the preparation enriched in oil rich in very long chain fatty acids, obtained according to the second process of the invention, hereinafter referred to as “third process of the invention ", which comprises:
  • step (i) obtaining a preparation enriched in oil rich in very long chain fatty acids wherein said preparation is carried out according to the process to obtain a preparation enriched in oil rich in very long chain fatty acids of the present invention ii) refine the oil rich in very long chain fatty acids obtained in step (i)
  • step (iii) convert the oil rich in very long chain fatty acids obtained in step (ii) into biolubricants.
  • biolubricant refers to a lubricant that is derived from biomass, such as animal, plant or microbial waste that is not toxic to animal life or aquatic life. and that can be degraded by the action of microorganisms in a relatively short period of time.
  • said biolubricants are obtained from a microbial biomass enriched in oil rich in very long chain fatty acids.
  • microbial biomass enriched in oil rich in very long chain fatty acids has been previously defined in the context of the first method of the invention and applies equally in the context of the third process of the invention.
  • the third process of the invention comprises obtaining a preparation enriched in oil rich in very long chain fatty acids according to the method of the second aspect of the invention.
  • the third process of the invention comprises refining the oil rich in very long chain fatty acids obtained from the second process of the invention.
  • refining refers to the process of purification of a chemical substance obtained many times from a natural resource.
  • Numerous methods are known in the state of the art for the refining of substances.
  • oils such as an oil rich in gondonic, erucic or nervous acid
  • a gas can also be refined in this way by cooling or compressing it until liquefaction.
  • Gases and liquids can also be refined by extraction with a solvent that dissolves the substance of interest or impurities.
  • the oil rich in very long chain fatty acids obtained according to the second process of the invention is refined by alcoholysis in acidic medium.
  • a catalyst can be used to improve the speed and the final yield.
  • the alcoholysis in acidic medium is carried out using an acid as catalyst. Acidification can be performed by using any acid such as, for example, hydrochloric acid or phosphoric acid.
  • the alcohol used in the reaction is preferably in excess between 5 and 40 times with respect to the microbial biomass from which the preparation enriched in oil rich in very long chain fatty acids is obtained.
  • the reaction is allowed to proceed with constant stirring for 20 to 36 hours at a temperature between 40 and 70 ° C.
  • the alcoholysis is carried out with alcohols having 1, 2, 3 or 4 carbons, with methanol being the most preferred alcohol.
  • the third process of the invention comprises converting the rich oil into refined very long chain fatty acids obtained in step ii) into biolubricants.
  • fatty acids such as gondioic acid, erucic acid or nerve acid
  • biolubricants include, without limitation the procedures shown in US8357643 B2. These methods are based on a stage of chemical modification of the fatty acids obtaining epoxy derivatives in the presence of a basic catalyst for obtaining a diester, followed by a hydrogenation stage that gives rise to mono alcohols and acylation that ultimately generates monoesters.
  • chemical modifications made to said free fatty acids include alkylation reactions, addition of a radical, acylation, eno reactions, aminoalkylation, co-oligomerization, hydroformylation, epoxidation and acyloxylation.
  • non-limiting the obtaining of biolubricants according to the third stage of said third process of the invention can be carried out by a first step of expoxidation of fatty acids and a second step where a reaction between said fatty acids modified with carboxylic anhydrides in the presence of a catalyst giving rise to a biolubricant.
  • Said third stage can also be carried out by a first epoxidation step of fatty acids, a second hydrogenation step of said fatty acids thus obtaining an intermediate with hydroxyl groups and a third step of acylation of said hydroxyl groups with an acylating agent resulting in obtaining a biolubricant.
  • basic catalysts comprise a tertiary amine such as triethylamine.
  • the present invention relates to the use of the microorganism of the invention, hereinafter "first use of the invention", to obtain a microbial biomass enriched in oil rich in very long chain fatty acids according to the first method of the invention.
  • the present invention relates to the use of the microorganism of the invention, hereinafter "second use of the invention", to obtain a preparation enriched in oil rich in very long chain fatty acids according to the second process of the invention .
  • the present invention relates to the use of the microorganism of the invention, hereinafter "third use of the invention", to obtain biolubricants according to the third process of the invention.
  • microorganism microbial biomass
  • biolubricants microbialants
  • Example 1 Construction of integrative cassettes in Rhodosporidium toruloides.
  • genes encoding enzymes with Ci 8 -ketoacyl-CoA synthase (elongase) activity were synthesized.
  • the sequences of these polypeptides are those shown in SEQ ID NO: 1-4 and 6.
  • the fragments obtained were cloned into a vector under the control of the glycerol-3-phosphate dehydrogenase promoter of R. toruloides and the Tnos terminator of Agrobacterium tumefaciens giving rise to pNEOL86-119-148-149.
  • the pNEOL85 vector contains another expression cassette containing the genetics resistance gene with codon use of R. toruloides (G418Rt) under the control of the promoter of the phosphoglycerate kinase of R. toruloides (pPGK47) and the T35S terminator of the mosaic virus of cauliflower.
  • the vector pNEOL1 11 is identical to the vector pNEOL85 except that its pPGK promoter is truncated and only contains about 580 bp (pPGK47-t).
  • the vector pNEOL112 is identical to the vector pNEOL11 1 except the selection marker is changed to a hygromycin resistance gene (hphRt).
  • the pNEOL120-136-154-155 vectors were cut with Pac ⁇ and a fragment of about 4 kb, bearing the different expression cassettes, cloned into the Ti plasmids pNEOL57 or pNEOL105, giving rise to the pNEOL126-136 integrative cassettes -163-164 ( Figure 1).
  • R. toruloides CECT 13085 was performed using the Agrobacterium tumefaciens-mediated transformation system (ATMT).
  • ATMT Agrobacterium tumefaciens-mediated transformation system
  • the pre-inoculum of A. tumefaciens was grown by carrying the integrative plasmid in the MM growth medium (prepared according to Hooykaas et al., 1979) and the pre-inoculum of R. toruloides CECT 13085 in YPD medium (extract of yeast 10g / L, glucose 20g / L, peptone 20g / L) for 24h.
  • MI medium is inoculated (prepared according to Bundock et al., 1995) supplemented in 200 ⁇ iring acetosyringone with an initial D0 6 6o of 0.5, and incubated 6h at 30 ° C with agitation of 250 rpm.
  • the strain R. toruloides CECT 13085 is inoculated in 10 mL of YPD with an initial D0 6 or 1.5. After 6h of incubation, 100 ⁇ of each culture and a co-culture is carried out on a 0.45 ⁇ nitrocellulose membrane in MI medium supplemented in 200 ⁇ acetosyringone, incubated at 25 ° C for 72h.
  • the co-culture or transformation mixture is collected with 2 ml_ of YPD medium and seeded in Petri dishes with YPD medium supplemented with cefotaxime (200 ⁇ g / mL) and geneticin (35 ⁇ g / mL) or hygromycin (40 ⁇ g / mL).
  • the transformants were chopped in YPD medium supplemented with geneticin (35 ⁇ g / mL) or hygromycin (40 ⁇ g / mL).
  • the integration of the cassettes containing the different elongases in the yeast genome was checked by PCR using specific oligonucleotides 021 + (SEQ ID NO: 7), 022- (SEQ ID NO: 8).
  • genomic DNA of the transformants was cut with a restriction enzyme (Kpnl) and in each case hybridized with a probe, amplified with oligonucleotides 065+ (SEQ ID NO: 9) and 065- (SEQ ID NO: 10 ), specific to the conserved domain of elongasas.
  • the probe was labeled according to the protocol described in the Roche kit (DIG High Prime DNA labeling and detection Starter kit I). Both hybridization and PCR assays confirmed the integration of expression cassettes in the genome of the different transformants.
  • Clones containing at least one copy of the AtFAERt gene (Arabidopsis thaliana) according to SEQ ID NO: 1, or the LaKCSRt gene (Lunaria annua) according to SEQ ID NO: 2 were grown on MB03-2 medium plates at 30 ° C for 2 days .
  • Table 1 shows the results of some of the clones analyzed and the parental strain CECT 13085. Approximately 0.5 g of cells were collected and the biomass was dried in an oven at 65 ° C for 24 hours. The oil was extracted with n-hexane and the fatty acid composition was determined by CG-FID.
  • the oil extracted from the selected clones showed the presence of gondoic acid (> 20% of the total fatty acids) as well as small amounts of erucic acid, all absent in the oil extracted from the parental strain.
  • the content of very long chain fatty acids (greater than 20 carbon atoms) in the oil increased between 6 and 7 times.
  • Table 1 Gondoic acid production. Composition of the oil (fatty acids) in recombinant clones expressing the AtFAERt gene or the LaKCSRt gene
  • Clone T126-25 was grown in 500 ml flasks containing 100 ml of the MB03-2 culture media (NH 4 N0 3 composition 0.7 g / L, CaCI 2 .2H 2 0 0.4 g / L, MgS0 4 .7H 2 0 0.4 g / L, KH 2 P0 4 0.75 g / L, corn macerated liquid 9.6 g / L at pH6, glycerin 1 10 g / L), MB03-1 1 (macerated corn liquid 9.6 g / L, sugars Wheat straw hydrolyzate 1 10 g / L, at pH 6.0) and MB03-12 (macerated corn liquid 9.6 g / L, beet molasses sugars 40 g / L, at pH 5.0).
  • the MB03-2 culture media NH 4 N0 3 composition 0.7 g / L, CaCI 2 .2H 2 0 0.4 g / L, MgS0 4 .
  • Example 4 Production of oil enriched in gondoic acid by expression of two ketoacyl-CoA synthases
  • Clones containing at least one copy of the AtFAERt (Arabidopsis thaliana) gene according to SEQ ID NO: 1, and at least one copy of the EloIRt gene gene (R. toruloides) according to SEQ ID NO: 6 were grown on MB03-2 medium plates at 30 ° C for 2 days.
  • Table 3 shows the results of some of the clones analyzed and the parental strain CECT 13085. Approximately 0.5 g of cells were collected and the biomass was dried in an oven at 65 ° C for 24 hours. The oil was extracted with n-hexane and the fatty acid composition was determined by CG-FID.
  • the oil extracted from the selected clones showed the presence of gondoic acid (> 20% of the total fatty acids) as well as small amounts of erucic acid, all absent in the oil extracted from the parental strain. Due to this change, the content of very long chain fatty acids in the oil increased between 7 and 10 times.
  • Table 3 Gondoic acid production. Composition of the oil (fatty acids) in recombinant clones expressing the AtFAERt gene and the EloIRt gene
  • T116-126-68 15.3 5.4 30.7 17.2 7.0 18.7 3.3 29.0 Clone T1 16-126-64 was grown in 500 ml flasks containing 100 ml of the culture media MB03-2, MB03-11 or MB03-12. The cultures grew at 250 rpm, 30 ° C for 96 h. Once the cultures were finished, the cells were collected by centrifugation and the biomass of each flask was dried in an oven at 65 ° C for 24 hours. Oil extraction and fatty acid composition were determined as indicated in Example 3. The results obtained (Table 4) confirmed the presence of gondoic acid (> 24% of total fatty acids) and erucic acid. The oil extracted from the parental strain did not show any of these fatty acids in its composition. In these cases the content of very long chain fatty acids in the oil increased more than 10 times.
  • Table 4 Gondoic acid production. Composition of the oil (fatty acids) in recombinant clones expressing the AtFAERt gene and the EloI Rt gene (liquid media)
  • Clones containing at least one copy of the CraFAERt gene from Crambe abyssinica according to SEQ ID NO: 4 were grown in MB03-2 medium plates at 30 ° C for 2 days.
  • Table 5 shows the results of some of the clones analyzed and the parental strain CECT 13085. Approximately 0.5 g of cells were collected and the biomass was dried in an oven at 65 ° C for 24 hours. The oil was extracted with n-hexane and the fatty acid composition was determined by CG-FID. The oil extracted from the selected clones showed the presence of erucic acid (> 20% of the total fatty acids), as well as small amounts of gondoic and nervous acid, all of them absent in the oil extracted from the parental strain. The very long chain fatty acid content in the oil was increased about 4-5 times.
  • Table 5 Erucic acid production. Composition of the oil (fatty acids) in recombinant clones expressing the CraFAERt gene
  • Example 6 Production of oil enriched in nerve acid.
  • Clones containing at least one copy of the CgKCSRt gene of Cardamine graeca according to SEQ ID NO: 3 were initially grown on MB03 -2 medium plates at 30 ° C for 2 days.
  • Table 6 shows the results of the analysis of some clones and the parental strain CECT 13085. Approximately 0.5 g of cells were collected and the biomass was dried in an oven at 65 ° C for 24 hours. The oil was extracted with n-hexane and the fatty acid composition was determined by CG-FID. The oil extracted from the selected clones showed the presence of nerve acid (4-19% of the total fatty acids), as well as small amounts of gondoic and erucic acid, all absent in the oil extracted from the parental strain. The very long chain fatty acid content (greater than 20 carbon atoms) in the oil was increased about 4-6 times.
  • C16 C16: C18: C18: 1A C18: 2A9
  • C20 C20: 1A1 C22: 1A1 C24: 1A1% AG> 0 1 0 9 12 0 1 3 5
  • C20 C16: C18: C18: 1A C18: 2A9
  • T164-90 18.7 2.8 2.8 30.5 19.4 6.6 4, 1 5.6 4.6 20.8 Clone T164-59 was grown in 500 ml flasks containing 100 ml of MB03-2 or MB03-12 medium. The cultures were grown at 250 rpm, 30 ° C for 96 h. The oil extraction and fatty acid composition were determined as indicated in example 3.

Abstract

La présente invention concerne la production d'acides microbiens à teneur élevée en acides gras à chaîne très longue par la culture d'un microorganisme de l'espèce Rhodosporidium toruloides, par insertion d'au moins un gène codant pour une enzyme à activité C183-cétoacétyl-CoA syntase qui permet de produire des huiles microbiennes à teneur élevée en acides gras à chaîne très longue en présence de différentes sources de carbone.
PCT/ES2016/070634 2015-09-08 2016-09-08 Production d'huiles microbiennes à teneur élevée en acides gras à chaîne très longue WO2017042413A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070117190A1 (en) * 2005-11-23 2007-05-24 Damude Howard G Delta-9 elongases and their use in making polyunsaturated fatty acids
WO2008061334A1 (fr) * 2006-11-21 2008-05-29 National Research Council Of Canada Gènes fae de lunaria annua, cardamine graeca et teesdalia nudicaulis et leur utilisation lors de la production d'acides nervoniques et eicosanoïques dans les huiles de graines
WO2012052468A2 (fr) * 2010-10-21 2012-04-26 Basf Plant Science Company Gmbh Nouvelles désaturases d'acides gras, élongases, composants d'allongement leurs utilisations
ES2526617A1 (es) * 2013-06-10 2015-01-13 Neol Biosolutions, S.A. Producción de aceites microbianos

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070117190A1 (en) * 2005-11-23 2007-05-24 Damude Howard G Delta-9 elongases and their use in making polyunsaturated fatty acids
WO2008061334A1 (fr) * 2006-11-21 2008-05-29 National Research Council Of Canada Gènes fae de lunaria annua, cardamine graeca et teesdalia nudicaulis et leur utilisation lors de la production d'acides nervoniques et eicosanoïques dans les huiles de graines
WO2012052468A2 (fr) * 2010-10-21 2012-04-26 Basf Plant Science Company Gmbh Nouvelles désaturases d'acides gras, élongases, composants d'allongement leurs utilisations
ES2526617A1 (es) * 2013-06-10 2015-01-13 Neol Biosolutions, S.A. Producción de aceites microbianos

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