High purity polyhydroxybutyrate (PHB) production and economic purification process from genetically modified bacteria

Abstract

The present invention provides genetically modified Gram-positive bacteria that efficiently produce polyhydroxyalkanoates (PHAs). Also provided in the present description is a DNA molecule encoding enzymes necessary for the synthesis of PHAs. The invention further provides methods of producing PHAs comprising cultivating a genetically modified Gram-positive bacterium that produces polyhydroxyalkanoates (PHAs) efficiently and procedure for purification of PHA from a culture of a genetically modified Gram-positive bacterium that produces polyhydroxyalkanoates (PHAs).

Description

[0001] Title of the invention:

[0002] High purity polyhydroxybutyrate (PHB) production and economic purification process from genetically modified bacteria.

[0003] Field of Invention:

[0004] The present invention relates to the production of polyhydroxybutyrate referred to as PHB from the fermentation of genetically modified B. subtilis strains that allow not only the accumulation of biopolymer granules using agro-industrial residues or by-products as part of the culture medium, but also their release to the medium thanks to the expression of heterologous lytic enzymes that fragile their membrane. This allows that the purification process does not require the use of organic solvents, surfactants, thermal or physical treatment, allowing for an economical and environmentally friendly industrial PHB production process.

[0005] Background of the invention:

[0006] It is well known today that the industry needs to produce biocompatible and biodegradable materials, therefore, the development of biodegradable plastic resins receives high global attention, mainly those produced using environmentally friendly clean technology. The potential market for these new materials is enormous, with the polyhydroxyalkanoates (PHAs) family being an important part of this group. These polyesters can be naturally produced by a large number of living organisms, as well as produced by genetically modified organisms. Some of the most representative PHAs that have found industrial applicability are PHB (poly-3-hydroxybutyrate), PHB- V (poly-hydroxybutyrate-co-hydroxyvalerate), P4H B (poly-4-hydroxybutyrate), P3HB4HB (poly-3-hydrobutyrate-co-4-hydroxybutyrate) and some medium chain PHAs, for example: PHHx (poly-hydroxyhexanoate). Depending on the stage of development of these polymers it is possible to use them from the packaging for shortuse hygiene products (with low grammage), as well as they can be used to manufacture containers and the packaging of agrochemicals, motor oils, disposable diapers, etc. Furthermore, when intrinsic biodegradability property is required, PHAs can be used as trash bags, fishing items, among other outdoor plastic products. In agro-industry they can be used in seedlings, reforestation tubes, greenhouse coverings and, mainly, in controlled-release systems for nutrients, fertilizers, herbicides and insecticides. In the field of biomedicine, PHAs can be used due to their total biocompatibility, such as controlled-release drug microcapsules, sutures, and fixation bolts for bone fractures. Since its biodegradation rate in vivo is very slow but continuous and complete, it shows an excellent potential to be used in reabsorbable prostheses. All of these commercial applications mostly require high purity PHAs in order to obtain the required plastic properties, and an efficient and cost-effective purification process on an industrial scale is indispensable.

[0007] Advances in the understanding of the biosynthetic pathways of PHAs make it possible to produce high amounts of PHAs, not only in wild-type microorganisms, but also in genetically modified microorganisms or plant cells. However, it remains difficult and challenging to develop an efficient and low-cost purification method from such cell biomass.

[0008] Cell biomass biopolymer extraction and purification processes using halogenated solvents are currently prohibitive as they are highly aggressive with the environment and health. Therefore, if any solvent is used in the extraction process, it should first of all meet the conditions of being environmentally friendly. In this regard, Brazilian patent BR9302312, among others, exhibits the use of non-halogenated solvents for the biopolymer extraction from the cell biomass, but many of them exhibit characteristics that make its manipulation difficult on an industrial scale (toxicity, explosiveness, etc.), in addition to its high cost. On the other hand, patent EPA-01455233 A2 describes as part of the purification method, the use of enzymes or surfactants to solubilize cell material and allow the PHA granules release. However, if high purity of the biopolymer is required the need for the use of solvents is a restriction in the present patent. Moreover, while low amounts of enzymes are added (1 % relative to the dry weight of biomass), they are expensive and cannot be reused, unlike what does happen when solvents are used. In addition, high dilutions of cell material are required, leading to high volumes of effluents.

[0009] In summary, an industrial PHA production process must consider, as it is well described in US patent number 9,045,595, the following components: i) microorganisms strains that show a high efficiency conversion of raw material into polymer, ii) with a simple and efficient production protocol, iii) low-cost, high-yield raw material and iv) a polymer extraction and purification process that enables a high purity product, completely preserving the original characteristics of the biopolymer, with high yielding and efficiency, using a process that is not environmentally aggressive.

[0010] In addition to the economic aspect, as it is an environmentally friendly product, the entire process must be compatible. It would not make sense to produce low environmental impact plastic if only non-renewable energy is used. An interesting possibility to this problem is to have the entire bioplastic production chain incorporated into agro-industry (Nonato, R.V, Mantelatto, P.E Rossell, C. E. V., “Integrated Production of Biodegradable Plastic (PHB), Sugar and Ethanol”, Appl. Microbiol. Biotech. 57:1-5, 2001 ).

[0011] Therefore, the present invention meets all the necessary requirements described herein. That is, the use of high yielding bacteria in production in PHB from cultures with agro-industrial residues and whose purification does not require halogenated solvents thanks to the intracellular expression of heterologous lytic enzymes. In this way, a global process economically competitive, sustainable and with a PHB of high purity and quality is obtained.

[0012] Considerations: US Patent number 9,045,595 also states a process that does not use halogenated solvents, but requires cold and heat microfiltration, etc. In comparison, the present invention comprises a simpler and more economical process, as it requires only water washing and treatment with an alkaline solution with a pH range between 7 and 14. On the other hand, US patent number 8,986,977 B2 proposes the use of enzymes that degrade the wall either intracellularly or extracellularly for the release and purification of lipid bodies contained in plant cells. However, in the previous art, the release and purification in microorganisms has not been carried out, nor has it been taught or described in the art that there may be a synergy effect observed in the process described herein due to the internal pressure generated by the accumulation of the molecule of interest to be purified.

[0013] Brief description of the invention: The present invention includes a process, already tested on an industrial scale, comprising the PHB production as a fermentation product of genetically modified bacteria that allow for high accumulation of granules from this biopolymer (or any intracellularly cumulative biomolecules insoluble in water, e.g.: PHAs, PLA) with a membrane sensitized by the expression of heterologous lytic enzymes. Advantageously, these bacteria only initiate their lysis process once the internal pressure generated by the granule accumulation acts synergistically with the sensitized wall by action of the intracellularly expressed lysines in the appropriate amount. This joint action allows the culture to begin to lyse only at reaching its maximum yield (it does not affect the growth curve) and allows PHB with a purity greater than 95% to be obtained with only an osmotic shock or mild alkaline treatment.

[0014] Fermentation is carried out from an initial inoculum with the genetically modified strain and optimized according to the raw material to be used, such as crude glycerin from the biodiesel production, light vinasse from the alcoholic fermentation from corn or sugar cane, molasses, clarified juice, hydrolysed collagen from tanneries, among others. It can be a closed or fed-batch process, depending on the type of raw material used as a culture medium. The end point of the fermentation process is given when the accumulated PHB is between 4 g / L- 50 g / L and the lysis process in the fermentation is at least 1 hour (24 to 72 h of fermentation time).

[0015] The purification process consists of a first centrifugation step for removal of the supernatant which will be reused in a subsequent fermentation. The wet biomass obtained is subjected to an osmotic shock versus water at room temperature (RT) and mild stirring for a period of 30 min to 2 hours. Alkali is then added, if necessary, in a range of pH 7 to 14, depending on the batch lysis degree and it is incubated at RT with mild stirring for 30 min to 4 hours. The sample undergoes a second centrifugation. The PHB wet biomass is washed with H2O to pH < 11. Subsequently, a third centrifugation is carried out. The wet solution obtained that contains the purified PHB undergoes spray drying, oven, or any other drying treatment that does not affect the molecular weight of the obtained PHB.

[0016] Brief Description of Drawings: Figure 1. Photograph taken at 100x magnification (optical microscope), corresponding to a late exponential phase culture of IMT591 strain (ATCC Accession No. PTA- 127642). Cells were stained with violet crystal. Accumulation of PHB granules is observed inside (translucent body) prior to the cell lysis process

[0017] Figure 2. Photograph taken at 100x magnification (optical microscope), corresponding to a stationary phase culture of IMT591 strain (ATCC Accession No. PTA-127642). Cells were stained with violet crystal. It is clearly observed that most bacteria underwent the cell lysis process, releasing the PHB granules (translucent body) into the medium.

[0018] Figure 3. PHB growth and production curve as reference for a 700 L fermentation.

[0019] Detailed description of the invention:

[0020] A non-sporulating B. subtilis strain was constructed to efficiently produce PHB using a wide range of raw materials as substrates, named IMT589. The heterologous expression of lytic enzymes was incorporated, allowing the use of a simple and economical biopolymer purification method for industrial production. One of these strains was named IMT591 (deposited with the ATCC having ATCC Accession No. PTA-127642) and shows the following modified genotype: sigE::erm; a / sS::Pspank- phaBCchAPI; amyE::P43-ply2-4.

[0021] Likewise, IMT675 strain was constructed, (deposited with the ATCC having ATCC Accession No. PTA-127641 ) similar to IMT591 but exhibiting increased expression of lytic enzymes, due to the coding genes ply2 and ply4 are under the control of the Pspank promoter, which has greater strength in comparison to P43. The genotype of IMT675 is the following: sigE::erm; alsS::Pspank-phaBCchAP1 ; amyE::P spank-p / y 2- 4.

[0022] IMT591 (ATCC Accession No. PTA-127642) is employed in the fermentation process leading to the PHB production as described below for a 50,000 litre production plant and its corresponding purification:

[0023] Culture propagation: Dilution factor 1 / 50:

[0024] From the LB-Agar plates (or any other solid media known in the state of art in which the strain grows correctly) the following inoculum is carried out:

[0025] Inoculum: 500 mL of minimum medium (or the medium of interest to be used as raw material) with the addition of the corresponding carbon source. Example: 1.5% Refined Glycerin, Crude Glycerin, Glucose, Sucrose, Molasses, Clarified Juice, etc.). It is cultured with stirring (100-300 rpm) in a range of 20-40°C, preferably 37°C. When the Absorbance at 600 nm (Abs) of the culture is > 12 (estimated time of 12 to 16 h), such culture is used to inoculate a tank (TK A) with 20 L of culture.

[0026] -TK A: 20 L of minimum medium (MM) + 1.5% Crude Glycerin (or the medium and carbon source to be used), AbSinitiai ~0.2-0.4. It is cultured in a range of 20 to 40°C with stirring to maintain dissolved oxygen above 10% dissolved oxygen (DO). When the Abs of the culture is > 12 (estimated time of 12 to 16 h), such culture is used to inoculate tank B (TK B) with 1000 L of culture.

[0027] Formulation 1 L of MM:

[0028] -TK B: 1000 L of MM + 1 .5% Crude Glycerin (or the medium and carbon source to be used), is inoculated with the TK A culture using the liters necessary to obtain an AbSinitiai ~0.2-0.4. It is cultured in a range of 20 to 40°C with stirring to maintain dissolved oxygen above 10%, pH 6.5. When the Abs of the culture is > 12 (estimated time of 8 to 10 h), such culture is used to inoculate tank C (TK C) with 50000 L of culture.

[0029] • 50000 L Fermentation (TKC):

[0030] 48000 L of MM + 1 .5 % Crude Glycerin (or the medium and carbon source to be used), AbSinitiai ~0.2-0.4. Once the bioreactor is inoculated, it will reach an initial volume of 50000 L and initiates the growth phase according to the following biomass reproduction curve (corresponding to assays at 700 L). During this period, the remaining glycerin ranges from 2 to 0.1 %.

[0031] According to Figure 3, the batch is fed by addition of Glycerin (or other carbon source) prepared in a range of 20 to 60%, starting at the optimal point of exponential growth given in an Abs of 20. This happens between 8 to 18 h after the beginning of the culture and ends once the addition of glycerin has been completed after about 50 h have elapsed since the start of the process. The addition of the 100 L of glycerin throughout the fed stage (approx. 34 h) is carried out according to the criterion of maintaining the remaining carbon source in the range of values from 1 to 0.1 %.

[0032] Fermentation conditions are:

[0033] Temperature: 37°C (controlled, range between 20 and 40°C) pH: 6.5 (controlled, range between 5.8 and 7.8) Aeration: 1 vvm

[0034] Dissolved O2 concentration: >1 % (this value depends on the raw material used for fermentation)

[0035] Foam - Controlled

[0036] Working pressure: 100 to 800 g / cm2

[0037] • Purification:

[0038] Once the fermentation is finished, the biomass is concentrated by separating it from the depleted culture medium using a vertical plate centrifuge, decanter or other system that allows the separation of biomass from the supernatant. The approximate time of this process is 1 hour. The supernatant of the separation process is mainly composed of the total dissolved solids (called salts, which also include fermentation metabolites, cellular debries) and carries some biomass and PHB. On the other hand, there is an intrinsic loss of the process, due to dead volumes within the system.

[0039] Osmotic Shock and Alkaline Treatment.

[0040] The objective of this step is to break, hydrolyze and solubilize whole cells and cell debris in order to increase the purity of the suspended solids, i.e. increase the ratio of PHB to biomass.

[0041] The treatment consists of two steps. The first is a process of osmotic shock with water (reverse osmosis quality) to exert stress and pressure on the cell wall of bacteria, causing them to rupture by osmotic pressure.

[0042] The residence time is 15 min to 1 h and continuous stirring is required. Ratio 10 to 30 L / kg biomass.

[0043] The second process consists of the addition of alkalis so as to obtain a solution in a pH range of 7 to 14, to continue with the desired hydrolysis and solubilization process. The residence time with continuous stirring is 30 min to 2 hours. Depending on the fermentation batch and its degree of previous lysis, this step may not be required.

[0044] Once the alkalinization has been completed, it is separated by centrifugation (approximate time 1 hour) in order to separate a supernatant rich in hydrolyzed cell debris from a pellet concentrated in PHB.

[0045] In this way, PHB of good quality and purity > 95% is obtained.

[0046] Biological deposits

[0047] Representative materials of the present invention were deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110, USA, on 18th October 2023. See, Table 1 , infra.

[0048] Table 1 The deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations thereunder (Budapest Treaty). This ensures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Georgina Reh, Leandro Nakamatsu, Gonzalo Lamberto, Gustavo Schujman; Bioceres S.A., and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. Section 122 and the Commissioner’s rules pursuant thereto (including 37 C.F.R. Section 1 .14 with particular reference to 886 OG 638).

[0049] The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions; the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

[0050] Examples:

[0051] Microorganism used for PHB production

[0052] For the construction of the IMT591 strain (deposited with the ATCC having ATCC Accession No. PTA-127642), the following genetic modifications were made to the prototrophic strain of B. subtilis PY79:

[0053] -SigE was deleted by transforming B. subtilis with a vector that interrupts said gene with an erythromycin resistance cassette. sigE encodes the Sigma E factor of RNA polymerase, specific for sporulation. Therefore, its deletion makes B. subtilis a nonsporulating microorganism.

[0054] -The Pspank-est-phaBACP1 construct, together with a spectinomycin resistance cassette, was integrated into the alsS locus: Pspank-est (168bp): modified version of the Hyperspank promoter (www. parts. iqem.orq / Part:B Ba K143015) to which the transcriptional stabilizer of the aprE gene was added in order to increase mRNA half-life. In addition, strain IMT591 (ATCC Accession No. PTA-127642) does not express Lacl repressor, making Pspank- est a very strong constituent promoter. phaA (1181 bp): [3 -Cupriavidus necator ketothiolase phaB (740 bp): Cupriavidus necator acetyl-CoA-reductase phaCch (1704pb): Chromobacterium sp LISM2 PHA polymerase phaP1 (579 bp): Cupriavidus necator phasin

[0055] - It was also integrated into the amyE the P43-ply2-ply4 construction, with a chloramphenicol resistance cassette:

[0056] P43 (99 bp): strong constitutive promoter in B. subtilis. Derived from the promoter region of the B. subtilis cdd gene, which encodes the enzyme cytidine deaminase (Wand and Doi, 1984. ply2 (480 bp): N-terminal catalytic domain of the prophage A Ba02 (PlyL) endolysin found in the B. anthracis genome (Low 2005; Low 2011 ).

[0057] Ply4 (570bp): N-terminal catalytic domain of the prophage Ba04 (PlyB) endolysin found in the B. anthracis genome (Low 2011 ).

[0058] List of other possible enzymes with lytic activity to be used:

[0059] PlyB: prophage of B. anthracis muramidase activity. (Low 2011 )

[0060] Ply21 : enzyme with amidase activity of the prophage TP21 of B. cereus (Lieh Yoon Low, 2005).

[0061] Phy11 : enzyme with amidase activity of prophage Staphylococus aureus (Donovan, D.M., 2006).

[0062] XlyA: enzyme with amidase activity of prophage Bacillus subtilis (Longchamp, P.F., 1994).

[0063] Cpl-1 : enzyme with muramidase activity of prophage Streptococcus pneumoniae (Sanz, J. M., 1992).

[0064] Cellosyl: enzyme with amidase activity of prophage Streptomyces coelicolor( Brau, B., 1991 ). Fermentation in fed batch using biodiesel-derived crude glycerin in minimal culture medium.

[0065] Strain used IMT591 (ATCC Accession No. PTA-127642). The fermenter is inoculated at an initial Absorbance (600 nm) of 0.2 to 0.4 containing a minimum culture medium (buffer phosphate and salts) and 2% (v / v) of initial glycerin (1 .75% crude glycerin and 0.25% refined glycerin, this ratio varies according to the NGOM (non-glycerol organic matter) of the crude glycerin. Fermentation conditions are constant temperature at 37°C, aeration 1 vvm, pH 6.5 (may be in the range between 6 and 7.5) maintained with pH sensor coupled to base pump containing NH4OH (optional NaOH 2 N), 15% Dissolved oxygen (DO) coupled in cascade to stirring between 150 to 1200 rpm. Once the glycerin has been consumed from the closed batch, feed with crude glycerin is carried out, maintaining its remaining value in the culture in a range of 0 to 1 % v / v. Fermentation was terminated after 72 h from started (may be in a range of 48 to 72 h) Total biomass produced 19.3 q / L initial culture and total PHB produced 15 q / L initial culture. With a glycerin to PHB bioconversion of 25.4%.

[0066] Fermentation in fed-batch using crude glycerin derived from biodiesel in culture medium containing 50% of supernatant from a previous fermentation.

[0067] Strain used IMT591 (ATCC Accession No. PTA-127642). The fermenter is inoculated at an initial Absorbance (600 nm) of 0.2 to 0.4 containing phosphate buffer and salts, 50% v / v of pre-fermentation supernatant (rich in cellular debries and organic acids such as, mainly, malate, acetate, succinate and lactate) and 2% (v / v) of initial glycerin (1 .75% of crude glycerin and 0.25% of refined glycerin, this ratio varies depending on the NGOM of the crude glycerin). Fermentation conditions are constant temperature at 37°C, aeration 1 vvm, pH 6.5 (may be in the range between 6 and 7.5) maintained with pH sensor coupled to base pump containing 15 % NH4OH (optional NaOH2 N), 15% Dissolved oxygen (DO) coupled in cascade to stirring between 150 to 1200 rpm. Once the glycerin has been consumed from the closed batch, feeding with crude glycerin is carried out, maintaining its remaining value in the culture in a range of 0 to 1 % v / v. Fermentation was terminated after 72 h (may be in a range of 48 to 72 h). Total biomass produced 21.6 g / L initial culture and total PHB produced 18.1 g / L initial culture. With a glycerin to PHB bioconversion of 36%. Fermentation in fed-batch using Vinasse from alcoholic fermentation of sugarcane together with molasses.

[0068] Strain used IMT591 (ATCC Accession No. PTA-127642). Composition of the fermentation medium contains 50% filtered Vinasse (this value can range between 50 to 90%) with the addition of 2.5% (w / v) molasses (this value can range between 2% and 5%). This culture medium is brought to pH 7.2 with NH4OH (NaOH can also be used) and then sterilized. The fermenter is inoculated at an initial Absorbance (600 nm) of 0.2 to 0.4. Fermentation conditions are constant temperature at 37 °C, aeration 0.2 vvm, pH in a range between 6.0 y 7.8 maintained with pH sensor coupled to base pump containing 15 % NH4OH (optional NaOH2 N), 0.1 to 5 % Dissolved oxygen (DO) coupled to cascade to stirring between 150 to 1200 rpm. Molasses feeding begins when culture pH reaches a value of 6.8, given by pulses such that the concentration of molasses in the culture is not more than 5% (w / v). Fermentation was terminated after 48 h (may be in a range of 30 to 48 h). Total biomass produced 9.15 g / L initial culture and total PHB produced 5.3 g / L initial culture. With a molasses to PHB bioconversion of 10.6%.

[0069] EXAMPLE 1 PHB purification (typical procedure on fermentation biomass strain IMT591, expressing lytic enzymes).

[0070] 100 mL of sample with 20.9 q biomass / L culture (IMT591 strain, ATCC Accession No. PTA-127642) and 12 g PHB / L, i.e. 57.4% of PHB.

[0071] The sample is centrifuged at 3200 g and at 25°C for 20 minutes. The obtained pellet is treated with osmotic shock with 25 mL H2O d / g biomass, stirring for 1 hour at RT and 270 rpm. After this step, 30 mL NaOH 0.1 N / g biomass is added, and the sample is stirred for 2 hours (alkaline treatment). The sample treated is centrifuged at 3200 xg and RT for 20 minutes. The supernatant is discarded, and the pellet is resuspended in 30 mL H20d / g biomass (washing) and stirring at 270 rpm at RT for 1 hour. The sample is centrifuged under the same conditions mentioned above and a second washing is performed on the pellet, repeating the procedure described. Finally, the pellet product of the second washing is allowed to dry in a stove at 60 °C until obtaining a constant weight. Under these conditions, a product with a purity of 99% and a PHB recovery of 91% is achieved relative to the initial PHB of the sample.

[0072] EXAMPLE 2 Purification of PHB without alkaline treatment on fermentation biomass strain IMT591, expressing lytic enzymes

[0073] 100 ml of sample with 15.25 g_biomass / L culture (cepa IMT591 , ATCC Accession No. PTA-127642) and 12.41 g PHB / L, i.e. 81.4% of PHB.

[0074] Samples are centrifuged at 3200 x g and RT for 20 minutes. The obtained pellets are treated with osmotic shock with 15 ml H2Od / g biomass, stirring for 3 hours with stirring at 270 rpm and RT. These samples do not undergo alkaline treatment. Samples are centrifuged at 3200 xg for 20 minutes. The supernatant is discarded and the pellet is resuspended in 15 mL H2Od / g biomass (washing) and stirring at 270 rpm at RT for 1 hour. The sample is centrifuged under the same conditions mentioned above and a second washing is performed on the pellet, repeating the procedure described. Finally, the pellet product of the second washing is allowed to dry in a stove at 60 °C until obtaining a constant weight.

[0075] Under these conditions, a product with a purity of 94% and a PHB recovery of 77% is achieved relative to the initial mass of the product.

[0076] EXAMPLE 3 Purification of PHB without osmotic shock on fermentation biomass strain IMT591, expressing lytic enzymes

[0077] 150 mL of sample with 32.87 q biomass / L culture (IMT591 strain. ATCC Accession No. PTA-127642) and 29.45 g PHB / L, i.e. 89.6 % of PHB.

[0078] The sample is centrifuged in the same condition as the above examples. An alkaline treatment is carried out on the pellet obtained by adding 50 mL NaOH 0.2 N Zg biomass (no osmotic shock is carried out), stirring for 1 hour on a stirrer at 270 rpm and 25°C. The sample treated is centrifuged at 3200 xg and 25 °C for 20 minutes. The supernatant is discarded, and the pellet is resuspended in 50 mL H20d / g biomass (washing) and stirring at 270 rpm at 25 °C for 1 hour. The sample is centrifuged under the same conditions as above. Here the process can be completed or the washing repeated. Finally, the washed pellet is allowed to dry in a stove at 60 °C until obtaining a constant weight. Under these conditions, a product with a purity of 91 to 95% (depending on whether 1 or 2 washings are performed, respectively) and a PHB recovery of 85% relative to the initial PHB is achieved.

[0079] EXAMPLE 4 PHB Purification - (pH alkalinization - single washing, on fermentation biomass strain IMT591, expressing lytic enzymes)

[0080] A 100 mL sample with 21.25 q biomass / L culture (IMT591, ATCC Accession No. PTA-127642) and 17.35 g PHB / L, i. e., the 81.6 % of PHB.

[0081] The sample is centrifuged at 3200 xg and 25 °C for 20 minutes. The pellet obtained is subjected to an osmotic shock treatment with 25 ml H20d / g biomass, stirring for 4 hours at 270 rpm and 25 °C. After this step, a volume of 5% (w / v) NaOH is added such that a pH=12 is reached, and the sample is stirred for an additional 2 hours at 270 rpm and 25°C (alkaline treatment). The sample treated is centrifuged at 3200 xg and 25 °C for 20 minutes. The supernatant is discarded, and the pellet is resuspended in 30 mL H2Od / g biomass (washing) and stirred at 270 rpm at 25 °C for 1 hour. The sample is centrifuged under the same conditions mentioned above, the pH of the washed supernatant is equal to 10, this supernatant is discarded, and the pellet is allowed to dry in a stove at 60 °C until constant weight is obtained.

[0082] Under these conditions, a product with a purity of 98% and a PHB recovery of 72% is achieved relative to the initial PHB of the sample.

[0083] EXAMPLE 5 PHB Purification (typical procedure on culture biomass strain IMT589, in the absence of heterologous lytic enzymes)

[0084] This example allows to validate the importance of the expression of lytic enzymes to obtain a high purity PHB using a purification method without the addition of solvents and / or detergents.

[0085] A sample of 26 ml with 6.3 g biomass / L culture (IMT589 strain, ATCC Accession No. PTA-127642) and 4.2 g PHB / L, i e 66.8 % of PHB.

[0086] The sample is centrifuged at 3200 xg and 25 °C for 20 minutes. The obtained pellet is treated with osmotic shock with 25 mL H2O d / g biomass, stirring for 1 hour at RT and 270 rpm. After this step, 30 mL NaOH 0.1 N / g biomass is added, and the sample is stirred for 2 hours (alkaline treatment). The sample treated is centrifuged at 3200 xg and RT for 20 minutes. The supernatant is discarded, and the pellet is resuspended in 30 mL H2Oa / g biomass (washing) and stirring at 270 rpm and RT for 1 hour. The sample is centrifuged under the same conditions mentioned above and a second washing is performed on the pellet, repeating the procedure described. Finally, the pellet product of the second washing is allowed to dry in a stove at 60 °C until obtaining a constant weight.

[0087] Under these conditions, a product with a purity of 85.5% and a PHB recovery of 50.6% is achieved relative to the initial PHB of the sample.

[0088] EXAMPLE 6 PHB Purification (typical procedure on culture biomass strain IMT675, expressing heterologous lytic enzymes from a stronger promoter than strain IMT 591)

[0089] This example allows to validate the importance and need for synergy between the turgence generated by the internal accumulation of biopolymer granules together with the action of the lytic enzymes to obtain a high purity PHB using a purification method without the addition of solvents and / or detergents.

[0090] A 100 mL of sample with 24.1 q biomass / L culture (IMT675 strain, ATCC Accession No. PTA-127641) and 12.4 g PHB / L, i.e. 51.4% of PHB.

[0091] The sample is centrifuged at 3200 g and at 25°C for 20 minutes. The obtained pellet is treated with osmotic shock with 25 mL H2O d / g biomass, stirring for 1 hour at RT and 270 rpm. After this step, 30 mL NaOH 0.2 N Zg biomass is added and the sample is stirred for 2 hours (alkaline treatment). The sample treated is centrifuged at 3200 g and RT for 20 minutes. The supernatant is discarded and the pellet is resuspended in 30 mL H2Od / g biomass (washing) and stirred at 270 rpm at RT for 1 hour. The sample is centrifuged under the same conditions mentioned above and a second washing is performed on the pellet, repeating the procedure described. Finally, the pellet product of the second washing is allowed to dry in a stove at 60 °C until obtaining a constant weight.

[0092] Under these conditions, a product with a purity of 75% and a PHB recovery of 92% is achieved relative to the initial PHB of the sample.

[0093] The strain IMT675 (ATCC Accession No. PTA-127641 ) used in this assay has the particularity of expressing lytic enzymes from a stronger promoter than the strain IMT591 (ATCC Accession No. PTA-127642), however, the increase in lytic enzymes results in premature cell lysis and does not allow good intracellular granule accumulation. This is reflected in a PHB / biomass ratio of < 55 %. In this example, the standard purification protocol used for strain IMT591 was used, however, unlike what was reported in examples 2.6.5-8, wherein the purity obtained is > 90%, in this case it was 75%. This decrease in the purity degree reflects the need for synergy and balance between internal pressure by macromolecule accumulation and the action of lytic enzymes on the wall of the microorganism.

[0094] Detailed in the following table are the sequences employed throughout the present invention.

Claims

Claims:

1. A genetically modified Gram-positive bacterium that efficiently produces polyhydroxyalkanoates (PHAs), wherein said bacterium comprises: a) a heterologous DNA sequence encoding the enzymes necessary for the synthesis of PHA, and b) a heterologous DNA sequence encoding an enzyme with lytic activity, wherein the microorganism has a mutation that makes it non-sporulating, and wherein said microorganism has a high conversion efficiency from raw material into polymer.

2. The genetically modified bacterium of claim 1 , wherein the bacterium is of Firmicutes phylum, including the genuses: Bacillus, Lactobacillus, Lactococcus, Streptococcus, Clostridium, among others.

3. The genetically modified bacterium of claim 1 , wherein the bacterium is Bacillus subtilis.

4. The genetically modified bacterium of claim 1 , wherein the bacterium produces PHAs selected from the group consisting of: poly-3-hydroxybutyrate (PHB), poly- hydroxybutyrate-co-hydroxyvalerate (PHB-V), poly-4hydroxybutyrate (P4HB), poly-3-hydrobutyrate-co-4-hydroxibutyrate (P3HB4HB), and medium chain PHAs such as poly-hydroxyhexanoate (PHHx).

5. The genetically modified bacterium of claim 4, wherein the bacterium produces PHB.

6. The genetically modified bacterium of claim 5, wherein the heterologous DNA sequence encoding the enzymes necessary for the synthesis of PHA comprises: a) a modified version of the Hyperspank promoter, b) a gene encoding [3-ketothiolase of Cupriavidus necator, c) a gene encoding acetyl-coA-reductase of Cupriavidus necator, d) a gene encoding PHA polymerase of Chromobacterium sp LISM2, and e) a gene encoding phasin of Cupriavidus necator.

7. The genetically modified bacterium of claim 6, wherein the heterologous DNA comprise SEQ ID NO: 1 , 3, 5, 7, 13-14.

8. The genetically modified bacterium of claim 1 , wherein the mutation that makes it non-sporulating is an insertion into sigE gene.

9. The genetically modified bacterium of claim 1 , wherein the enzyme with lytic activity is selected from the group consisting of: Ply2, Ply21 , Ply-4, Phil 1 , PlyB, XlyA, Cpl- 1 , Cellosyl and others.

10. The genetically modified bacterium of claim 8, wherein the enzyme with lytic activity is ply2, of SEQ ID NO: 10.11 . The genetically modified bacterium of claim 1 , wherein the bacterium is capable of producing PHAs using as raw material: crude glycerin, vinasse, molasses, cell debris, organic acids such as malate, acetate, succinate, and lactate, or any combination thereof; protein derivatives, dairy derivatives (whey protein, lactose, etc.).

12. The genetically modified bacterium of claim 1 , wherein the microorganism is selected from: IMT591 (ATCC Accession number: PTA-127642) and IMT675 (ATCC Accession number PTA-127641 ).

13. A recombinant nucleic acid molecule comprising: a) a modified version of the Hyperspank promoter, b) a gene encoding [3-ketothiolase of Cupriavidus necator, c) a gene encoding acetyl-coA-reductase of Cupriavidus necator, d) a gene encoding PHA polymerase of Chromobacterium sp LISM2, and / or e) a gene encoding phasin of Cupriavidus necator, wherein the recombinant nucleic acid molecule encodes the enzymes necessary for the synthesis of PHAs.

14. The recombinant nucleic acid molecule of claim 13, wherein the heterologous DNA comprises SEQ ID NO: 1 , 3, 5, 7, 13-14.

15. A vector comprising the nucleic acid molecule of claim 14.

16. The vector of claim 15 which is used to transform the microorganism of claim 1 .

17. A PHA production method comprising efficiently culturing a genetically modified Gram-positive bacteria that produces polyhydroxyalkanoates (PHAs), wherein said bacteria comprises: a heterologous DNA sequence encoding the enzymes necessary for the synthesis of PHAs, and a heterologous DNA sequence encoding an enzyme with lytic activity, wherein the microorganism has a mutation that makesit non-sporulating together with at least one raw material under conditions necessary for PHAs to be produced.

18. The method of claim 17, wherein the bacterium is of genus: Bacillus, Lactobacillus, Lactococcus, Streptococcus, Clostridium, among others.

19. The method of claim 18, wherein the bacterium is Bacillus subtilis.

20. The method of claim 18, wherein the bacterium produces PHAs selected from the group consisting of: poly-3-hydroxybutyrate (PHB), poly-hydroxybutyrate-co- hydroxyvalerate (PHB-V), poly-4hydroxybutyrate (P4HB), poly-3-hydroxibutyrate- co-4-hydroxibutyrate (P3HB4HB), and medium chain PHAs such as polyhydroxyhexanoate (PHHx).

21. The method of claim 20, wherein the bacterium produces PHB.

22. The method of claim 21 , wherein the heterologous DNA sequence encoding the enzymes necessary for the synthesis of PHB comprises: a) a modified version of the Hyperspank promoter, b) a gene encoding [3-ketothiolase of Cupriavidus necator, c) a gene encoding acetyl-coA-reductase of Cupriavidus necator, d) a gene encoding PHA polymerase of Chromobacterium sp LISM2, and / or e) a gene encoding phasin of Cupriavidus necator.

23. The method of claim 22, wherein the heterologous DNA sequence comprises SEQ ID NO: 1 , 3, 5, 7, 13-14.

24. The method of claim 23, wherein the bacterium is selected from: IMT591 (ATCC Accession number: PTA-127642) and IMT675 (ATCC Accession number PTA- 127641 ).

25. The method of claim 26, wherein the enzyme with catalytic activity is selected from the group consisting of: Ply2, Ply21 , Ply4, Phil 1 , PlyB, XlyA, Cpl-1 , Cellosyl and others.

26. The method of claim 25, wherein the enzyme with lytic activity is ply2, of SEQ ID NO: 10.

27. The method of claim 17, wherein the raw material is selected from the group consisting of: crude glycerin, vinasse, molasses, cellular debries, organic acids such as malate, acetate, succinate, and lactate, or any combination thereof; protein derivatives, dairy derivatives (whey proteins, lactose, etc.).

28. A procedure for PHA purification from a culture of a genetically modified Grampositive bacteria producing polyhydroxyalkanoates (PHAs) comprising: a. centrifuging a PHA containing culture, b. performing an osmotic shock and / or performing an alkaline treatment, and c. washing.

29. A solvent-free polyhydroxybutyrate (PHB) obtained by the procedure of claim 28.

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