FR3101357A1 - PROCESS FOR PRODUCING 1,2,4-BUTANETRIOL FROM A PLANT - Google Patents

Abstract

Disclosed is a natural process for the production of 1,2,4-butanetriol from Cakile maritima characterized in that the plant is subjected to salt stress during its growing period to increase the production of 1,2,4 -butanetriol by said plant. Figure to be published with the abstract: FIGURE 2.

Description

METHOD FOR PRODUCING 1,2,4-BUTANETRIOL FROM A PLANT

The present invention lies in the field of the production of 1, 2, 4-butanetriol, a molecule of industrial interest. The present invention relates more particularly to a process for the production of 1,2,4-butanetriol from halophyte plants, in particular Cakile maritima .

1,2,4-Butanetriol is a basic chemical that has a wide variety of uses and in different areas. As an important chemical, butanetriol has versatile applications in many fields and has attracted considerable interest in recent years. For example, it can be used in the production of solvents and in the synthesis of various pharmaceutical products, such as anti-viral and cholesterol-lowering drugs, among others (Crestor® and Zetia®). Butanetriol is also the direct precursor to the manufacture of butanetriol trinitrate, an excellent energy plasticizer to replace nitroglycerin, which is less volatile, more thermally stable and has better low temperature properties. Thus, 1, 2, 4-butanetriol is an industrially useful compound.

Current methods of producing 1,2,4-butanetriol use petroleum-derived raw materials and produce environmentally harmful by-products. The most common way to produce this chemical is by using sodium borohydride (NaBH4) to chemically reduce malic acid to 1,2,4-butanetriol. However, the process generates a large amount of borate salts. The elimination of these salts causes pollution. Chromite and rubidium can also be used as catalysts for the production of 1,2,4-butanetriol, but these methods require high temperatures and pressures, as well as toxic by-products (Campbell, C. J. & Hinshaw, J. C. inventors Thiokol Corporation, assigned. Making 1,2,4-butanetriol by hydroformylation of glycidol. United States patent US 4410744. 1983 Oct 18.).

In recent years, the use of biomass for the production of value-added chemicals has attracted considerable interest. Xylose is the second most abundant sugar in nature and a major constituent of hemicellulose in lignocellulosic biomass. A number of wild type and genetically engineered microbial strains have been isolated or engineered to ferment xylose for the production of ethanol, xylitol, succinate, lactate and other important products. Using xylose from biomass to produce bio-based chemicals has many advantages over traditional petrochemical processes because it is made from renewable resources, has mild processing conditions, and reduces environmental pollution. . Researchers also explored new routes for the production of butanetriol and ultimately developed a new process to produce butanetriol from xylose by microbial conversion.

Thus, the production of butanetriol today is mainly carried out in fermenters by genetically modified bacterial cultures of Escherichia coli (Wang et al. 2018, Bioresource technology; Zhang et al. 2016, Enzyme and microbial technology). These cultures are generally optimized to synthesize as much butanetriol as possible from a precursor, xylose, up to concentrations of 4g/L after 72 hours of culture (Cao et al. 2015, Scientific reports). These production methods in bioreactors are limited by side effects, in particular the accumulation of toxic compounds (Zingaro et al. 2012, Metabolic engineering).

Surprisingly, the inventors have developed a new natural production process, under controlled conditions from the halophyte plant Cakile maritima . The method according to the present invention offers a natural alternative solution at the expense of the conventional methods of the prior art based essentially on chemical methods or from genetically modified bacteria or plants.

Furthermore, to our knowledge, there is no prior art describing a method for producing 1,2,4-butanetriol from a plant, in particular Cakile maritima.

[Brief description of the invention]

An object of the invention is to allow natural production, respectful of the environment and produced on marginal (saline) land, thus avoiding competition with food crops. The invention also aims to offer, in fact, a high added value valuation in the context of soil remediation culture.

The invention therefore makes it possible to develop an alternative and natural process with the ambition of limiting the impact on the environment such as the pollution generated by production and allowing a culture of remediation of saline soils.

According to a first aspect, the invention relates to a process for the production of 1,2,4-butanetriol from the Cakile maritima plant, characterized in that said plant is exposed to salt stress during its cultivation, preferably during its period. growth to increase the production of 1,2,4-butanetriol.

According to a preferred embodiment, the exposure to salt stress comprises the addition of salts to the culture medium of a Cakile maritima plant, preferably in the form of a concentrated saline solution, so that the culture medium has a salt concentration of at least 50 mM, of at least 100 mM, preferentially of at least 200 mM, more preferentially of at least 400 mM and even more preferentially of at least 500 mM.

In other words, exposing the plant to salt stress includes adding a concentrated aqueous saline solution to the culture medium of at least 50 mM, at least 100 mM, preferably at least least 200mM, more preferably at least 400mM and even more preferably at least 500mM.

According to another preferred embodiment, the exposure to salt stress comprises the addition of salts to a culture medium every other day, preferably a concentrated aqueous salt solution is added to the culture medium every other day.

Preferably, Cakile maritima is watered for at least 10 days, preferably at least 20 days, from the start of germination of said plant.

Therefore, exposure to salt stress is maintained in the culture medium for at least 10 days, preferably at least 20 days, preferably the concentrated aqueous saline solution is added to the culture medium for at least 10 days, preferably at least 20 days from the start of germination of said plant.

According to a method of the invention, the culture is carried out on a whole plant or in cell culture. According to a preferred mode, the culture on a whole plant is carried out in a greenhouse. According to a preferred mode, the cell culture is carried out in a bioreactor.

When the culture is carried out on the whole plant, Cakile maritima is watered from the start of germination with the concentrated aqueous saline solution, preferably when the plant has developed the first leaf stage, even more preferably from the "4 leaf" leaf stage. ".

Advantageously, exposure to salt stress is initiated from germination, preferably when the plant has developed the first leaf stage, even more preferably from the "4-leaf" leaf stage.

According to another embodiment, the invention relates to a process for the production of butanetriol making it possible, in particular, to desalinate or to remedy salt and/or polluted soils. Thus, the process according to the invention is characterized in that the plant is cultivated in salty and/or polluted soils. According to another embodiment of the present invention, the Cakile maritima plant is harvested after the growth period, preferably the butanetriol is extracted after harvesting said plant. Advantageously, the method according to the invention is characterized in that the Cakile maritima plant is harvested after the growth period and the butanetriol is extracted.

The invention is particularly suitable for the natural production of 1,2,4-butanetriol while respecting the environmental conditions, in particular in the context of soil remediation culture (desalination, soil depollution).

[Description of the invention]

Other objectives, characteristics and advantages will emerge from the detailed description which follows with reference to the drawings given by way of illustration and not limitation, among which:

FIG. 1 represents the relative abundance (relative level) of butanetriol present in Cakile maritima after 10 days of treatment according to a possible embodiment of the invention;

FIG. 2 represents the relative abundance (relative level) of butanetriol present in Cakile maritima after 20 days of treatment according to a possible embodiment of the invention;

There is therefore a need for a natural process making it possible to increase the production of 1,2,4-butanetriol from plants, in particular Cakile maritima .

Cakile maritima is a halophyte plant, able to complete its life cycle up to concentrations of 500 mM NaCl. This plant is found in nature on the coasts of the whole world and in particular on the French coasts and bordering the Mediterranean. In the context of the invention, the inventors have more particularly studied an ecotype originating from the region of Raoued in Tunisia. The inventors have been busy collecting the seeds in this region and sowing them in order to reproduce them and obtain a greater quantity of seeds.

By "butanetriol" is meant within the meaning of the invention "1,2,4-butanetriol" or "1,2,4-Trihydroxybutane" or "2-Deoxyerthritol" or "Butane-1,2,4-Triol (CAS Number: 3068-00-6) of Formula I:

According to a first aspect, the invention relates to a process for the production of 1,2,4-butanetriol from the Cakile maritima plant, characterized in that said plant is exposed to salt stress during its growth period to increase the production 1,2,4-butanetriol. Thus, a salt stress is induced on said plant via prolonged exposure or not to a saline solution, during the growth period of said plant, to increase the production of 1,2,4-butanetriol. Advantageously, the method comprises a final step of harvesting Cakile maritima at the end of the growth period with a view to collecting the 1,2,4-butanetriol of interest produced and synthesized naturally by Cakile maritima under salt stress conditions.

More particularly, after the harvesting of the seeds of interest and their reproduction in order to obtain a large quantity of Cakile maritima seeds, the Cakile maritima seeds were removed from their pods and planted individually in a moist soil composed of 90% of compost and 10% lava rock or basalt. The pots were then transferred to a greenhouse and the plants were maintained under ambient temperature and light conditions for the duration of the experiment. In particular, an average high temperature of 22°C and an average low temperature of 17-21°C, with a light cycle of 16L:8D and this during the growth period necessary for the plant. The leaves are harvested after at least 10 days of treatment, preferably at least 20 days of treatment.

The present invention also relates to a method for producing 1,2,4-butanetriol from the Cakile maritima plant, said method comprising the steps of:

1. sowing seeds of Cakile maritima ,
2. exposure to salt stress from the germination of the plant and during its growth period to increase the production of 1,2,4-butanetriol, and
3. after at least 10 days of treatment, advantageously at least 20 days of treatment, Cakile maritima is harvested in order to obtain the 1,2,4-butanetriol of interest.

Within the meaning of the invention, the term “salt stress” is understood to mean a condition in which an excess of ions is applied essentially, but not exclusively, to the Na ⁺ and Cl ^– ions in the rhizosphere and in the water. Salt stress triggers both osmotic stress and ionic stress and is often accompanied by a significant drop in water potential. Salt stress can directly or indirectly affect the physiological status of plants by changing plant metabolism, growth and development. The vast majority of salt stress is caused by sodium salts, particularly NaCl. Therefore, the terms halophytes and glycophytes essentially refer to the stresses caused by an excess of Na ⁺ .

Surprisingly, the inventors have thus identified that this change in metabolism significantly modifies the quantity of butanetriol produced by said plant.

By salt stress is meant the condition in which the plant is placed with the addition of an aqueous saline solution of at least 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mm, 170mm, 180mm, 190mm, 200mm, 210mm, 220mm, 230mm, 240mm, 250mm, 260mm, 270mm, 280mm, 290mm, 300mm, 310mm, 330mm, 330mm, 340mm, 350mm, 360mm, 370mm, 380mm, 390mm, 400mm, 410mM, 420mM, 430mM, 440mM, 450mM, 460mM, 470mM, 480mM, 490mM, 500mM.

According to a preferred embodiment, the exposure to salt stress comprises the addition of salts to the culture medium of a Cakile maritima plant, preferably in the form of a concentrated saline solution, so that the culture medium has a salt concentration of at least 50 mM, of at least 100 mM, preferentially of at least 200 mM, more preferentially of at least 400 mM and even more preferentially of at least 500 mM.

By "culture medium" within the meaning of the invention, is meant a suitable culture medium known to those skilled in the art for cell culture and in particular culture in a bioreactor such as Murashige and Skoog medium (MS, Murashige and al., 1962) or a suitable culture medium known to those skilled in the art for culture in a greenhouse or in the ground. Consequently, the addition of a concentrated aqueous saline solution to the culture medium can, for example, be irrigation with a solution comprising water and salt, advantageously NaCl, on the leaf system and/or root system of the plants grown in greenhouses or in the ground. Of course, watering can be automatic or natural. By natural watering is meant watering induced naturally by the climate and the weather, for example an oceanic climate by the sea which is naturally rich in NaCl. For cell culture, the addition of a concentrated aqueous saline solution in the culture medium can for example be the culture medium which comprises a saline solution of at least 50 mM, advantageously the culture medium is enriched with a solution at least 50mM saline for at least 10 days of treatment.

In the context of the present invention, watering in the presence of aqueous saline solution begins from the "4-leaf" growth stage.

However, according to another embodiment, Cakile maritima is watered from germination, preferably when the plant has developed the first leaf stage, even more preferably when the plant has reached the “4 leaf” growth stage.

Thus, the concentrated aqueous saline solution is added to the growing medium from germination, preferably when the plant has developed the first leaf stage, even more preferably from the "4-leaf" leaf stage.

By “4-leaf growth stage”, or “4-leaf plants” is meant within the meaning of the invention the moment when the plant comprises 4 leaves. The observation of growth stages are few and based on the number of leaves. Thus, the main stage allowing leaf development includes a ^1st stage where the cotyledons are completely unfolded, a ^2nd stage where the first pair of leaves is unfolded (stage 2 opposite leaves), a ^3rd stage or the second pair of leaves is unfolded (4-leaf stage) and a ^4th stage where the first alternate leaf is unfolded (5-leaf stage). Consequently, the watering begins within the meaning of the invention when the second pair of leaves is spread out.

Once growth has reached the "4-leaf" stage, plants are watered every 2 days with varying concentrations of aqueous saline solution, particularly NaCl solution. Plant treatment lasts at least 10 days, preferably at least 20 days. Within the meaning of the invention, the term "treatment of at least 10 days" means a treatment of 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days and 20 days. In particular, within the meaning of the invention, the duration of plant treatment of 10 days is understood to mean the moment when the plant has reached the "4-leaf" growth stage, followed by watering for 10 days. According to a similar procedure, the treatment lasts at least 20 days from the 4-leaf stage of growth.

Leaf samples are taken at 10 days and at 20 days of treatment. Sheets 1 and 3 will then be analyzed at 10 days and sheets 3 and 5 at 20 days.

Thus, the method according to the invention comprises the addition of a concentrated aqueous saline solution to the culture medium for at least 10 days, preferably at least 20 days. The method according to the invention also comprises the addition of a concentrated aqueous saline solution to the culture medium every two days.

In particular, 4-leaf plants are irrigated for a period of at least 20 days with 100 mL of 0 mM to 500 mM NaCl solutions every other day.

According to another particularly advantageous embodiment of the invention, the method is characterized in that the culture is carried out on a whole plant or in cell culture.

Advantageously, the culture on a whole plant is carried out in a greenhouse. However, and as an alternative, cultivation can be carried out in the open ground, in particular on marginal soils or soils unsuitable for conventional cultivation. Cultivation on marginal land also avoids competition with food crops.

By "marginal land" is meant, within the meaning of the invention, polluted, saline land or land unsuitable for food cultivation.

Thus, the method according to the invention is characterized in that the plant is cultivated directly in the culture medium, said culture medium can be naturally concentrated in saline solution and/or said culture medium can be concentrated by the addition of saline solution.

According to another embodiment, the culture is carried out in cell culture, preferably in a bioreactor. This mode is particularly advantageous in order to produce a large quantity of 1,2,4-butanetriol under controlled conditions and a confined space relatively cheap and taking up little space.

The establishment of a culture in cell suspension of the halophyte Cakile maritima has in particular been described by the inventors (Ben Hamed et al. 2014).

Cultures of Cakile maritima were initiated from an aerial part of 14-day-old light seedlings. The seeds were immersed in 70% ethanol for 1 min, then rinsed with sterile distilled water, immersed in chlorine bleach for 10 min and after being rinsed three times (5 min each) with sterile distilled water. The seeds were placed in a petri dish containing Murashige medium and Skoog, including vitamins (MS, Murashige et al., 1962), with 30 gL ^-1 of sucrose and 8 gL ^-1 of agar. The pH was adjusted to 5.8 with KOH. Stem segments were thinly sliced and then placed on solid callus inducing medium (CIM), containing 6.2 gL ^-1 Gamborg B5 (Gamborg et al., 1968) supplemented with 20 g glucose, 8 gL ^{- 1} agar and growth regulators, 9.06 µM of 2.4 D and 0.46 µM of kinetin. The pH was adjusted to 5.7 with KOH. After 2-3 weeks, calluses appeared on the sides of the segments. When the size of the calluses exceeded 1 cm, they were divided and transferred to the callus-inducing medium.

Establishment cell suspension cultures of Cakile maritima are based on approximately 5 g of calli which have been transferred to 125 mL flasks containing fresh MS medium supplemented with 30 gL ^-1 of sucrose. The pH was adjusted to 5.7 with KOH. Flasks were incubated on a rotary shaker at 120 rpm and kept at 22°C in the dark. Feeding the cultures with fresh medium was carried out at intervals of 10-14 days, during which the suspensions were left under agitation (rotary agitator at 120 rpm). This procedure was repeated for about 8 weeks. Then, the suspension was subcultured every 7 days by transferring 20 ml of culture into 50 ml of fresh medium in a 250 ml Erlenmeyer.

Finally, salt treatment for cultured cells relies on adding 10 mL of NaCl solutions to the culture medium to achieve the desired concentration. Old cells were subcultured in the supplemented medium and cultured for 7 days in a bioreactor.

Regarding the cell suspension cultures of Cakile maritima , the inventors have already shown that the previously generated cell lines retain the halophytic characteristics of the whole plant (Ben Hamed et al. 2014, Advances in Horticultural Science; 2016, Plant Science ).

According to another aspect, the invention relates to a process for the production of butanetriol which makes it possible in particular to desalinate or remedy salt and/or polluted soils. Advantageously, said method comprises a step in which the Cakile maritima plant is harvested after the growth period allowing the export of the accumulated pollutants. Therefore, the valorization of the use of Cakile maritima for the desalination or the remediation of polluted soils in addition to that of other metabolites of economic interest is of interest.

Other aspects and advantages of the invention will be disclosed in the following examples and should be considered illustrative and not limiting.

EXAMPLES:

The inventors have characterized the capacity of Cakile maritima to produce butanetriol under saline stress conditions on whole plants grown in greenhouses. Furthermore, the inventors also master the greenhouse cultivation of the plant as well as the saline treatments. The inventors have also developed a cell culture of this plant (Ben-Hamed et al. 2014).

Cakile maritima is a halophyte plant capable of completing its life cycle in a medium containing up to 500mM of NaCl. C. maritima lives on the coasts of the whole world and in particular in France and all around the Mediterranean.

The inventors carried out as part of their research work, a study of the metabolome in order to decode the changes in the metabolism of the leaves of C. maritima under saline conditions over time. The inventors applied salt concentrations from 0 to 500mM NaCl and analyzed the metabolic content of leaves from leaf stage 1 to 5. Using Mev software, the inventors performed a Wilcoxson test and generated heat maps for each statistical test. The inventors calculated a factor change for statistically relevant metabolites. The inventors have also observed relative abundance changes in primary and secondary metabolites, such as alkaloids and antioxidants, including 1,2,4-butanetriol (butanetriol).

Surprisingly and unknown to date, the inventors have discovered the presence of 1,2,4-butanetriol as a metabolite when Cakile maritima is cultivated in the presence of salt concentration.

Thus, at 10 days, treated and untreated plants produced butanetriol at a basal level (Fig. 1). On the other hand and completely unexpectedly, after 20 days, the salt treatment increases the production of butanetriol in a dose-dependent manner (Fig. 2). Thus, the results show a strong relative increase (around 20 times) in the quantity of butanetriol under salt stress conditions.

To our knowledge, the natural production of butanetriol has so far been detected at a basal level only in 2 plants: wheat (Tang et al. 2018, Physiology and biochemistry of plants) and maize (Zhu et al. 2016, Applied Soil Ecology). In this context, the present invention has a double interest: i) Cakile maritima offers an alternative to the production of butanetriol by GMOs, and ii) the production of Cakile maritima can be induced by salt stress in a dose-dependent manner.

Claims (10)

Process for the production of 1,2,4-butanetriol from the Cakile maritima plant, characterized in that said plant is exposed to salt stress during its cultivation, preferably during its growth period to increase the production of 1,2 ,4-butanetriol. Method according to claim 1, in which the exposure to salt stress comprises the addition of salts to a culture medium of a Cakile maritima plant, preferably in the form of a concentrated salt solution, so that the culture medium has a saline concentration of at least 100 mM, preferably of at least 200 mM. A method according to any preceding claim, wherein exposure to salt stress comprises adding salts to a culture medium every other day. A method according to any preceding claim, wherein the exposure to salt stress is maintained in the culture medium for at least 10 days, preferably at least 20 days. A method according to any preceding claim, wherein the cultivation is carried out on a whole plant or in cell culture. A method according to claim 5, wherein the whole plant cultivation is carried out in a greenhouse. A method according to claim 5, wherein the cell culture is carried out in a bioreactor. Method according to any one of claims 1 to 6, in which the exposure to salt stress is initiated from germination, preferably when the plant has developed the first leaf stage, even more preferably from leaf stage "4 leaves ". Process according to Claim 8, characterized in that the plant is cultivated in salty and/or polluted soils. A method according to claim 9, wherein the Cakile maritima plant is harvested after the growth period and the butanetriol is extracted.