EP3011010A1 - Verfahren zur herstellung von lipiden durch mikroorganismen und verwendung dieser lipide - Google Patents

Verfahren zur herstellung von lipiden durch mikroorganismen und verwendung dieser lipide

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Publication number
EP3011010A1
EP3011010A1 EP14731653.3A EP14731653A EP3011010A1 EP 3011010 A1 EP3011010 A1 EP 3011010A1 EP 14731653 A EP14731653 A EP 14731653A EP 3011010 A1 EP3011010 A1 EP 3011010A1
Authority
EP
European Patent Office
Prior art keywords
bacteria
glycerol
phosphate
medium
lipids
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14731653.3A
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English (en)
French (fr)
Inventor
France Thevenieau
Marie-Joëlle Virolle
Thierry Dulermo
Maxime VERGNE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite Paris Sud Paris 11
Avril SARL
Original Assignee
Universite Paris Sud Paris 11
Avril SARL
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Application filed by Universite Paris Sud Paris 11, Avril SARL filed Critical Universite Paris Sud Paris 11
Priority to EP14731653.3A priority Critical patent/EP3011010A1/de
Publication of EP3011010A1 publication Critical patent/EP3011010A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
    • C07C69/22—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety
    • C07C69/30—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety esterified with trihydroxylic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/52—Esters of acyclic unsaturated carboxylic acids having the esterified carboxyl group bound to an acyclic carbon atom
    • C07C69/533—Monocarboxylic acid esters having only one carbon-to-carbon double bond
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
    • C10L1/00—Liquid carbonaceous fuels
    • C10L1/02—Liquid carbonaceous fuels essentially based on components consisting of carbon, hydrogen, and oxygen only
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
    • C10L2200/00—Components of fuel compositions
    • C10L2200/04—Organic compounds
    • C10L2200/0461—Fractions defined by their origin
    • C10L2200/0469—Renewables or materials of biological origin
    • C10L2200/0476—Biodiesel, i.e. defined lower alkyl esters of fatty acids first generation biodiesel
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
    • C10L2290/00—Fuel preparation or upgrading, processes or apparatus therefore, comprising specific process steps or apparatus units
    • C10L2290/26—Composting, fermenting or anaerobic digestion fuel components or materials from which fuels are prepared

Definitions

  • the present invention relates to the technical field of the production of lipids by microorganisms, and in particular the use of said lipids.
  • Biodiesel can be made from oleaginous crops, animal fats or recycled fats.
  • Campbell 2008 and Strobel et al. 2008 described the optimization of algal and mushroom growing conditions in different types of bioreactors to maximize lipid and fatty acid yields for biofuel refining.
  • An alternative to photosynthetic lipid production is to use heterotrophic organisms that produce lipids from organic molecules (such as sugars) without light.
  • Oils from single-cell organisms have traditionally been used as special products, for example in health foods, and not as basic chemicals.
  • WO 2009/034217 has described a fermentation method for producing paraffins, fatty acids and alcohols using waste and microorganisms.
  • Application WO 2009/046375 describes the conversion of polysaccharides from biomass into monosaccharides, oligosaccharides, and their transformation into biofuels by using recombinant microorganisms comprising exogenous genes which enable said microorganism to develop on the polysaccharide as the sole source of carbon.
  • US application 2009/0064567 describes the production of biological oils by heterotrophic fermentation of microorganisms using raw materials containing cellulose as the main source of carbon.
  • the application WO2009 / 011480 A1 describes the production of biological oils of cellulosic material depolymerized by microalgae and fungi.
  • WO 2009/009391 A2 describes the preparation of fatty esters by first producing an alcohol composition and supplying this product to a host for the production of a fatty ester.
  • US2011294173 discloses a method for producing lipids from bacteria of the genus Streptomyces by providing as a carbon source organic waste.
  • the invention aims to overcome these disadvantages.
  • One of the aims of the invention is to provide a process for the production of lipids which is inexpensive and which has a high yield.
  • Another object of the invention is to provide culture media for carrying out the process from microorganisms.
  • the description relates to the use of glycerol, or a C3-C5 sugar as a major source of carbon when culturing Actinomycete bacteria for the production of lipids by said bacteria, said bacteria being cultured in a first medium enriched in phosphate and / or nitrogen, and then cultured in a second culture medium depleted of phosphate and / or nitrogen.
  • the invention relates to the use of glycerol, or a C3-C5 sugar as a major source of carbon when culturing Actinomycete bacteria for the production of lipids by said bacteria, said bacteria being cultured in a first phosphate-enriched medium, then cultured in a second phosphate-depleted culture medium.
  • the invention is based on the surprising finding made by the inventors that actinomycetes bacteria grown in the presence of glycerol as a carbon source accumulate a large amount of reserve lipids.
  • the bacteria in order to produce a large quantity of lipids, are first cultured in a medium enriched in nitrogen and / or in phosphate for a certain time (preculture).
  • the sources of phosphate or nitrogen are organic or mineral sources. This first crop is made for a short time and is considered a "preculture”.
  • the bacteria are then transferred to a second medium that still contains glycerol as the major source of carbon, but depleted of nitrogen and / or phosphate. This second culture is the main culture.
  • the nutrients will be called nitrogen and organic or inorganic phosphate.
  • the inorganic or mineral terms will be uniformly used to designate nutrients that are not organic.
  • the culture will be carried out in a nitrogen-depleted environment
  • the culture will be carried out in a medium depleted in inorganic or organic phosphate
  • the culture will be carried out in an environment depleted of inorganic or organic phosphate and nitrogen.
  • the preculture medium of 2.5 to 50 mM of inorganic phosphate, preferably 3 to 40 mM, more particularly 4 to 30 mM, more particularly of 5 to 20 mM, especially 5 to 10 mM inorganic phosphate.
  • the organic phosphate added in such a pre-culture medium is made using the following compounds: phosphate carried by nucleotides derived from nucleic acids such as ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or from adenosine triphosphate (ATP), guanosine triphosphate (GTP), yeast extract ... this list is not exhaustive, and the skilled person will be able to determine the appropriate source of organic phosphate.
  • the inorganic phosphate used in the pre-culture medium is made by means of the following soluble compounds: K 2 HPO 4 / KH 2 PO 4 OR of HPC Nal -1 PC
  • the phosphate concentration inorganic varies from 4mM to 20mM, especially the concentration is about 5mM.
  • the concentration of glycerol in the pre-culture and the culture does not exceed 2M, advantageously 1.5M, in particular 1M.
  • lipid production by Actinomycetes is correlated with the concentration of glycerol present in the culture and pre-culture medium. Dose-dependent or "dose-dependent" lipid production of glycerol will be considered to be up to 2M glycerol.
  • glycerol as the major carbon source of the invention is more advantageous than the conventional use of glucose. Indeed, as shown in the examples below, the lipid production by the actinomycetes is dose dependent, or concentration, glycerol, while high glucose concentrations have the effect of inhibiting the growth and production or accumulation of lipids.
  • the term "major source of carbon” means an amount of carbon compound (s) metabolizable by bacteria and for producing energy, especially in the form of adenosine triphosphate (ATP). ), and allowing the biosynthesis of the organic molecules necessary for the growth and multiplication of said bacteria.
  • the majority source of carbon represents more than 90% of the carbon input in the culture or pre-culture medium.
  • C3-C5 sugars sugars comprising 3, or 4 or 5 carbons. These are at the same time aldoses (polyalcohols comprising an aldehyde function) or ketoses (polyalcohols comprising a ketone function), said C3, C4 and C5 sugars being in their linear or cyclic form, as appropriate, and possibly modified.
  • the lipids thus produced by actinomycetes using glycerol or a C3-C5 sugar are produced in the form of a composition comprising a mixture of fatty acids, said mixture comprising essentially triacylglycerols or TAG.
  • composition essentially comprising triacylglycerols is meant an oleaginous composition comprising more than 75% of trialkylglycerols, in particular more than 80%, in particular more than 85%, more particularly more than 90%, relative to the total amount of lipid. contained in said composition.
  • composition also includes, in a minor manner, free fatty acids, monoacylglycerols and diacylglycerols.
  • the actinomycetes producing lipids according to the invention are essentially bacteria belonging to the following genera: Streptomyces, Rhodococcus, Amycolatopsis, Actinomyces, Arthrobacter, Corynebacterium, Frankia, Micrococcus, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, etc.
  • the invention also relates to the use as defined above, wherein the cultivation in the first medium is carried out for a period of 15 to 120 hours.
  • the preculture is advantageously carried out for a time of about 15 to 120 hours, in particular from 20 to 90 hours, more particularly from 24 to 60 hours, in particular from 36 to 48 hours.
  • the preculture can thus be carried out during 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35.
  • the culture, in the nutrient-poor medium, as defined above, is carried out for a time of about 15 to 120 hours, preferably 20 to 80 hours, especially 72 hours.
  • the concentration of said phosphate varies from 0.5 mM to 2 mM, in particular is 1 mM.
  • the invention relates to the previously defined use, wherein said C3-C5 sugar is a triose, a tetrose or a pentose.
  • the invention also relates to the use as defined above, in which the C 3 -C 5 sugars are: glyceraldehyde, erythrose, threose, ribose, arabinose, xylose , lyxose, dihydroxyacetone, erythrulose, ribulose and xylulose.
  • the C 3 -C 5 sugars are: glyceraldehyde, erythrose, threose, ribose, arabinose, xylose , lyxose, dihydroxyacetone, erythrulose, ribulose and xylulose.
  • the invention relates to the above-mentioned use, wherein said bacteria are actinomycetes of the genus Streptomyces.
  • the invention relates to the aforementioned use, wherein the glycerol is selected from refined glycerol and unrefined glycerol.
  • the unrefined glycerol is advantageous, because of the production costs, it is also advantageous to use essentially pure or refined glycerol, that is to say enriched to at least 90%, especially 95%, in particular. 99%, especially 100%.
  • epichlorohydrin is the most important; it involves the chlorination of propylene to give allyl chloride which is oxidized with hypochlorite to dichlorohydrin and which reacts with a strong base to give epichlorohydrin. Epichlorohydrin is then hydrolysed to give glycerol.
  • Glycerol can also be produced by fermentation processes, for example involving yeasts, from monosaccharides by:
  • glycerol is produced industrially according to two main processes: the saponification of fatty substances or the transesterification of vegetable oils.
  • the saponification reaction is a reaction that produces soap and glycerol from fat and soda.
  • the glycerol resulting from the saponification is very pure (> 99%) and is therefore mainly used for pharmaceutical and cosmetic applications.
  • Glycerol or glycerin
  • VOMEs vegetable oil methyl esters
  • Transesterification is a three-step, reversible series reaction in which triglycerides are converted to diglycerides, converted to monoglycerides, and monoglycerides are converted to esters (biodiesel) and glycerol (co-product).
  • the oils or fats react with a short chain alcohol (usually methanol) in the presence of a catalyst.
  • a short chain alcohol usually methanol
  • This reaction can be carried out by homogeneous catalysis, with catalysts soluble in the reaction medium, or by heterogeneous catalysis, with catalysts totally insoluble in the reagents.
  • homogeneous catalysis is the technique most commonly used in biodiesel production processes. Transesterification can be performed by basic or acidic catalysis. A greater reactivity is generally obtained in basic medium.
  • Titanium alkoxides Ti (OBu) 4 , Ti (OPr) 4 ...,
  • Oxides of various metals such as Sn, Mg, Zn, Ti, Pb ...
  • Acid catalysts are rarely used because of their lower reactivity and the high risks of corrosion in industrial plants.
  • the alcoholates or metal oxides are mainly used for the synthesis of esters of heavy alcohols from different cuts of fatty acid methyl esters.
  • Soda in methanolic solution or sodium methylate are the catalysts selected for the production of biodiesel.
  • the chemical composition of unrefined glycerol varies depending on the production process.
  • components such as methanol, various salts or potassium chloride may be present therein with varying contents in the final product.
  • glycerol represents from 63 to 99% of the total product.
  • the methanol content ranges from 0 to about 25%.
  • the contents of phosphorus and potassium are between 1 and 2% of the dry matter.
  • Sodium is present at 1% of the dry matter.
  • Heterogeneous catalysis has significant advantages in terms of respecting the environment which limits discharges, and a neutral and salt or water-free glycerol results from such a process, for example that described in patent FR-B-2,752. 242.
  • the absence of salts in the reaction products does not, unlike homogeneous catalysis, require purification treatments.
  • the crude glycerol product can be purified or refined by treatment with activated carbon to remove organic impurities, by alkaline treatment to remove unreacted glycerol esters, and by ion exchange to remove salts.
  • the glycerol obtained by a distillation series has a high purity (> 99.5%).
  • the invention relates to the above-mentioned use, wherein said bacteria are lipogenic bacteria.
  • lipogenic bacteria is meant in the invention bacteria which are capable of naturally producing at least 20% of their biomass in lipids.
  • Such bacterial strains are advantageous because they make it possible, when they are cultured according to the method of the invention, to produce large quantities of lipids, representing more than 50% of their biomass.
  • Advantageous lipogenic strains of the invention are the following strains: Streptomyces antibioticus 3137, Streptomyces peuceius, Streptomyces rimosus 2535, Streptomyces coelicolor ATCC 19832, Streptomyces coelicolor ATCC21666, Streptomyces coelicolor Variant M 145, Streptomyces coelicolor Variant M145M1155, Streptomyces coelicolor Variant M145M1146, Streptomyces coelicolor Variant M145M1141, Streptomyces coelicolor Variant M ⁇ 45 ⁇ '1148, Streptomyces coelicolor Variant M145M1142 and Streptomyces coelicolor Variant M145M1144, Streptomyces linolmensis NRRL2936, Streptomyces exfoliatus, Streptomyces cealestius ATCC 15084, Streptomyces lividans 13
  • Another advantageous embodiment of the invention relates to the aforementioned use, wherein said lipogenic bacteria are genetically modified by substitution, deletion or insertion of at least one nucleic acid of their genome, so that they accumulate more lipids. than the original strain.
  • AbsA1 / AbsA2 of the biosynthetic pathway of the peptidic antibiotic "Calcium Dependent Antibiotic” led to an increase in the accumulation of triacylglycerols (TAG) in Streptomyces, under the conditions as described above (ie according to the method of the invention).
  • TAG triacylglycerols
  • strain of Streptomyces coelicolor M145 in which the four major pathways of antibiotic biosynthesis have been deleted (CPK, CDA, RED and ACT) and where two point mutations A262G and C271T have been introduced into the gene.
  • rpsL SC04659, 30S ribosomal protein S12
  • Variant strain M145: M1155 has increased lipid production by compared to M145 and this especially in the presence of glucose. In the presence of glycerol, the accumulation of lipids is strong even in the strain of origin M145 and the various introduced genetic modifications have a much lower impact than in glucose.
  • S. coelicolor strain M145 accumulates about 30% / 6% of its biomass in TAG when grown in R2YE glycerol / 0.1M glucose for 96h, respectively.
  • strain varying from M145, M1155, described in Gomez-Escribano and Bibb, Microbial Biotechnology (2011) 4 (2), 207-215 accumulates 40% of its biomass in TAG when it is cultured in R2YE glycerol 0.2M medium. , in condition of hypercompensation.
  • rpoB and / or rpsL genes can be mutated (by point mutation). These mutations have a positive effect both on the accumulation of reserve lipids and on the production of antibiotics (especially polyketide type) suggesting that these mutations have a positive effect on the generation of acetylCoA.
  • genes or gene combinations involved in the production of reserve lipids can also be mutated:
  • bacterial strains with gene modifications increasing the availability of precursors for lipid synthesis.
  • intermediate metabolites such as glycerol 3-phosphate (G3P) or else acetyl and malonyl coenzyme A (acetylCoA, malonylCoA).
  • bacterial strains with gene modifications increasing the availability of precursors for lipid synthesis.
  • intermediate metabolites such as glycerol 3-phosphate (G3P) or acetyl coenzyme A (acetyl CoA).
  • Glycerol can be metabolized by two potentially competing pathways, both of which lead to the synthesis of acetylCoA:
  • glycerol dehydrogenase SC02598 / SC06754
  • DHA kinase SCO0580, SCO0581 to SCO0576 operon, with divergence regulator, SCO0582
  • SCO7073 SCO7071 operon.
  • the genes coding for the enzymes of the glyoxylate pathway may be advantageous to reduce the expression of the genes coding for the enzymes of the glyoxylate pathway.
  • the SCO0982 gene and the neighboring genes may be deleted or mutated so that their products are no longer synthesized or are no longer functional.
  • the description also relates to a set of culture media comprising glycerol, or a C3-C5 sugar, as a majority source of carbon, said set of culture media comprising:
  • first culture medium enriched with inorganic phosphate and / or nitrogen
  • second culture medium that is poor in inorganic phosphate and / or in nitrogen
  • the description relates to a set of culture media comprising glycerol, or a C3-C5 sugar, as a majority source of carbon, said set of culture media comprising:
  • first culture medium enriched with inorganic phosphate and / or nitrogen
  • second culture medium that is poor in inorganic phosphate and / or in nitrogen
  • the invention also relates to a set of culture media comprising glycerol, or a C3-C5 sugar, as a majority source of carbon, said set of culture media comprising:
  • the description also relates to a set of culture media comprising glycerol, or a C3-C5 sugar, as a majority source of carbon, said set of culture media comprising:
  • culture media are media commonly used by those skilled in the art to allow the growth of actinomycetes bacteria.
  • the HT, YEME, MP5, SL11, R2 or R2YE and modified YEME media may be used.
  • the compositions of different advantageous media are described in the example section, hereinafter.
  • the first and the second culture medium are of the same nature.
  • the first medium is R2YE medium
  • the second medium is preferably R2YE medium, possibly slightly modified, including no longer including sucrose.
  • first medium of a different nature it may be appropriate to choose a first medium of a different nature from the second medium.
  • the description also relates to an assembly comprising:
  • said first and second culture media comprising as majority source of glycerol carbon, or a C3-C5 sugar, in particular a sugar chosen from glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxosis, dihydroxyacetone, erythrulose, ribulose and xylulose and
  • the invention furthermore relates to an assembly comprising:
  • said first and second culture media comprising as majority source of glycerol carbon, or a C3-C5 sugar, in particular a sugar chosen from glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxosis, dihydroxyacetone, erythrulose, ribulose and xylulose and
  • the description also relates to an assembly comprising:
  • said first and second culture media comprising as majority source of glycerol carbon, or a C3-C5 sugar, in particular a sugar chosen from glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxosis, dihydroxyacetone, erythrulose, ribulose and xylulose and
  • the Actinomycetes bacteria are in particular lipogenic strains, in particular streptomyces, chosen in particular from Streptomyces antibioticus 3137, Streptomyces peuceius, Streptomyces rimosus 2535, Streptomyces coelicolor ATCC 19832, Streptomyces coelicolor ATCC21666, Streptomyces coelicolor Variant M 145, Streptomyces coelicolor Variant M145M1155, Streptomyces coelicolor Variant M145M1146, Streptomyces coelicolor Variant M145M1141, Streptomyces coelicolor Variant M 145M 1148, Streptomyces coelicolor Variant M145M1142 and Streptomyces coelicolor Variant M145M1144, Streptomyces linolmensis NRRL2936, Streptomyces exfoliatus, Streptomyces cealestius ATCC
  • the disclosure further relates to a method for producing lipids by Actinomycete bacteria, said method comprising
  • said first and second culture media comprising as a majority / main carbon source of glycerol, or a C3-C5 sugar.
  • the invention relates to a process for producing lipids by Actinomycete bacteria, said process comprising
  • said first and second culture media comprising as a majority / main carbon source of glycerol, or a C3-C5 sugar.
  • the description relates to a process for producing lipids by Actinomycete bacteria, said process comprising
  • said first and second culture media comprising as a majority / main carbon source of glycerol, or a C3-C5 sugar.
  • the invention relates to a method as defined above, in which said bacteria are Actinomycetes of the family Streptomycetes.
  • the invention relates to a method as defined above, wherein said bacteria are lipogenic bacteria, in particular bacteria genetically modified by substitution, deletion or insertion of at least one nucleic acid of their genome, so that they accumulate more fat than the original strain.
  • the invention furthermore relates to a lipid composition comprising lipids that can be obtained by the process as defined above.
  • the percentages being expressed by weight of the total fatty acids of the composition.
  • the fatty acids are predominantly in the form of TAG, that is to say that at least 50% of the fatty acids are in the form of TAG.
  • the fatty acids are as follows:
  • 14-methylpentadecanoic acid isoC16: 0 / isohexadecanoic acid
  • 14-methylpentadecenoic acid iso C16: 1 / iso hexadecenoic acid
  • the invention furthermore relates to the use of the aforementioned process for the manufacture of lubricants, surfactants, coatings (paints, inks, etc.), solvents, food ingredients or as synthesis intermediates for oleochemicals.
  • biodiesel by known trans-esterification processes, for example a trans-esterification process of the triglycerides present in the composition according to the invention or the microorganism extract according to the invention by methanol in the presence of a catalyst, to obtain methyl esters of fatty acids and glycerol.
  • the biodiesel obtained can be used in mixture with fossil fuel or pure diesel fuel.
  • biokerosene by means of a hydrogen treatment.
  • This process consists of a first step aimed at removing the oxygen present in the charges and converting them into a section composed of paraffinic hydrocarbons and a so-called decarboxylation pathway, which leads to hydrocarbons having one carbon atom less than the initial fatty chain, and is accompanied by the formation of CO and C0 2 .
  • the unsaturations of the fatty chains are completely hydrogenated, and the glycerol group is converted into propane.
  • secondary reactions such as shift and methanation reactions can lead to the formation of CO and methane.
  • the isomerizing hydrocracking stage makes it possible to crack parraffinic diesel chains in branched kerosene and in naphtha.
  • the kerosene yield on diesel is about 60% (40% naphtha).
  • the overall yield kerosene on crude oil is of the order of 55%.
  • the description also relates to a process for producing biofuel, lubricants, surfactants, coatings (paints, inks, etc.), solvents, and synthesis intermediates from lipids derived from bacteria Actinomycete, said process comprising
  • said first and second culture media comprising as a major source of glycerol carbon, or a C3-C5 sugar.
  • the invention also relates to a process for manufacturing biofuel, lubricants, surfactants, coatings (paints, inks, etc.), solvents, and synthesis intermediates from lipids derived from Actinomycete bacteria, said process comprising:
  • said first and second culture media comprising as a major source of glycerol carbon, or a C3-C5 sugar.
  • the lipid composition obtained according to the process of the invention can be used to manufacture a biofuel using the process described in the application WO2005093015
  • the process for producing a lipid composition that can be used as a biofuel or as a fuel component from the composition obtained by the process of the invention comprises the said process
  • At least one trans-esterification step in which the composition obtained by the process according to the invention is reacted by heterogeneous catalysis with at least one primary monoalcohol chosen from methanol and ethanol, to give, on the one hand, at least one methyl and / or ethyl ester of the fatty acid (s) of the starting (or more) triglyceride (s) and, secondly, glycerol, these products being free of by-products; and
  • an acetalisation step in which the glycerol is reacted with at least one compound chosen from aldehydes, ketones and acetals derived from aldehydes or ketones.
  • catalysis Two types of catalysis can be envisaged for transesterification of a vegetable oil into methyl (or ethyl) esters from heterogeneous catalysts: catalysis in a batch reactor or continuous catalysis using the fixed bed principle. Generally, one works continuously in fixed bed.
  • Figure 1 is a graph showing the percentage proportion of TAG accumulated by strains.
  • the strains tested are as follows: A: S.antibioticus 3137 II, B: S.peucetius, C: S.rimosus 2535 J11, D: S.coelicolor VariantM145M1155, E: S.lincolnensis NRRL 2936, F: S .exfoliatus, G: S.avermitilis NRRL 8165, H: S.coelicolor M145 Variant M1146, I: S.coelicolor M145 Variant M1141, J: S.coelicolor M145 Variant M1148, K: S.cealestis ATCC 15084, L: S.
  • Figures 2A-C show TAG accumulation by Streptomyces lividans TK24.
  • Figure 2A shows a photo of electron microscopy of streptomyces lividans mycelium grown for 48 hours, from spores, on R2YE medium in the presence of glycerol and inorganic phosphate (5 mM).
  • Figure 2B shows a photo of electron microscopy of streptomyces lividans mycelium from the step described in Figure 2A, and cultured for 48 hours on a fresh R2YE medium in the presence of glycerol and depleted in inorganic phosphate (5 mM).
  • Figure 2C shows a photo of electron microscopy of streptomyces lividans mycelium from the step described in Figure 2A, and cultured for 48 hours on a fresh R2YE medium in the presence of glycerol and depleted in inorganic phosphate (1 mM).
  • the presence of accumulated lipids is identified by the presence of white lipid vesicles.
  • FIG. 3 represents a graph showing the carbonyl band ratio of the TAG / protein amide band I, which corresponds to the percentage of TAG relative to the dry weight, of different streptomyces strains (1: S. coelicolor M145, 2: S. coelicolor M1146, 3: S. coelicolor M1155, 4: S. antibioticus and 5: S. rimosus) cultured for 72 h on R2YE medium in the presence of 0.1 M glucose (bars). white) or in the presence of 0.2M glycerol (black bars).
  • FIG. 4 represents a graph showing the carbonyl band ratio of the TAG / protein amide band I, which corresponds to the percentage of TAG relative to the dry weight, of a strain of Streptomyces (S.lividans TK24) cultivated in the presence of various sources of carbon (C3 or C5) (1: 0.2M glycerol, 2: 0.17M xylose and 3: 0.17M arabinose) for 72 hours on R2YE medium. The same amounts of carbon equivalent were used.
  • C3 or C5 sources of carbon
  • FIGS. 5A-C show the fatty acid composition of the lipids accumulated under the conditions of the invention, during a culture in the presence of glucose, glycerol, or refined glycerol.
  • FIG. 5A represents a graph showing the proportion, in g per mg of dry biomass, of each of the indicated fatty acids, after culturing for 96h in the presence of 0M glucose (white bars), 0.1M glucose (black bars) and 0.2M glycerol (bars with hatching) of a strain of Streptomyces.
  • FIG. 5B represents a graph showing the proportion, in g per mg of dry biomass, of each of the indicated fatty acids, after culturing for 96 h in the presence of 0M of refined glycerol (white bars), 0.1 M of glycerol (black bars ) and 0.2M glycerol (bars with hatching) of a strain of Streptomyces.
  • FIG. 5C represents a graph showing the proportion, in g per mg of dry biomass, of each of the indicated fatty acids, after culturing for 96h in the presence of 0M of refined glycerol (white bars), 0.1M of refined glycerol (bars). black) and 0.2M refined glycerol (bars with hatching) of a strain of Streptomyces.
  • Figures 6A-B illustrate the advantageous effect of mutations on the production of lipids in R2YE glucose medium.
  • FIG. 6A schematically shows the genes involved in the degradation of lipids by the glyoxylate route, which route is essentially present in oleaginous microorganisms.
  • Figure 6B is a graph showing the accumulation of TAG in strains of Streptomyces lividans having a deletion of at least one gene from the glyoxylate pathway.
  • FIG. 7 is a graph showing the carbonyl band ratio of the TAG / protein amide band I, which corresponds to the percentage of TAG relative to the dry weight, of a strain of Streptomyces (S.coeiicolo MS) cultured on a medium comprising carbon source of glucose (white columns) or glycerol (black columns), the carbon sources being at concentrations of 1: 18g / L, 2 .: 45g / L, 3 .: 68g / L and 4 .: 90g / L, for 96h on a medium R2YE devoid of sucrose.
  • the following culture media can be used in the context of the invention. All of the culture media described below are supplemented with C3-C5 sugars, glucose or glycerol: glucose (10 g / l or 18 g / l) or glycerol (0.2 mol / l and 18 g, 4 g / L).
  • the HT medium comprises, for one liter: 1 g of yeast extract (Yeast extract-Difco), 1 g of beef extract (Difco), 10 g of white dextrin (Prolabo), 2 g of NZ amine type A, 20 mg of CoCl 2 , 7H 2 O.
  • DALP culture medium 3 g of yeast extract, 5 g of bacto-peptone, 3 g of malt extract, and 5 mM of MgCl 2 .
  • the DALP medium comprises for one liter: 10 g of bacto-peptone, 5 g of yeast extract and 10 g of white dextrin. The pH is adjusted to 7.0.
  • the MP5 medium comprises for one liter: 7 g of yeast extract, 5 g of NaCl, 36 ml of glycerol, 20.9 g of MOPS. The pH is adjusted to 7.5.
  • the medium SL1 1 comprises for one liter: 25 g of white dextrin, 12.5 g of yeast extract, 1 g of MgSO 4 , 1 g of KH 2 PO 3 , 20.9 g of MOPS. The pH is adjusted to 7.1.
  • Modified YEME culture medium :
  • the modified YEME medium comprises for one liter: 3 g of yeast extract, 5 g of bactopeptone, 3 g of malt extract.
  • the samples taken are then centrifuged, the bacterial pellet is frozen at -80 ° C. and lyophilized in order to obtain the dry mass. It is used for TAG content analysis by Fourier Transformed Infra Red Spectroscopy (FTIRS).
  • FIRS Fourier Transformed Infra Red Spectroscopy
  • a first Petri dish of a sporulation medium (SFM: Soy flour 20g, Mannitol 20g, Bacto agar 15g, tap water qs 1L) is seeded from spores in order to obtain a bacterial mat. This petri dish is incubated for 5 to 10 days until sporulation of the strain.
  • SFM Soy flour 20g, Mannitol 20g, Bacto agar 15g, tap water qs 1L
  • the culture dish consists of 8mL of R2YE medium, covered with a cellophane. This box is seeded with a wooden stick. 20 ⁇ of water are deposited on the cellophane, the rod having previously touched the SFM box containing the sporulated strain is saturated in "fresh spores" and then immersed in the 20 ⁇ of water present on the R2YE box and spread over the entire cellophane surface. The culture dishes are then incubated 72 to 96h to 28 in the dark.
  • the bacteria were recovered by scraping the cellophane then frozen at -80 ⁇ C and lyophilized for dry weight analysis and content of TAG by FTIRS.
  • streptomyces strains considered as lipogenic that is to say strains that produce more than 20% of their biomass in TAG.
  • the bacteria were spread on the surface of a cellophane deposited on medium
  • the inventors compared the accumulation of TAG under conditions of hypercompensation in Pi of different strains of streptomyces, in the presence of glucose (0.1 M) or glycerol (0.2M).
  • the bacteria cultured according to the process of the invention accumulate more TAG than the same bacteria grown in the presence of glucose.
  • the inventors compared the accumulation of TAG under Streptomyces P1 hypercompensation conditions, coelicolor in the presence of C5 (0.17mM) or glycerol (0.2M) sugars.
  • the cells are lyophilized and then crushed to have a powder that is directly analyzable to the FTIR spectrometer as described for example in [Dean et al. 2010 Bioresource Technology, 101 (12), 4499-4507 ⁇ .
  • the bacterium is able to accumulate significant amounts of TAG including with C5 sugars.
  • the inventors have also identified the fatty acid composition accumulated by the bacteria cultured under the conditions of the invention, in the presence of crude glycerol (FIG. 5B) or of refined glycerol (FIG. 5C), the glycerol being present at a concentration varying from 0 to 0.2M.
  • the same culture in the presence of glucose is used as a control ( Figure 5A).
  • the quantification of fatty acids accumulated as TAG in bacteria grown in the presence of unrefined glycerol is as follows: per 100 g of dry biomass:
  • Example 8 Hyperconpensation in a medium comprising glycerol and having a deletion of genes for the degradation of fatty acids.
  • Bacteria (S. coelicolor M145) with deletions of different genes from the glyoxylate pathway were cultured under the conditions of the invention in the presence of glycerol.

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