EP3011010A1

EP3011010A1 - Method for the production of lipids by microorganisms, and use of said lipids

Info

Publication number: EP3011010A1
Application number: EP14731653.3A
Authority: EP
Inventors: France Thevenieau; Marie-Joëlle Virolle; Thierry Dulermo; Maxime VERGNE
Original assignee: Universite Paris Sud Paris 11; Avril SARL
Current assignee: Universite Paris Sud Paris 11; Avril SARL
Priority date: 2013-06-21
Filing date: 2014-06-20
Publication date: 2016-04-27
Also published as: CN105849250A; WO2014202778A1; US20160369308A1

Abstract

The invention relates to the use of glycerol, or a C3-C5 sugar, as the main carbon source during the culturing of actinomyces bacteria for the production of lipids by said bacteria.

Description

Process for the production of lipids by microorganisms, and the use of said lipids

The present invention relates to the technical field of the production of lipids by microorganisms, and in particular the use of said lipids.

The use of renewable biological materials for biofuel production is generally motivated by reduced impacts on climate and food production, securing fuel supply, and other economic factors.

Biological resources that can not be used for food or livestock and that can be made in an environmentally friendly way, are of growing interest. Biodiesel can be made from oleaginous crops, animal fats or recycled fats.

Since it is known that algae and certain microorganisms are capable of producing and / or accumulating lipids, their use as an oil source for biodiesel has also been suggested.

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.

This kind of production is unfortunately at relatively low yields and the end product becomes expensive.

A new generation of processes using less expensive raw materials for the production of lipids by heterotrophic microorganisms has been proposed in some recent patent publications.

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.

Furthermore, 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.

These documents, however, do not solve a major problem: the yield of lipids produced by the various microorganisms.

Also, the reduction of the production costs and the increase of the quantity of lipid remain problems not yet solved.

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. Also, 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.

In the invention, in order to produce a large quantity of lipids, the bacteria are first cultured in a medium enriched in nitrogen and / or in phosphate for a certain time (preculture). In the invention 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.

In a uniform manner, the nutrients will be called nitrogen and organic or inorganic phosphate. Also, in the invention, the inorganic or mineral terms will be uniformly used to designate nutrients that are not organic.

It is advantageous to perform the preculture in a medium enriched in nitrogen and / or phosphate, and to carry out the culture in a depleted medium of the same nutrients. As well :

if the preculture is enriched in nitrogen, the culture will be carried out in a nitrogen-depleted environment,

if the preculture is enriched in inorganic or organic phosphate, the culture will be carried out in a medium depleted in inorganic or organic phosphate, and

if the preculture is enriched in inorganic or organic phosphate and in nitrogen, the culture will be carried out in an environment depleted of inorganic or organic phosphate and nitrogen.

The culture in a rich medium then in a nutrient-depleted environment is known from the state of the art as "hypercompensation".

In the context of the hypercompensation in phosphate, it is advantageously added to 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 In the pre-culture medium, the phosphate concentration inorganic varies from 4mM to 20mM, especially the concentration is about 5mM.

Advantageously, the concentration of glycerol in the pre-culture and the culture does not exceed 2M, advantageously 1.5M, in particular 1M.

In the examples below, it is shown that 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.

The use of 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.

In the invention, 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.

In the invention, the term "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.

In the invention, 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.

By "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.

The 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. In an advantageous embodiment, 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. , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 , 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 hours.

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.

When the culture medium is depleted in inorganic phosphate, the concentration of said phosphate varies from 0.5 mM to 2 mM, in particular is 1 mM.

In an advantageous embodiment, the invention relates to the previously defined use, wherein said C3-C5 sugar is a triose, a tetrose or a pentose.

In another advantageous embodiment, 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.

In yet another advantageous embodiment, the invention relates to the above-mentioned use, wherein said bacteria are actinomycetes of the genus Streptomyces.

In yet another advantageous embodiment, the invention relates to the aforementioned use, wherein the glycerol is selected from refined glycerol and unrefined glycerol.

Although 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%.

Various methods are known for producing glycerol.

The historical synthesis of glycerin or glycerol is due to Wurtz, from allyl tribromide. However, this synthesis is not complete because the allyl tribromide is itself prepared from glycerine. The total synthesis is due to Charles Friedel and Silva from propylene.

Various synthetic routes from propylene exist. The process of 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:

(1) formation of a complex between the acetaldehyde and bisulphite ions thereby retarding ethanol production and restoring the oxidoreduction balance in the synthesis of glycerol, or

(2) cultures of growing microorganisms at pH values near or above 7, or

(3) using osmotolerant microorganisms.

However, today, 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, is also produced by the transesterification of vegetable oils, of which it is a co-product, in the production of vegetable oil methyl esters (VOMEs) which serve as fuels under the name of biodiesel.

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).

During the transesterification reaction, the oils or fats react with a short chain alcohol (usually methanol) in the presence of a catalyst. 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.

At present, 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.

Three major classes of catalysts exist:

- basic catalysts:

· Hydroxides, alkoxides or soaps of alkali or alkaline earth metals (Li, Na,

K, Ca, Ba, Cs ...),

• amines from the guanidine family, for example;

- acid catalysts:

• mineral acids: HCI, H ₂ S0 ₄ ,

· Sulphonic acids,

• ion exchange resins (strong acid),

• zeolites;

- the other catalysts:

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. Thus, components such as methanol, various salts or potassium chloride may be present therein with varying contents in the final product.

In general, whatever the production method used, 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.

Other techniques make it possible to carry out this reaction using innovative technologies such as microwave heating, enzymatic catalysis or even using supercritical methanol.

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%).

In yet another advantageous embodiment, the invention relates to the above-mentioned use, wherein said bacteria are lipogenic bacteria. By "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 Ί45Μ '1148, Streptomyces coelicolor Variant M145M1142 and Streptomyces coelicolor Variant M145M1144, Streptomyces linolmensis NRRL2936, Streptomyces exfoliatus, Streptomyces cealestius ATCC 15084, Streptomyces lividans 1326, Streptomyces actuosus, Streptomyces ambofaciens OS MOROCCO J9 and J5, Streptomyces reticuli DSM 40776, Streptomyces parvulus 2283FB, Streptomyces Venezuela 5110, Streptomyces noursei and Streptomyces cinabarinnus.

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.

For example, the inventors have shown that the deletion of the two components

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).

The inventors have also found that the 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.

The 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.

In view of the knowledge of the genomes of bacteria, and the knowledge of those skilled in the biosynthetic pathways of antibiotics, the skilled person can easily determine genes or gene sequences that must be targeted.

Moreover, the 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.

In addition, the genes or gene combinations involved in the production of reserve lipids can also be mutated:

the genes involved in the increase of the incoming glycerol flux (transport), its conversion to glycerol 3P (precursor of TAGs) and its catabolism in acetylCoA (enzymatic and / or signaling / regulation functions),

the genes involved the biosynthesis of TAGs, biosynthesis of fatty acids and their charge on the glycerol backbone (enzymatic and / or signaling / regulation functions),

the genes involved in the degradation of TAGs.

It is also advantageous in the context of the above-mentioned use to use bacterial strains with gene modifications increasing the availability of precursors for lipid synthesis. Thus, for example, it is advantageous to have bacterial strains accumulating intermediate metabolites such as glycerol 3-phosphate (G3P) or else acetyl and malonyl coenzyme A (acetylCoA, malonylCoA).

It is furthermore advantageous in the context of the above-mentioned use to use bacterial strains with gene modifications increasing the availability of precursors for lipid synthesis. Also, for example, it is advantageous to have bacterial strains accumulating intermediate metabolites such as glycerol 3-phosphate (G3P) or acetyl coenzyme A (acetyl CoA).

In addition, it is advantageous, within the framework of the above-mentioned use, to use bacterial strains exhibiting gene modifications increasing the activity of the enzymatic systems involved in the generation of precursors (acetyl and malonylCoA) and the biosynthesis of TAGs among which may be mentioned the increase in the availability of co-factors essential to the enzymatic activity (biotin etc.) or the intensity of post-translational modifications (phosphorylation, acetylation, glycosylation, etc.) having a positive effect on the activity of enzymes involved in the generation of precursors and the biosynthesis of TAG.

Glycerol can be metabolized by two potentially competing pathways, both of which lead to the synthesis of acetylCoA:

the glycerol kinase route, Gly3P DH (operon SC01659 to SC01661 under the control of the GylR repressor, SC01658) and SC04774 (putative glycerol 3P dehydrogenase). This route leads to the manufacture of Gly3P necessary for the biosynthesis of TAGs.

To increase the pool of G3P, it is possible in particular to over-express genes SC01659 (glycerol transporter) and SCO1660 (glycerol kinase) or to inactivate SC04774 which is supposed to convert Gly3P into DHAP which enters glycolysis.

the glycerol dehydrogenase (SC02598 / SC06754), DHA kinase (SCO0580, SCO0581 to SCO0576 operon, with divergence regulator, SCO0582) and / or SCO7073 to SCO7071 operon.

It is also possible to increase the catabolism of glycerol by the glyDH / DHA Kinase pathway to increase the acetylCoA pool.

In all cases, special attention will be paid to the redox equilibrium of the cell (the generated NADH must be reoxidized).

To increase the acetylCoA pool, it is also possible to increase the expression of the genes encoding the enzymes of glycolysis and / or the activity of the corresponding enzymes by playing on possible post-translational regulations and / or on the abundance of cofactors necessary for the activity of these enzymes. Of particular interest will be the enzymes of the lower part of glycolysis and in particular the Pyruvate Dehydrogenase complex.

To increase the production of lipids by bacteria, it is also advantageous to carry out genetic modifications in said bacteria to reduce the degradation of fatty acids.

As an illustration, it may be advantageous to reduce the expression of the genes coding for the enzymes of the glyoxylate pathway. In particular, 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 aforementioned examples of gene modifications are for illustrative purposes only and should not be construed as limiting the scope of the invention. The skilled person, with his general knowledge of the field in question is able to determine the most appropriate modifications.

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:

a first culture medium enriched with inorganic phosphate and / or nitrogen, and a second culture medium that is poor in inorganic phosphate and / or in nitrogen.

In addition, 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:

Advantageously, 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:

a first culture medium enriched with mineral phosphate, and

a second culture medium which is poor in inorganic phosphate.

Advantageously, 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:

a first culture medium enriched in nitrogen, and

a second culture medium which is poor in nitrogen.

In the invention, culture media are media commonly used by those skilled in the art to allow the growth of actinomycetes bacteria.

By way of example, in the context of the cultivation of Streptomycetes, 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.

Advantageously, in the invention the first and the second culture medium are of the same nature. Also, if the first medium is R2YE medium, the second medium is preferably R2YE medium, possibly slightly modified, including no longer including sucrose.

In certain aspects of the invention, it may be appropriate to choose a first medium of a different nature from the second medium. Those skilled in the art, depending on the bacterial strains used, will be able to choose the most appropriate media.

The description also relates to an assembly comprising:

a first culture medium enriched in phosphate and / or nitrogen, and

a second culture medium that is poor in phosphate and / or nitrogen,

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

at least one strain of Actinomycetes bacteria.

Advantageously, the invention furthermore relates to an assembly comprising:

a first culture medium enriched in phosphate, and

a second phosphate-poor culture medium,

at least one strain of Actinomycetes bacteria.

Advantageously, the description also relates to an assembly comprising:

a first culture medium enriched in nitrogen, and

a second nitrogen-poor culture medium,

at least one strain of Actinomycetes bacteria.

As mentioned previously, 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 15084, Streptomyces lividans 1326, Streptomyces actuosus, Streptomyces ambofaciens OS MOROCCO J9 and J5, Streptomyces reticuli DSM 40776, Streptomyces parvulus 2283FB, Streptomyces Venezuela 5110, Streptomyces noursei and Streptomyces cinabarinnus.

The disclosure further relates to a method for producing lipids by Actinomycete bacteria, said method comprising

a step of culturing said bacteria in a first medium enriched in phosphate and / or nitrogen, and

a step of culturing said bacteria in a second medium that is poor in phosphate and / or nitrogen,

said first and second culture media comprising as a majority / main carbon source of glycerol, or a C3-C5 sugar.

Advantageously, the invention relates to a process for producing lipids by Actinomycete bacteria, said process comprising

a step of culturing said bacteria in a first phosphate-enriched medium, and

a step of culturing said bacteria in a second medium that is poor in phosphate,

Advantageously, the description relates to a process for producing lipids by Actinomycete bacteria, said process comprising

a step of culturing said bacteria in a first nitrogen-enriched medium, and

a step of culturing said bacteria in a second medium that is poor in nitrogen,

Advantageously, the invention relates to a method as defined above, in which said bacteria are Actinomycetes of the family Streptomycetes.

In an advantageous embodiment, 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.

An advantageous composition of the invention obtainable by the above-mentioned method comprises, or essentially consists of, or consists of the following fatty acids:

from 6.7% to 7.7% of iso C14: 0,

- from 3.1% to 4% C14: 1,

- from 9.8 to 1 1% C15: 0 anteiso,

from 0.1% to 0.7% of C15: 0,

from 61.5% to 62.5% of isoCI 6: 0,

from 0.5% to 1.5% of isoCl 6: 1,

- from 3.1% to 4% C16: 0,

from 0.1% to 7.7% C16: 1,

from 0.5% to 1.5% of C17: 0,

from 0.5% to 1.5% of C17: 1,

from 1, 1% to 2.1% of isoCl 7: 0, and

- from 6.7% to 7.7% of anteisoCI 7: 0,

the percentages being expressed by weight of the total fatty acids of the composition. Furthermore, in the composition according to the invention, 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.

In the invention, the fatty acids are as follows:

12-methyl tridecanoic acid (iso C14: O / iso tetradecanoic acid),

tetradecenoic acid (C14: 1),

12-methyl tetradecanoic acid (C15: 0 anteoiso / anteiso pentadecanoic acid),

pentadecanoic acid (C15: 0),

14-methylpentadecanoic acid (isoC16: 0 / isohexadecanoic acid), 14-methylpentadecenoic acid (iso C16: 1 / iso hexadecenoic acid),

hexadecanoic acid (C16: 0),

hexadecenoic acid (C16: 1),

- 15-methyl hexadecanoic acid (iso C17: 0 / isoheptadecanoic acid),

heptadecanoic acid (C17: 0),

heptadecenoic acid C17: 1, and

- 14-methyl hexadecanoic acid (C17: 0 anteiso / anteisoheptadecanoic 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.

It is possible, for example, to obtain 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.

It is also possible to produce 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 . In all cases, the unsaturations of the fatty chains are completely hydrogenated, and the glycerol group is converted into propane. In addition, secondary reactions such as shift and methanation reactions can lead to the formation of CO and methane. On the sulphide type catalysts conventionally used in hydrotreatment, the two routes of elimination of oxygen (hydrogenation and decarboxylation) coexist. The hydrogen consumptions corresponding to this hydrodeoxygenation step depend on the nature of the oil or animal fats of departure, in particular the average number of unsaturations per chain and the length of the hydrocarbon fatty chains.

Then, 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). In the state of the art, the overall yield kerosene on crude oil is of the order of 55%.

By trans-esterification or chemical or enzymatic hydrolysis and / or associated with other chemical processes, it is also possible to obtain lubricants, solvents and surfactants or food ingredients from the above-mentioned process.

All of these techniques are well known to those skilled in the art.

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

a step of culturing the bacteria in a first medium enriched in phosphate and / or in nitrogen, and

a step of culturing said bacteria in a second medium depleted of phosphate and / or nitrogen,

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:

a step of culturing the bacteria in a first medium enriched in phosphate, and

a step of culturing said bacteria in a second medium depleted of phosphate,

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

As an indication, 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 etherification step in which the glycerol is reacted with at least one olefinic hydrocarbon of 4 to 12 carbon atoms; and or

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.

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.

The aforementioned method is given by way of example and should not be considered as limiting. The skilled person will be able to use any other similar method with the help of his general knowledge in the field.

The invention will be better understood with the help of the examples and the following seven figures. DESCRIPTION OF THE FIGURES

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. lividans 1326, M: S.coelicolor M145 Variant M1142, N: S.actuosus, O: S. ambofaciens. BONE. MOROCCO J 9, P: S.reticuli DSIVI 40776, Q: S.ambofaciens.05.MAROC J 5, R: S.parvulus 2283 FB, S: S.coelicolor Variant M145 M1144, T: S.coelicolor M145, U: S.coelicolor ATCC19832, V: S.venezuela 5110J II, W: S.noursei, X: S.cinabarinnus ATCC 23617, Y: S.coelicolor ATCC 21666 and Z: S.venezuelae ATCC 15439.

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.

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.

EXAMPLES

Example 1: Culture media

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).

Culture medium HT:

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.

Modified R2YE culture medium:

K ₂ SO ₄ (1.4.10-3 mol / L and 0.25 g / L) MM = 174 g / mol

MgCl ₂ 6H ₂ O (5.10-2 mol / L and 10.12 g / L) MW = 203 g / mol

Amino acids of casein (0.1 g / L)

CaCl ₂ 2H ₂ O (2.10-2 mol / L and 2.94 g / L) MM = 147 g / mol

L-proline (2.6 × 10 -2 mol / L and 3 g / L) MM = 1 15 g / mol

TES buffer (2.5 × 10 -2 mol / L and 5.73 g / L) MM = 2304 g / mol

Trace element (2 mL / L)

Yeast Extract (5 g / L)

NaOH (5.10-3 mol / L and 0.2 g / L) MM = 30 g / mol

Stock Solution of trace elements R2YE:

ZnCl ₂ 40 mg / L; FeCl ₃ .6H ₂ O 200 mg / L; CuCl ₂ .2H ₂ 0 10 mg / L; MnCl ₂ .4H ₂ 0 10 mg / L; Na ₂ B ₄ O ₇ .10H ₂ O 10 mg / L; (NH ₄ ) ₆ Mo ₇ 0 ₂ 4.4H ₂ 0 10 mg / L

Synthetic medium Medium 194:

TES 22.8 mM;

2.0 mM Citric acid, H2O;

2.2 mM NaCl;

KH ₂ P0 ₄ 1 1, 0 mM;

(NH ₄ ) ₂ SO ₄ 43.0 mM;

MgSO ₄ .7H ₂ O 1, 7 mM;

0.2 mM CaCl ₂ .2H ₂ O;

FeS0 ₄ .7H ₂ 0 137.6 μΜ;

CuSO ₄ .5H ₂ O 12.0 μΜ;

ZnS0 ₄ .7H ₂ 0 1 1, 7 μΜ;

MnSO ₄ .H ₂ 0 33.9 μΜ; Na ₂ MoO ₄ .2H ₂ O 1, 6 μΜ;

CoCl ₂ .6H ₂ O 3.2 μΜ;

KI 1, 5 μM;

AICI ₃ .6H ₂ O 1, 3 μM;

Η ₃ Β0 ₃ 2.5 μΜ;

NiCI ₂ .6H ₂ O 1, 6 μM;

Thiamine-HCl 21.0 μM (7 mg / L);

Biotin 1, 2 μΜ (0.30 mg / L);

Riboflavin 5.0 μΜ (2 mg / L);

Pantothenic Calcium Acid 8.4 μΜ (2 mg / L);

Folic acid 0.6 μΜ (0.25 mg / L);

P-amino-benzoic acid 1, 8 μΜ (0.25 mg / L);

Pyridoxine-HCI 9.7 μΜ (2 mg / L);

Nicotinamide 16.3 μΜ (2 mg / L);

Nicotinic acid 0.5 μΜ (0.0625 mg / L);

Antifoam 204 100.0 μ _;

Pluronic F68 10% 0.5 ml_ / L

YEME culture medium:

3 g of yeast extract, 5 g of bacto-peptone, 3 g of malt extract, and 5 mM of MgCl ₂ . DALP culture medium:

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.

MP5 culture medium:

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.

Culture medium SL1 1:

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.

Example 2: Culture conditions

liquid cultures (50 ml flask, 2L fermenter)

Pre-culture inoculated with 10 ⁶ spores / mL and incubated 48h at 28 ° C pH 7, shaking 180-200 rpm (rotations per minute) The seeded crop with a pre-culture volume representing 2.5% of the final volume of the crop. The culture was incubated 72-96 hours at 28 ^<€ pH 7 180-200 rpm for flask (for fermentor: p0 ₂ 70%, stirring 400-600 rpm)

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).

Solid cultures (petri dishes)

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.

The culture dish consists of 8mL of R2YE medium, covered with a cellophane. This box is seeded with a wooden stick. 20 μί 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 μί 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.

At the end of the culture, 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.

Examples 3: lipogenic strains

The inventors have identified streptomyces strains considered as lipogenic, that is to say strains that produce more than 20% of their biomass in TAG.

The results are shown in Figure 1.

Example 4 Hyperconpensation in Glycerol Medium

In order to determine the proportion of TAGs accumulated by the microorganisms, the inventors tested the Streptomyces lividans strain.

The bacteria were spread on the surface of a cellophane deposited on medium

Solid R2YE containing glycerol (0.2M) and 5mM inorganic phosphate (Pi) for 48h. Observation by electron microscopy of the bacteria shows that they contain very few lipid vesicles (FIG. 2A).

At the end of the 48h, the bacteria are displaced on the surface of a new solid R2YE medium containing glycerol (0.2M) and either 5 mM Pi (FIG. 2B) or 1 mM Pi

(Figure 2C). Observation by electron microscopy shows that the bacteria which have been subjected to a phosphate limitation (transfer from 5mM to 1mM Pi) very strongly accumulate TAG (hyper-compensation phenomenon), while those which have not been subjected to a Such limitation (transfer from 5mM to 5mM) does not show this phenomenon and accumulate a smaller amount of TAG.

Similar results are obtained in the context of a hypercompensation in nitrogen.

These results are confirmed with the strain Streptomyces coelicolor M1 155, as shown in FIG. 7. For this strain (M1 155), it is found that in a dose-dependent manner, the bacteria grown on R2YE / glycerol accumulate more TAG than those cultured in vitro. R2YE / glucose.

Example 5 Hyperconpensation in Glycerol Medium and in Glucose

In order to validate the above results, 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 results are shown in Figure 3.

It is found that regardless of the strain used, the bacteria cultured according to the process of the invention accumulate more TAG than the same bacteria grown in the presence of glucose.

Example 6 Hyperconpensation in Medium Comprising C5 Sucs

In order to validate the previous results, 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 results are shown in Figure 4.

It is found that the bacterium is able to accumulate significant amounts of TAG including with C5 sugars.

EXAMPLE 7 Hyperconpensation in Medium Comprising Glycerol Refined or Not

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). These results show a greater accumulation when the glycerol source is not refined.

Moreover, these results show that the TAGs which accumulate when the bacteria are cultured using the process according to the invention, present a large amount of iso C 16: 0 fatty acid, and to a lesser extent a significant amount. C15: 0.

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:

isoC14: 0: 3.5g,

isoC14: 1: 1, 75g,

anteisoC15: 0: 5g,

C15: 0: 0.1 g,

isoC16: 0: 30g,

isoC16: 1: 0.5g,

C16: 0: 1, 75g,

C16: 1: 0.25g,

isoC17: 0: 0.8g,

anteisoC17: 0: 3.5g,

C17: 0: 0.5g, and

C17: 1: 0.5g.

Example 8 Hyperconpensation in a medium comprising glycerol and having a deletion of genes for the degradation of fatty acids.

In order to increase the TAG content of the bacteria, the inventors have tested the impact of modifications of the fatty acid degradation pathways.

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.

The results are shown in Figure 6B.

It is found that bacteria with deletion of the SCO0981, SCO0982, SCO0983 and SCO0984 genes (ICL4 in Figure 6B) accumulate up to more than 200% more TAG than bacteria with no such deletions.

These results show that the process according to the invention, combined with gene modifications of fatty acid metabolism pathways, considerably increases the accumulation of fatty acids by bacteria.

Claims

1. 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, then cultured in a second culture medium low in phosphate.

2. Use according to claim 1, wherein said sugar is a triose, a tétrose or a pentose.

3. Use according to claim 1 or claim 2, wherein said bacteria are Acticnomycetes of the genus Streptomyces.

4. Use according to any one of claims 1 to 3, wherein the glycerol is selected from refined glycerol and unrefined glycerol.

5. Use according to any one of claims 1 to 4, wherein said bacteria are lipogenic bacteria.

6. Use according to claim 5, wherein said lipogenic bacteria are genetically modified by substitution, deletion or insertion of at least one nucleic acid of their genome, so that they do not produce substantially antibiotic.

7. A set of culture media comprising glycerol, or a C3-C5 sugar, as a major source of carbon, said set of culture media comprising:

a first culture medium enriched in phosphate, and

a second culture medium which is poor in phosphate.

8. Set comprising

a first culture medium enriched in phosphate, and

a second phosphate-poor culture medium,

said first and second culture media comprising as a major source of glycerol carbon, or a C3-C5 sugar, and

at least one strain of Actinomycetes bacteria.

9. Process for producing lipids with Actinomycete bacteria, said method comprising

a step of culturing bacteria in a first phosphate-enriched medium, and

a step of culturing said bacteria in a second medium depleted of phosphate,

The method of claim 9, wherein said bacteria are Acticnomycetes of the family Streptomycetes.

11. The method of claim 9 or claim 10, 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 do not produce substantially more antibiotic.

Lipid composition comprising lipids obtainable by the process as defined in any one of claims 9 to 11.

13. Use of the method according to any one of claims 9 to 11 for the manufacture of lubricants, surfactants, coatings, solvents, food ingredients or synthetic intermediates for oleochemistry.

A method of manufacturing lubricants, surfactants, coatings, solvents, food ingredients or synthetic intermediates for oleochemistry from lipids derived from Actinomycete bacteria, said method comprising

a step of culturing said bacteria in a first phosphate-enriched medium, and

a step of culturing said bacteria in a second medium that is poor in phosphate,