ES2608968B1 - PRODUCTION OF MICROBIAL OILS WITH HIGH CONTENT IN VERY LONG CHAIN FATTY ACIDS - Google Patents

Abstract

Production of microbial oils with high content of very long chain fatty acids. # The present invention relates to the production of microbial oils with high content of very long chain fatty acids by culturing a microorganism of the Rhodosporidium toruloides species, by the insertion of at least one gene that encodes an enzyme with C {sub, 18} 3-ketoacetyl-CoA synthase activity that allows to produce microbial oils high in very long chain fatty acids in the presence of different carbon sources.

Description

PRODUCTION OF MICROBIAL OILS WITH HIGH CONTENT IN ACIDS VERY LONG CHAIN FATS

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

Oleochemicals constitute a class of aliphatic molecules derived from lipids. Oleochemicals are used in a wide range of applications that include transport 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.

Currently, most oleochemicals are produced from relatively cheap lipid sources such as vegetable oils and animal fats. The growing demand for oleochemicals, and in particular biodiesel, has led to an increase in the production of vegetable crops and has raised concerns about the sustainability and environmental impact of oilseed seed production.

Therefore, the interest in finding alternative raw materials for the production of oleochemicals, such as used oils, fatty fatty residues, or lipids of microbial origin (algae or other microorganisms) has grown significantly during the last decade. Of all of them, 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 Plleger. 2013. Curr Opin Biolechnol,

24: 1044-1053.).

Vegetable oils are the most used raw materials for the production of oleochemicals. The use of vegetable oils can be divided into three categories: food, biodiesel and oleochemicals. Of the 157 million tons consumed in 2012-2013, 77% were used in food production, 12% in biodiesel production and the remaining 11% in oleochemical production (OECD. 2014. Oilseeds: World markets and trade).

As a consequence of the growing demand for oleochemicals, the production of vegetable oils has increased steadily (14%) from 149 million tons in 2010-2011 to 170 million tons in 2013-2104, and forecasts indicate that it will still grow more, up to 28% more in the year 2023 compared to 2013-2014 figures. Other estimates (Carlsson et al. 2011. Eur J Lipid Sci Technol, 113: 812-831) indicate that in 2030 the global oil needs will triple (390 MT / year) due to population increase and higher consumption caloric (150 MT / year for human consumption) and, above all, for the aforementioned increase in the use of oils for industrial applications (240 MT / year). This increase in production will not be possible with current oil crops. Therefore, the production of oils and their derivatives (fatty acids, fatty alcohols, methyl esters, etc.) by microorganisms can be a good alternative source for the future obtaining of these products from sustainable and renewable raw materials.

Very long chain fatty acids (AGCML), those that contain more than 18 carbon atoms, are commonly present in seeds and waxes of plants belonging to the Cruciferaceae, Limnanthaceae, Simmondsiaceae and Tropaeolaceae families. Erucic acid (cis-docosa-13-enoic acid, 22: 1 D.13) is the most abundant AGCML in seed oil of several species such as the HEAR varieties (from English, high erucic acid rapeseed) of Brassica napus, B. napa or B. carinata, in which it accounts for 45-55% of the total fatty acids. However, its cultivation is very restricted since it is conditioned by climatic conditions and, for example, they grow very poorly in hot and dry areas.

Erucic acid is of great interest since it is a valuable raw material with more than 1,000 patented industrial applications. At present, 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. There are many other intended applications for erucic acid and its hydrogenated derivative, behenic acid, such as high temperature lubricants, high molecular weight anionic surfactants, viscoelastic surfactanles, 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 .6. 15) is another strategic AGCML that exists in nature as a product of elongation of oleic acid (18: 1 .ó.9), being its immediate precursor erucic acid. Nerve acid has been identified in seed oils from very few plants such as Lunaria annua (silver plant), 80ra90 officinalis (borage), Cannabis saliva (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.

Therefore, there is great market potential for the expansion and development of new renewable sources of AGCML-rich oils such as erucic and nervous acid among others.

Two potential solutions include the production of lipids in photoautotrophic algae or the conversion of plant biomass using genetically modified microorganisms. With respect to the first, and although numerous advances have been made, there are still numerous challenges (Wijffels and Barbosa. 2010. Science, 329: 796-799). On the other hand, 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 and 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 CO2_ However, both types are technically difficult to handle and their cultivation processes and of growth 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. Similarly, the exploitation of oleaginous filamentous fungi as production organisms has also been impeded by the lack of efficient transformation techniques.

There is therefore a need in the state of the art to achieve high performance, production and productivity of oils of microbial origin rich in AGCML and to achieve that these processes can become competitive with those used today.

SUMMARY OF THE INVENTION

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 e18 3-ketoacyl-CoA synthase activity that allows to produce oils with a high fatty acid content of very long chain (equal to or greater than 20 carbon atoms). The production of oil by means of the use of said microorganism is above 50 gIL And the content of very long chain fatty acids is greater than 35% of the total fatty acids present.

Therefore, in a first aspect, the present invention relates to a microorganism of the genetically modified Rhodosporidium toruloides species with at least one gene encoding an enzyme with e18 3-ketoacetyl-CoA synthase activity.

In another aspect, 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 carbon source and at least one nitrogen source under conditions suitable for the growth of said microorganism; Y

ii) separate the microbial biomass from the culture broth.

In another aspect, 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.

In another aspect, the present invention relates to a process for obtaining a preparation 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 carbon source and at least one nitrogen source under conditions suitable for the growth of said microorganism;

ii) separate the microbial biomass from the culture broth; and iii) extracting the oil rich in very long chain fatty acids from the microbial biomass obtained in step (ii).

In another aspect, 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 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); and iii) convert the oil rich in very long chain fatty acids obtained in step (ii) into biolubricants.

Finally, 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 an oil-rich preparation rich in very long chain fatty acids. The present invention also relates to the use of the microorganism of the invention to obtain biolubricants.

5 BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Integrative cassettes in Rhodosporidium toruloides

Detailed description of the invention

In a first aspect, the present invention relates to a microorganism, hereinafter "microorganism of the invention", of the genetically modified Rhodosporidium toru {oides species with at least one gene encoding an enzyme with e'8 3-ketoacil activity -CoA sinlas8.

The term "gene", as used herein, refers to a deoxyribonucleotide chain that encodes a protein. In a particular embodiment of the invention, said term refers to a deoxyribonucleotide chain that encodes an enzyme with Cle 3-ketoacyl-CoA synthase activity.

The term "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.

Specifically, the microorganism of the invention is a modified microorganism.

25 genetically The term "genetically modified microorganism" as used herein, refers to a microorganism whose genetic material has been altered using genetic engineering techniques. Accordingly, said genetically modified microorganism expresses an enzyme that has an activity C, e 3-ketoacyl-CoA synthase, compared to a corresponding unmodified control microorganism thereof.

30 strain In the context of the present invention, the enzyme having C activity, and 3-ketoacyl CoA synthase may be encoded by a gene encoding said enzyme in another organism.

Generally, the gene encoding an enzyme having C'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. After being introduced into the microorganism, the gene can be expressed exogenously if it is expressed in a vector / plasmid in the microorganism (i.e. 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

5 in the state of the art. Appropriate techniques that allow genetic transformation in yeasts include but are not limited to:

Spheroplast transformation that involves eliminating the yeast cell wall

and contacting the spheroplasts with the plasmid in the presence of PEG.

Transformation with L¡ +, which involves the treatment of yeast cells with

10 monovalent alkaline cations (Na +, K +, Rb +, Cs + and L¡ +) in combination with PEG to stimulate the uptake of AON by intact cells. Gene bombardment that involves bombing cells with microprojectiles coated with the exogenous AON. Electroporation, which involves administering electrical pulses to yeasts that

15 produces the opening of pores in the membrane of spheroplasts and intact yeast cells. Agrobacterium tumefaciens-mediated transformation (ATMT) is based on the use of the gene transfer capacity that A. tumefaciens naturally possesses.

20 The transformants are grown in a suitable nutrient medium and under selection conditions to ensure retention of the endogenous AON. The insertion of the gene encoding a C18 3-ketoacyl-CoA synthase in said transformants can be determined by any appropriate molecular biology technique, for example, by Southern

25 blot or PCR. Conventional methods of detecting and quantifying the expression of a gene can be found, for example, in Sambrook et al., 2001. "Molecular cloning: a Laboratory Manual", 3, ed., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3.

The term "Cle 3-ketoacyl-coA synthase", "acyl-Coa elongasa", "beta-ketoacil CoA synthase", "3oxoacil CoA synthase very long chain", or "fatty acid elongase" as it is

30 used herein, refers to a polypeptide belonging to the E.C. family of enzymes. 2.3.1 .199 and which catalyzes the addition of two carbons to saturated fatty acids

or unsaturated 18-26 carbons at their carboxyl end. Thus, the enzyme with Cle 3-ketoacyl-CoA synthase activity of the invention is capable of elongating saturated or unsaturated fatty acids of at least 18 carbons to two carbon atoms to give rise to

35 the corresponding fatty acids. In a particular and preferred embodiment of the invention, said enzyme elongates saturated or saturated fatty acids in two carbon atoms whose hydrocarbon chain comprises 18 to 26 carbons.

Preferably, the enzyme with e'8 3-ketoacyl-CoA synthase activity catalyzes the aforementioned elongation reaction using as a single substrate monounsaturated fatty acids of at least 18 carbons. In a particular and preferred embodiment of the invention, 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.

In another particular and preferred embodiment of the invention, 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.

In another particular and preferred embodiment of the invention, 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

However, enzymes with C18 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 shorter length (for example, fatty acids of 14-16 carbon atoms). Thus, enzymes with C18 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 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 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 specificity against fatty acids of length 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, 11, 12, 13, 14, 15, 16 or 17 carbons. The term "specificity", as used herein, refers to the efficiency with which the enzyme transforms a particular substrate into the reaction product. The term "greater specificity," as used herein, 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. of 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, 11, 12, 13, 14 or 15 carbons. In a preferred embodiment, it is understood that an enzyme with C18 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 at the same number of unsaturations as oleic acid.

In another preferred embodiment, it is understood that an enzyme with C18 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 at the same number of unsaturations as gondoic acid.

In another preferred embodiment, it is understood that an enzyme with C18 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 at the same number of unsaturations as erucic acid.

In another preferred embodiment, it is understood that an enzyme with C18 3-ketoacyl-CoA activity without rate 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 that present at the same number of unsaturations as arachidic acid.

The specificity of an enzyme can be determined by measuring the specificity constant (KcatlKm). The person skilled in the art knows that the preferred substrates for enzymes are those for which the Km values are low and the Kcat values are high compared to substrates for which it shows no specificity or for which it shows a lower specificity. The determination of the specificity of the enzyme with C18 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 the maximum speed (Km). By way of illustration, the specificity of the enzyme with e18 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 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 velocity versus at the ratio of the concentration of the fatty acid of 20 carbons (product of elongation of the fatty acid of 18 carbons) / the concentration of the fatty acid of 18 carbons (elongation product of the fatty acid of 16 carbons) at which the reaction speed 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.

The term "fatty acids", as used herein, 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. In this last group, we can also differentiate between monounsaturated fatty acids or MUFA (from English "monounsaturated fatty acid '") and polyunsaturated fatty acids or PUFA (from English "polyunsaluraled rally acid').

The term "oleic acid" or "cis-S-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

C, sH340 2

The term "araguidic 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 C2oH400 2.

The term "gondoic 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 C2oH3802

The term "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 C22H4202.

The term "nervous 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 C24H460 2.

In a particular embodiment of the invention, the gene encoding the enzyme having C18 3-ketoacyl-CoA synthase activity has codons optimized for expression in the recombinant microorganism, that is in R. toruloides.

The term "optimized codons," as used herein, refers to the alteration of codons in nucleic acid molecules to reflect the use of codons typical of the host organism without altering the AON-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 database, data source: NCBI-GenBank). In this context of the invention, the genetically modified microorganism is grown under suitable conditions that allow the expression of the gene having Cle 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

Preferably, said gene is introduced into a replicative AON expression or construction vector (cassette) that allows expression of the gene encoding an enzyme with C18 3-ketoacyl-CoA synthase activity according to the present invention and that includes a transcriptional unit comprising 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 e18 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. 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. AON regions are operatively linked when they are functionally related to each other. For example, the AON for a signal peptide is operably linked to the AON 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 operatively 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.

In the case of eukaryotic cells, these normally comprise 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-f05 phosphate dehydrogenase (GPD) and the 3-phosphoglycerate kinase (GPK) promoter, the MRP7 promoter and the alcohol oxidase promoter (AOX1). 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 feramonas (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. 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 (GAL 1).

Preferably, in those cases where heterologous protein is suspected of being toxic to the host cell, 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. In a preferred embodiment, the gene encoding enzymes with C'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 control. of a constitutive promoter. When constructing suitable expression plasmids, 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. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase-2, isocytochrome e, 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 containing a promoter, origin of replication and termination compatible with yeast, is suitable.

5 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 can be called 21-1 such as the one that appears in episomal plasmids (YEp or

10 episomal yeast 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.

15 Single-copy autonomous plasmids: plasmids that contain the ARS1 autonomous replication sequence and a cenrometer sequence (CEN4). Plasmids of this type include centromeric plasmids (YCps or yeast centromeric plasmids). Integration plasmids: plasmids that can be integrated into the genome of the

20 host cell. Plasmids of this type include integration plasmids (YIPs)

or yeast integration plasmids). Examples of plasmid-based vectors of this type are pRS303, pRS304, pRS305 or pRS306 and the like.

In an even more particular and preferred embodiment of the invention, said genes that

25 encode C18 3-ketoacyl-CoA synthase enzymes are expressed under control of the glycerol-3-phosphate dehydrogenase promoter of R. toruloides.

Preferred embodiments of the invention contemplate the use of terminator sequences. The term "terminator sequence", as used in the present invention, refers to an AON sequence located at the end of a transcriptional unit 30 signaling the termination of transcription. Terminators are untranslated AON 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

terminator of the nopaline synthase gene 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.

5 10 l S

As mentioned above, the invention contemplates embodiments in which the vectors used for the expression of the gene encoding an enzyme with C18 3-ketoacyl-CoA synthase activity in R. toruJoides comprise a genetic marker, such as a gene which confers resistance to an antibiotic. If desired, the expression of said gene can be optimized for expression in R. toruJoides by the use of promoter, terminator and codon sequences optimized for yeast expression. Examples of such sequences have been mentioned above herein. In a preferred embodiment, said promoter and terminator sequences are different from the promoters and terminators used for the correct expression of the gene encoding the enzyme with C16 3-ketoacyl-CoA synthase activity in the microorganism of the invention.

In a particular embodiment, the gene encoding an enzyme with e16 3-ketoacyl Coa synthase activity comprises at least one sequence selected from SEa ID NO: 1, SEa ID NO: 2, SEO ID NO: 3, SEa ID NO: 4 and variants functionally equivalent thereof.

twenty

In a particular embodiment, the gene encoding an enzyme with C16 3-ketoacylCoA synthase activity is the AtFAERt gene of Arabidopsis thaliana whose enzyme comprises the sequence shown in SEa ID NO: 1 or a functionally equivalent variant thereof.

2S

In another particular embodiment, the gene encoding an enzyme with C16 3-ketoacyl-CoA synthase activity is the LaKCS gene of Lunaria annua cu and the enzyme comprises the sequence shown in SEO ID NO: 2 or a functionally equivalent variant thereof.

In another particular embodiment, the gene encoding an enzyme with C16 3-ketoacylCoA synthase activity is the CgKCS gene of Cardamine graeca whose enzyme comprises the sequence shown in SEO ID NO: 3 or a functionally equivalent variant thereof.

30

In another particular embodiment, the gene encoding an enzyme with C16 3-ketoacylCoA synthase activity is the CraFAERt gene from Crambe abyssinica whose enzyme comprises the sequence shown in SEa ID NO: 4 or a functionally equivalent variant thereof. 16

The term ~ is "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 the said encoded gene or polypeptide occurs naturally. Thus, the term 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 C18 3-ketoacyl-CoA synthase activity shown in SEO 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. By way of illustration, 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.

Functionally equivalent variants of the C18 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 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 one 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with respect to SEO ID sequences NO: 1-4 of the C18 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 (Altschul SF et al. Basic Local Alignment Search Too !. J Mol Biol. 1990 Oet 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.

In a preferred embodiment of the invention, said gene encoding an enzyme with e18 3-ketoacyl-CoA synthase activity consists of the sequence shown in SEQ ID NO: 1.

In another preferred embodiment of the invention, said gene encoding an enzyme with e18 3-ketoacyl-CoA synthase activity consists of the sequence shown in SEO ID NO: 2.

In another preferred embodiment of the invention, said gene encoding an enzyme with C18 3-ketoacyl-CoA synthase activity consists of the sequence shown in SEO ID NO: 3.

In another preferred embodiment of the invention, said gene encoding an enzyme with C18 3-ketoacyl-CoA synthase activity consists of the sequence shown in SEO ID NO: 4.

In a particular embodiment, the microorganism of the invention refers to a mutant of the Rhodosporidium toruloides strain CECT 13085. The term "strain", as used herein, 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 biomass hydrolysates without de-specifying and / or has the ability to target xylose beacon. 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 ES2526617 A 1.

In a particular embodiment, 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 at the delta-12 position of a chain fatty acid. very long. The sequence of the R. toruloides FA02 protein is deposited in the GenBank database (version of March 20, 2015) under the number EMS18237 .1. In a preferred embodiment

of the invention, said gene encodes an enzyme with delta-12 desalurase activity whose sequence is shown in the sequence SEQ ID NO: 5.

The expression "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. According to the present invention, 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.

The term "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. In a preferred embodiment of the invention, 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 that allows to detect the enzymatic activity of FAD2. By way of illustration, 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.

The term "mutation" refers to substitutions, insertions or deletions that occur at the level of the nucleotide sequence. By "insertion" is meant the gain of one or more nucleotides. Within the insertion there are duplications that consist of the repetition of a segment of DNA inside a sequence, which can occur three (triplication) or more times. By "deletion" as used herein, the loss of one or more nucleotides is understood. Deletions can be total or partial. The term "total deletion" of the sequence of a gene, as used in the

This invention refers to the loss of 100% of the nucleotides that constitute the nucleotide sequence of said gene. The term "partial deletion of the sequence of a gene, as used in the present invention, 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 As the person skilled in the art knows, there are appropriate methods in the state of the art to perform 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.

In an even more particular embodiment, 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.

The term uKU70 ~, as used herein 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 term "KU70 gene is not functional," as used herein, refers to the said gene encoding a KU70 protein whose binding capacity to damaged DNA and / or whose ability to recruit proteins involved in repair of said DNA is diminished with respect to the ability to perform said function by a protein encoded by said functional KU70 gene. According to the present invention, 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.

In another preferred embodiment according to the present invention, the expression "KU70 gene is not functional" also means that said gene is absent in the master gene of the microorganism of the invention, resulting from a total deletion of the sequence of said gene. . In a preferred embodiment of the invention, the microorganism of the invention has the KU70 gene completely deleted. The determination of the functionality of the KU70 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 the deletion of the

enzymatic activity of KU70, for example, by means of techniques to detect

sensitivity to agents that cause damage to the AON.

The term "KUBO", as used herein, refers to the R. toruloides gene that encodes a protein that participates in non-homologous recombination during the DNA repair process. The KU80 sequence of R. toruloides is deposited in the GenBank data base (May 27, 2014 version) under number KF850471.

The expression "KU80 gene is not to function, as used herein, refers to said gene encoding a KU80 protein whose binding capacity to damaged DNA and / or whose ability to recruit proteins involved in the Repair of said DNA is diminished with respect to the ability to perform said function by a protein encoded by said functional KUBO gene.According to the present invention, the microorganism of the invention has a non-functional KU80 gene if the capacity mentioned above it 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 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.

In another preferred embodiment according to the present invention, the expression "KUBO 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. In a preferred embodiment of the invention, the microorganism of the invention has the entire deleted KUBO gene. The determination of the functionality of the KUBO 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.

In another particular and preferred embodiment, the microorganism of the invention comprises a gene that encodes an enzyme with C'6 3-ketoacyl CoA synthase activity.

The term "C16 3-ketoacyl-coA synthase", as used herein, refers to a polypeptide belonging to the E.C. family of enzymes. 2.3.1.199 and which catalyzes the addition of two carbons to a 16-carbon fatty acid at its carboxyl end. Thus, the enzyme with C16 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.

Preferably, the enzyme with C16 3-ketoacyl-CoA synthase activity catalyzes the aforementioned elongation reaction using 16 carbon fatty acids as the sole substrate. In a particular and preferred embodiment of the invention, 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. However, enzymes with C16 3-ketoacylCoA 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 on fatty acids of 16 carbon atoms than over carbon atoms of greater (for example, fatty acids of 18 carbon atoms) or of shorter length (for example, fatty acids of 14 carbon atoms) Thus, enzymes with C16 3-ketoacyl-CoA synthase activity suitable for use in the The present invention includes 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 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 least 40 times, at least 50 times, at least 60 times, at least 70 times, to l 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 16-carbon fatty acid at its 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.

The terms "specificity", "greater specificity" as well as techniques that allow to determine the specificity of an enzyme, have been previously defined herein. In a preferred embodiment, it is understood that an enzyme with C16 3-ketoacyl-CoA synthase activity is specific against a substrate of 16 carbon atoms if its substrate specificity is greater than that observed against a substrate with a different number of carbon atoms and present at the same number of unsaturations as the substrate of 16 carbon atoms.

In a more particular and preferred embodiment of the invention, said gene encoding an enzyme with e16 3-ketoacyl-CoA synthase activity is the R. loruloides EloR1 gene whose enzyme comprises the sequence shown in SEO ID NO: 6 or a functionally variant equivalent thereof. The term "functionally equivalent variant" has been defined earlier in this document. Specifically, the functionally equivalent variants of said el6 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. By way of illustration, 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 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.

Functionally equivalent variants of said C'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 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 SEO ID NO: 6 sequence of said C16 3-Ketoacyl-CoA synthase indicated above.

In an even more particular and preferred embodiment of the invention, said gene encoding an enzyme with C16 3-ketoacyl-CoA synthase activity consists of SEO ID NO: 6

Procedure to obtain a microbial biomass rich in oil rich in very long chain fatty acids

In another aspect, 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 i) culturing the microorganism of the invention in a culture medium that

35 comprises at least one source of carbon and at least one source of

nitrogen, under conditions suitable for the growth of said

microorganism, and ii) separate the microbial biomass from the culture broth,

The term "microbial biomass", as used in the present invention, 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. As a renewable energy source, biomass can be used directly or indirectly, after conversion into another type of product such as biofuel. In the particular case of the present invention, microbial biomass is enriched in oil rich in very long chain fatty acids.

The term "oil", as used herein, 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. In addition, said oil may comprise free fatty acids, and other fat-soluble substances such as phospholipids, sphingolipids or sterols. Typically, the oil is composed of triacylglycerols in more than 90% of the total composition.

The term "microbial biomass enriched in oil rich in very long chain fatty acids", as used in the present invention, 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.

In a particular embodiment of the invention, 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.

In a more particular embodiment, 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. .

In an even more particular embodiment of the invention, gondoic acid represents at least 20% (w / w) with respect to the fatty acid tolal of said microorganism. In another even more particular embodiment of the invention, erucic acid represents at least 20% (w / w) with respect to the total fatty acids of said microorganism. In another even more particular embodiment of the invention, the nervous acid represents at least 5% (w / w) with respect to the total fatty acids of said microorganism. Techniques for determining the fatty acid content mentioned above in the context of the first aspect of the invention.

The term "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.

In a first stage, 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.

The term "cultivate", as used in the present invention, refers to the process of planting, maintaining and causing microorganisms to develop on suitable culture media.

The term "culture medium ~, as used in the present invention, refers to a liquid, semi-solid or solid medium that has the necessary nutrients to allow, under favorable conditions of pH, temperature and oxygenation, growth of microorganisms In a particular embodiment, the culture medium is a liquid medium Culture media suitable for cultivating microorganisms are widely known in the art Among other nutrients, the culture medium comprises a carbon source and a source of Non-limiting examples of suitable liquid culture media for carrying out the first process of the invention include MB032 medium (composition NH4N03 0.7 gIL, CaCI2.2H20 0.4 gIL, MgS04 .7H20 0.4 gIL, KH 2P04 0.75 gIL, macerated liquid corn 9.6 giL pH 6.0, glycerin 110 giL), medium MB03-11 (macerated corn liquid 9.6 giL, wheat straw hydrolyzate sugars 110 giL, pH 6.0) And medium MB03-12 (liqu Macerated from corn 9.6 gIL, sugars from beet molasses 40 gIL, pH 5.0). In a particular embodiment, the culture medium is a solid medium. Non-limiting examples of solid culture media suitable for carrying out the first process of the invention include MB03-2 medium (composition NH4N03 0.7 gIL, CaCI2 .2H20 0.4 gIL, MgS04.7H20 0.4 gIL, KH2P04 0.75 gIL, macerated liquid corn 9.6 gIL pH 6.0, glycerin 110 gIL, 2% agar).

In a particular embodiment, the carbon source is a lignocellulosic biomass hydrolyzate.

The term "biomass hydrolyzate", as used herein, 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

The term "saccharification" or uhydrolysis of biomass, "as used herein, refers to the production of fermentable sugars from polysaccharides.

The term "fermentable sugar", as used herein, 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.

The terms "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. Examples of 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 of wheat, barley, barley straw, hay, rice, rice straw, millet, paper waste, paper, pulp, woody or herbaceous processing waste, fruit or vegetable pulp, grain distillate products, herbs, husks of rice, cotton, hemp, flax, sisal, 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. In some embodiments, 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, canola 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 wood hard, such as poplar wood, softwood or a combination thereof).

In some embodiments, 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. In some embodiments, the biomass comprises a kind of fiber while in other alternative embodiments, the biomass comprises a mixture of fibers that originate from different biomass. In some embodiments, the biomass may also comprise transgenic plants that express ligninase and / or cellulases.

The term "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 biomass. In some embodiments, 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%. In some embodiments, the suspension is washed or treated with acid before pretreatment. In some embodiments, 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 "pretreated" when it has undergone physical and / or chemical procedures to facilitate saccharification. In some embodiments, 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.

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 inland surface area reducing the degree of polymerization and cellulose crislalinity, causing the separation of 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. The removal of solvents from the system is necessary, since they inhibit the growth of organisms.

Treatment with ionic liquids (for example, with a solution of sodium chloride) 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 160-260oC (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. , CyJJamcyces sp. , NeocaJJimastix sp., Orpinomyces sp., Piromyces sp., Sporotrichum thermophile, Scytalidium thermophiJJu, Thermonospora cubata, RhodosporiJJum rubrum, CeJJulomonas fimi, Clostridium stercocarium, BaciIJus polymyxa, Pyrococcus furiosus, Acidothermus ceJJulotycus, Saccharophagus degradans, etc.

As the person skilled in the art will understand, lignocellulosic biomass hydrolyzate can be obtained from different plant origins or by-products thereof. The term "by-product", as used in the present invention, refers to the product resulting from subjecting said plant to physical and / or chemical procedures. In a particular embodiment, 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. In a preferred embodiment, said hydrolyzate comes from wheat straw. In another preferred embodiment, said hydrolyzate

It comes from cane bagasse. In another preferred embodiment, said hydrolyzate comes from empty bunches of oil palm.

Preferably, 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.

In another particular embodiment, 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. As the person skilled in the art will understand, the proportion of hydrolyzate and glycerol may vary so that the growth and lipid production conditions of the microorganism of the invention are optimal. Thus, preferably 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.

In another particular embodiment, the carbon source is selected from the group consisting of glucose, glycerol, glycerin, molasses, xylose, arabinose, mannose, fructose, acetate, starches and combinations thereof. In a preferred embodiment, the carbon source is glucose. In another preferred embodiment, the carbon source is xylose. In a more preferred embodiment, the concentration of xylose in the culture medium is 20 gIl. In another more preferred embodiment, the concentration of xylose in the medium is 40 gIl.

In another particular embodiment, 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. . In a preferred embodiment, the nitrogen source is an ammonium salt, preferably ammonium chloride.

In another particular embodiment, the culture medium comprises solid inhibitors. The term "solid inhibitors", as used in the present invention, refers to compounds that inhibit microbial metabolism and negatively affect the growth of the organism. In a more particular embodiment, 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.

Additionally, if desired, 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. Typically, 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). In preferred embodiments, the microorganism of the invention is genetically engineered to favor the accumulation of very long chain fatty acids with at least one gene encoding an enzyme with C18 3-ketoacyl-CoA synthase activity and, with the deletion of the FAD2 and , additionally with a gene encoding an enzyme with C'6 3-ketoacylCoA synthase activity.

Methods for the cultivation of microorganisms are standard in the art and are widely known to those skilled in the art. 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.

The term "conditions suitable for the growth of the microorganism of the invention", as used in the present 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.

In a particular embodiment, the conditions suitable for the growth of said microorganism of step i) comprise

a temperature in a range between 18 oC and 37 oC, preferably between 23 oC and 32 oC, more preferably between 28 oC and 30De, a dissolved oxygen concentration of at least 20%, and / or constant agitation

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.

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. The person skilled in the art will understand that it is possible to carry out 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). The person skilled in the art will understand that it is possible to carry out 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).

Once the desired amount of biomass and / or the intracellular amount of oil rich in desired long chain fatty acids has been reached, in a second stage, 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.

In a particular embodiment, 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, decantation and combinations thereof.

In a particular embodiment, the first process of the invention further comprises drying the microbial biomass obtained in the second stage.

Microbial biomass

In another aspect, 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". The term "microbial biomass" has been described in the second aspect of the invention and is applied equally in the present aspect.

In a particular embodiment, 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.

In a particular embodiment, 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.

In a more particular embodiment, 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. .

In an even more particular embodiment of the invention, gondoic acid represents at least 20% (w / w) with respect to the total fatty acids of said microorganism. In another even more particular embodiment of the invention, erucic acid represents at least 20% (w / w) with respect to the total fatty acids of said microorganism. In another even more particular embodiment of the invention, the nervous acid represents at least 5% (w / w) with respect to the total fatty acids of said microorganism. Techniques for

Determining the fatty acid content mentioned above in the context of the first aspect of the invention.

Procedure to obtain a preparation enriched in oil rich in very long chain fatty acids

In another aspect, 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:

i) culturing the microorganism of the invention in a culture medium that

comprises at least one source of carbon and at least one source of

nitrogen, under conditions suitable for the growth of said

microorganism,

ii) separate the microbial biomass from the culture broth,

iii) extract the oil rich in very long chain fatty acids from biomass

microbial obtained in step (ii).

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. The terms "microorganism of the invention", "cultivate", culture medium "," suitable conditions for the growth of the microorganism of the invention "," microbial biomass "as well as the particular and preferred embodiments of said terms have been detailed above and they are interpreted in the same way in the context of the second process of the invention.

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.

The term "culture broth", as used herein, refers to the culture medium obtained after cultivation of the microorganism of the invention and which comprises 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.

In a particular embodiment, the mechanical extraction method is performed using screw press, French press or ball mill.

Thus, in another particular embodiment, the solid-liquid extraction method is performed using a water immiscible organic solvent. The term "organic solvent", as used herein, refers to a substance that dissolves a solute whose molecules contain carbon atoms. The term "water immiscible organic solvent", as used herein, refers to an organic solvent with little or no ability to mix with water. Illustrative non-limiting examples of 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 . Thus, in a preferred embodiment, 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.

In a particular embodiment, the second process of the invention further comprises drying the microbial biomass obtained in the second stage.

Procedure for obtaining biolubricants The preparation enriched in 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.

Therefore, in another aspect, 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:

i) obtain a preparation enriched in oil rich in fatty acids of

very long chain where such obtaining is carried out according to the

procedure 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 the

stage (i)

iii) convert the oil rich in very long chain fatty acids obtained in the

stage (ii) in biolubricants.

The term "biolubricant", as used in the present invention, 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. In a preferred embodiment of the invention said biolubricants are obtained from a microbial biomass enriched in oil rich in very long chain fatty acids. The term "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.

In a first stage, 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.

In a second stage, 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.

The term "refining" or "refining", as used in the present invention, 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

substance refining For example, the refining of oils, such as an oil rich in gondonic, erucic or nervous acid, is often achieved through distillation or fractionation. 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.

In a particular embodiment, 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. In this reaction 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. Typically, the reaction is allowed to proceed with constant stirring for 20 to 36 hours at a temperature between 40 and 70 ° C. Preferably, the alcoholysis is carried out with alcohols having 1, 2, 3 or 4 carbons, with methanol being the most preferred alcohol.

In a third stage, the third process of the invention comprises converting the rich oil into refined very long chain fatty acids obtained in step ii) into biolubricants. The person skilled in the art will understand that there are numerous processes in the art for converting fatty acids, such as gondonic acid, erucic acid or nerve acid, into biolubricants that include, without limitation the procedures shown in US8357643 82. Such methods are based on a stage of chemical modification of 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.

Typically, chemical modifications made to said free fatty acids include alkylation reactions, addition of a radical, acylation, eno reactions, aminoalkylation, co-oligomerization, hydroformylation, epoxidation and yyloxylation.

By way of illustration, non-limiting obtaining 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 is carried out 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 means of a first epoxidation step of the fatty acids, a second hydrogenation step of said fatty acids, thus obtaining an intermediate with hydroxyl groups and a third acylation step of said hydroxyl groups with an acyl agent having as a result obtaining a biolubricant. Typically, basic catalysts comprise a tertiary amine such as triethylamine.

Uses of the invention

In another aspect, 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.

In another aspect, 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 .

In another aspect, 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.

The terms "microorganism», "microbial biomass", "biolubricants" and their particularities have been described in the context of the microorganism and the first process of the invention, and are applicable to the first, second and third use of the invention. Likewise, the particular and preferred embodiments of the microorganism and of the first, second and third methods of the invention are equally applicable.

The invention is described in detail below by means of the following examples, which are to be interpreted as merely illustrative and not limiting the scope of the invention.

EXAMPLES

Example 1: Construction of integrative cassettes in Rhodosporidium toruJoides. From the sequences deposited in Genbank, genes encoding enzymes with C1s-ketoacyl-CoA synthase activity (elonga5a) were synthesized. The sequences of these polypeptides are those shown in SEO ID NO: 1-4 and 6. The fragments obtained were cloned into a vector under the control of the glycerol-3-phosphate dehydrogenase promoter.

R. toruloides and the Tn05 terminator of Agrobacterium tumefaciens resulting in pNEOL86119-148-149. Next, these vectors were digested with the mitochondrial endonuclease I-Scel and each expression cassette, containing the promoter, the elongase gene and Tnos, was cloned into the vectors pNEOL85, pNEOL111 or pNEOL112, originating the vectors pNEOL120-136- 154-155. The pNEOL85 vector contains another expression cassette containing the genetics resistance gene with codon use of R. toruloides (G418Rt) under the control of the phosphoglycerate kinase promoter of R. toruloides (pPGK47) and the T35S terminator of the mosaic virus of cauliflower. The pNEOL111 vector is identical to the pNEOL85 vector unless its pPGK promoter is truncated and only contains about 580 bp (pPGK47-t). The vector pNEOL112 is identical to the vector pNEOL111 unless the selection marker is changed to a hygromycin resistance gene (hphRt). Finally, the pNE0L120-136-154-155 vectors were cut with Pacl and a fragment of about 4 kb, bearing the different expression cassettes, cloned into the Ti plasmids pNEOL57 or pNEOL105, giving rise to the integrative cassettes pNEOL 126- 136-163-164 (Figure 1).

Example 2: Transformation of R. toruloides The transformation of R. toruloides CECT 13085 was performed using the Agrobacterium tumefaciens-mediated transformation system (ATMT). For this, the pre-inoculum of A. tumefaciens was grown by carrying the integrative plasmid in the MM growth medium (prepared according to Hooykaas and cO /., 1979) and the pre-inoculum of R. toru / oides CECT 13085 in YPD medium (yeast extract 10g / L, glucose 20g / L, peptone 20g / L) for 24h. From the pre-inoculum of Agrobacterium, 10 mL of MI medium is inoculated (prepared according to Bundock et al., 1995) supplemented in acetosiringone at 200¡1M with an initial D0660 of 0.5, and incubated 6h at 30DC, with 250 rpm agitation. In parallel, strain R. is inoculated.

CECT 13085 toruloids in 10 mL of YPD with an initial 00600 of 1.5. After 6h of incubation, 1001-11 of each culture are collected and a ca-culture is carried out on a 0.45 IJm nitrocellulose membrane in MI medium supplemented in 200 IJM acetosyringone, incubated at 25 ° C for 72h. Next, the ca-culture or transformation mixture is

5 collect 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 I-Ig / mL) or

10 hygromycin (40 IJgfmL). The integration of the cassettes containing the different elongases in the yeast genome was checked by PCR using specific oligonucleotides 021 + (SEO ID NO: 7), 022- (SEO ID NO: 8).

The presence of the cassettes in the yeast genome was also checked by

15 hybridization. For this, genomic DNA of the transformants was cut with a restriction enzyme (Kpnl) and in each case they hybridized with a probe, amplified with oligonucleotides 065+ (SEO ID NO: 9) and 065- (SEO ID NO: 10 ), specifies the conserved domain of elongasas. The probe was labeled according to the protocol described in the Rache kit (DfG High Prime ONA labeling and detection Starter kit 1). Both the trials of

20 hybridization such as PCR 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 SEO ID NO: 1, or the LaKCSRt gene (Lunaria annua) according to SEO ID NO: 2 were grown on 25 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

30 (> 20% of the total fatty acids) as well as small amounts of erucic acid, all of them 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

C1600 IC1800 IC180 1 ~ 91 C1802 ~ 9,121 C20D IC20: 1 ~ 11 1 C2n ~ 13

% AG;:.: (20

-

CECT 13085 21.2 10.0 42.2 19.9 5.3 0.0 0.0 5.3

T126-3

17.3 2.3 28.8 16.0 8.9 22.1 4.6 35.6

T126-4

16.8 2.1 27.9 14.0 9.0 24.3 5.9 39.2

AtFAERt

S126-7 13.5 1.5 23.5 15.9 11.2 25.0 9.3 45.5

T126-1O

18.0 2.5 29.7 16.6 9.1 20.7 3.5 33.2

T126-25

16.0 3.6 28.8 15.3 6.9 22.4 7.2 36.4

T126-28

12.3 0.0 28.2 21.0 10.3 21.2 7.0 38.5

S163-15

15.0 3.5 27.8 16.9 5.9 20.6 4.2 30.7

S163-18

17.0 3.5 29.2 14.1 5.1 21.0 3.9 30.1

T163-27

16.7 3.9 30.1 14.5 5.1 20.3 3.5 28.9

LaKC5Rt

S163-28

15.7 3.6 28.2 15.9 5.5 21.0 4.1 30.6

S163-36

15.2 3.7 28.5 15.9 5.6 20.8 4.2 30.6

T163-39

14.2 3.1 26.5 15.2 5.8 22.9 5.6 30.6

5 Clan T126-25 was grown in 500 mL flasks containing 100 mL of MB03-2 culture media (NH4N0 3 0.7 gIL, CaCI2.2H20 0.4 gIL, MgS04 .7H20 0 .4 gIL, KH, P0 4 0.75 giL, corn macerated liquid 9.6 giL at pH6, glycerin 110 giL), MB03-11 (macerated corn liquid 9.6 gIL, wheat straw hydrolyzate sugars 110 gIL, at pH 6.0) and MB03-12 (macerated corn liquid 9.6 gIL, beet molasses sugars 40

10 gIL, at pH 5.0). 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. From the biomass of each flask, the oil content was determined by gravimetry by extraction with n-hexane. The fatty acid composition of the oils was determined by CG-FID. The results obtained

15 (Table 2) again confirmed the presence of gondoic acid (> 20% of total fatty acids) and small amounts of erucic acid. On the contrary, 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 2: Gondoic acid production. Composition of the oil (fatty acids) in recombinant clones expressing the AtFAERt gene (liquid media)

C14: IC16: IC16: IC18: IC18: 1 IC18: 2 ~ 9, IC20: IC20: 111 IC22: 1 ~ O O 1 O ~ 9 12 O 11 13

% AG ~ C20

MB032

CECT 13085 T126-25 1.4 27.9 1.1 7.0 51.7 9.7 1.3 0.0 0.0 1.3 22.4 2.7 2.6 35.9 7.7 1.2 22, 6 2.7 1.3 26.5

MB0311

CECT 13085 T126-25 1.6 28.0 0.8 7.9 53.4 6.7 1.4 0.0 0.0 1.1 16.9 1.8 1.8 36.3 7.0 1.9 24, 8 8.3 1.4 35.0

MB0312

CECT 13085 T126-25 1.2 25.0 0.6 4.6 55.7 10.6 2.4 0.0 0.0 0.9 17.5 1.4 1.7 35.5 13.1 3.3 20, 2 5.1 2.4 28.6

Example 4: Production of oil enriched in gondoic acid by expression

5 of two ketoacyl-CoA synthases Clones containing at least one copy of the AtFAERt gene (Arabidopsis thaliana) according to SEO ID NQ: 1, and at least one copy of the gene of the Elo1Rt gene (R. toruloides) according to SEO ID NO: 6 were grown in 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.

10 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 strain

15 parental 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 Elo1Rt gene

C16: 0 I C18: 0 I C18: 1 ~ 9 I C18: 2 ~ 9.12 I C20: 0

IC20: 11111 I C22: 1 ~ 13 % AG> (20

CECT 13085 T116-1 26-26 T116-126-37 T116-126-38 T116-126-40 T116-126-64 T116-126-68

22.8 12.1 43.5 17.5 17.9 13.7 4.7 31.2 16.5 6.9 12.9 4.7 31.0 15.9 6.8 13.5 5, 5 30.8 16.5 6.6 13.1 5.4 31.6 16.5 6.7 10.4 2.5 25.9 16.4 8.2 15.3 5.4 30.7 17 , 2 7.0 0.0 0.0 21.1 3.9 21.3 4.6 20.4 3.9 20.1 3.8 22.0 12.4 18.7 3.3 3.9 31.9 32.7 30.9 30.6 42.6 29.0

The T116-126-64 clan was grown in 500 mL flasks containing 100 mL of culture media MB03-2, MB03-11 or MB03-12. The cultures grew at 250 rpm, 30 ° C for 96

h. After the cultures, the cells were collected by centrifugation and the biomass of each flask was dried in an oven at 65 ° e for 24 hours. Oil extraction and

The fatty acid composition was 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 Elo1 Rt gene (liquid media)

CECT 13085MB03-2 T116-12664 CECT MB0313085 11 T116-12664 CECT MB0313085 12 T116-12664

C1 4: IC16: IC16: IC18: IC18: 1 or 1 C18: ~ 9, IC20: IC20: 1l'r.1 IC22: 1l'r.1 OO 1 O 9 12 O 1 3 1.4 28.6 1 , 2 6.7 52.4 8.6 1.1 0.0 0.0 1.2 19.9 1.3 2.8 32.6 7.3 1.6 24.2 4.5 1.7 28.8 0.8 7.7 53.4 6.3 1.3 0.0 0.0 0.9 12.8 1.3 1.2 33.9 7.3 2.2 24.8 9, 2 1.3 26.4 0.7 4.8 55.7 9.0 2.1 0.0 0.0 0.9 14.9 1, 1 1.2 32.0 10.7 3.3 23 , 3 7.8 % AG ~ C20 1.1 30.3 1.3 36.1 2.1 34.4

Example 5: Production of oil enriched in erucic acid

15 Clones containing at least one copy of the CraFAERt gene from Crambe abyssinica according to SEO ID NOA 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 acid composition

20 fatty was determined by CG-Flo. 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 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.

25 Table 5: Erucic acid production. Composition of the oil (fatty acids) in recombinant clones expressing the CraFAERt gene

C16: 1 C16: 1 C18: 1 C18: 1 ~ 1C18: 2 ~ 9, 11 C20: 1 C20: 1 ~ 11 (22: 1111 1 C24: 1 ~ 1 01092 01 3 5

% AG ~ (20

CEa 13085 T170-13 T170-15 T170-16 T170-25 T170-33 T170-39

24.3 3.5 4.0 43.5 16.7 8.0 0.0 0.0 0.0 14.8 10.0 1.6 21.9 12.0 9.8 2.5 24, 0 2.2 18.1 8.2 1.6 26.5 13.5 9.2 3.8 17.7 1.3 14.8 9.9 1.4 21.8 12.1 8.9 2 , 8 24.7 2.4 16.9 8.8 1.6 24.8 12.9 9.1 3.3 19.7 1.8 17.5 8.0 1.7 26.0 13.2 9.0 3.6 17.9 1.8 13.9 10.4 1.6 20.5 13.9 9.5 2.4 23.9 2.7 8.0 38.5 32.1 38.8 33.9 32.2 38.5

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 in MB03 · -2 medium plates at 30 ° C for 2 days.

5 Table 6 shows the results of the analysis of some clones and of the parental strain CECT 13085. Approximately 0.5 9 of cells were collected and the biomass was dried in an oven at 6Soe 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 acids

10 fatty), as well as small amounts of gondoic and erucic acid, all of them 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. Table 6: Nerve acid production. Composition of the oil (fatty acids) in recombinant clones expressing the CgKCSRt gene

% AG ~

6: IC ~ 6: IC68: IC1 ~: 1fi IC18:; M, 1 IC ~ O: IC20 ~ M1 IC22 ~ M1 IC24 ~ 1M I

C6C20 CECT

21.6 1.1 6.8 42.5 21.2 5.6 0.0 0.0 0.0 5, 6

13085 S164-28

16.3 2.6 2.4 29.3 23.1 9.2 3.2 5.1 4.3 21.7 T164 -31

17.0 2.7 2.0 30.2 18.9 7.6 2.8 5.7 7.7 23.8 T164-59

12.5 6.9 1.4 23.2 17.6 7.1 2.0 5.6 19.1 33.9 T164 -86

17.4 3.1 2.5 29.2 19.9 7.2 3.8 5.9 5.8 22.7 Q164-88

15.6 3.3 2.2 27.4 23.0 8.4 3.1 5.7 6.4 23.6 T164-90

18.7 2.8 2.8 30, S 19.4 6.6 4.1 5.6 4.6 20.8

Clone T1 64-59 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.

The results obtained (Table 7) confirmed the presence of nerve acid (more than one

5% of total fatty acids) as well as similar amounts of gondoic acids and

erect. The oil extracted from the parental strain did not show any of these fatty acids in

His composition. In these cases the content of very long chain fatty acids in the

Oil was increased more than 10 times.

Table 7: Nerve acid production. Composition of oil (fatty acids) in Recombinant clones expressing the CgKCSRt gene (liquid media)

C16: IC16: IC18: IC18: 1 IC18: 2 / \ 9, IC20: IC20: 1 / \ IC22: lI \ IC24: 111 010 / \ 912 01113 15

% AG ~ C20

MB032

CECT 13085 Tl64-S9 29.1 1.4 7.6 50.7 8.6 1.1 0.0 0.0 0.0 25.1 2.1 3.7 35.7 8.1 1.5 6.0 7, 8 5.4 1.1 20.6

MB0312

CECT 13085 Tl64-S9 0.0 25.8 0.0 6.0 55.5 8.9 2.5 0.0 0.0 23.6 1.5 2.3 39.4 10.8 3.3 4.9 5, 5 5.9 2.5 19.7

Claims (29)

one.

A microorganism of the genetically modified Rhodosporidium toruloides species

with at least one gene encoding an enzyme with 3-ketoacyl-CoA synthase activity.

s

2. A microorganism according to claim 1, wherein said gene is expressed under

control of a constitutive promoter and / or where said gene contains cadons

optimized for expression in R. toruloides.

3.

A microorganism according to claims 1 or 2, wherein said enzyme with

10

3-ketoacyl-CoA synthase activity comprises at least one selected sequence

of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and variants

functionally equivalent thereof.

Four.

A microorganism according to claims 1 to 3, wherein the microorganism of the

fifteen

Rhodosporidium toruloides species in which the gene encoding a

enzyme with C'8 3-ketoacyl-CoA synthase activity belongs to the strain R. toruJoides

CECT 13085.

twenty

5. A microorganism according to any one of claims 1 to 4, wherein said

microorganism additionally comprises a gene that encodes an enzyme with

C'6 3-ketoacyl-CoA synthase activity.

6.

A microorganism according to claim 5, wherein said gene encoding a

25

Enzyme with C'6 3-ketoacyl-CoA synthase activity comprises the sequence SEQ ID NO: 6

or a functionally equivalent variant thereof.

7.

A procedure to obtain a microbial biomass enriched in oil rich in

very long chain fatty acids comprising:

30

i) culturing a microorganism according to any one of claims 1 to 6 in a

culture medium comprising at least one carbon source and at least

a source of nitrogen under conditions suitable for the growth of said

microorganism; Y

46

ii) separate the microbial biomass from the culture broth.

8.

Process according to claim 7, wherein the carbon source is selected from the group consisting of glucose, sucrose, glycerol, molasses, xylose, arabinose, mannose, fructose, acetate and combinations thereof.

9.

Process according to claim 8 wherein the carbon source is a lignocellulosic biomass hydrolyzate.

10.

Method according to claim 9, wherein the hydrolyzate is obtained from wheat straw, sugar cane bagasse, corn bagasse, empty bunches of oil palm, oil palm pruning, oil palm fiber, vine pruning, pruning Olive tree and combinations thereof.

eleven.

Method according to claim 8, wherein the carbon source is glucose.

12.

Process according to claim 8, wherein the carbon source is xylose.

13.

Process according to any one of claims 7 to 12, wherein the nitrogen source is selected from the group consisting of yeast extract, peptone, macerated corn liquid, urea, sodium glutamate, inorganic nitrogen sources and combinations thereof.

14.

Process according to claim 13, wherein the nitrogen source is an ammonium salt, preferably ammonium chloride.

fifteen.

Process according to any of claims 7 to 14, wherein the culture medium contains solid inhibitors selected from: acetic acid, formic acid, levulinic acid, cumaric acid, ferulic acid, succinic acid, 4-hydroxybenzaldehyde, vanillin, vanillic acid, syringaldehyde, acid 4-hydroxybenzoic, catechol, guaiac, syringic acid, furfural, 5-hydroxymethylfulfural and combinations thereof.

16.

Process according to any one of claims 7 to 15, wherein the conditions suitable for the growth of said microorganism of step (i) comprise temperature in a range between 18 ° C and 37 ° C dissolved oxygen concentration of at least 20% , and / or constant agitation.

17.

Method according to any of claims 7 to 16 wherein step (ii) is performed by a method selected from the group consisting of filtration, microfiltration, centrifugation and combinations thereof.

18.

Method according to any of claims 7 to 17, further comprising drying the microbial biomass of step (ii).

19.

Process according to any of claims 7 to 18, wherein said very long chain fatty acids represent at least 30% (w / w) with respect to the total fatty acids of said microorganism.

twenty.

Process according to claim 19, wherein 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, eicosadiene acid, docosadienoic acid, or combinations of the same.

twenty-one . Process according to claim 20, wherein said gondoic acid represents at least 20% (w / w) with respect to the total fatty acids of said microorganism.

22

Method according to claim 20, wherein said erucic acid represents at least 20% (w / w) with respect to the total fatty acids of said microorganism.

2. 3.

Method according to claim 20, wherein said nerve acid represents at least 5% (w / w) with respect to the total fatty acids of said microorganism.

24.

Microbial biomass enriched in oil rich in very long chain fatty acids obtained according to the method of any of claims 7 to 23.

25.

Method for obtaining a preparation enriched in oil rich in very long chain fatty acids comprising:

26.

Process according to claim 25, wherein the extraction of step (iii) is carried out by mechanical methods, by solid-liquid extraction methods or combinations thereof.

27.

Method according to claim 26, wherein the method of mechanical extraction is performed using screw press, French press or ball mill.

28.

Process according to claim 26, wherein the solid-liquid extraction method is performed using a water immiscible organic solvent.

29.

Process according to claim 28, wherein 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.

30

Process according to any of claims 25 to 29, further comprising drying the microbial biomass of step (ii).

i)

culturing a microorganism according to any one of claims 1 to 6 in a

culture medium comprising at least one carbon source and at least

a source of nitrogen under conditions suitable for the growth of said

microorganism;

ii)

separate the microbial biomass from the culture broth; Y

Ii)

extract the rich oil in Very long chain biomass fatty acids

microbial obtained in step (ii).

31. Use of the microorganism according to claims 1 to 6 to obtain a microbial biomass enriched in oil rich in very long chain fatty acids, to obtain a preparation enriched in very long chain fatty acids or to obtain biolubricants