WO2011086211A1 - Système d'autolyse cellulaire pour le traitement de la biomasse bactérienne dans la production de polyhydroxyalkanoates chez pseudomonas putida kt2440 - Google Patents

Système d'autolyse cellulaire pour le traitement de la biomasse bactérienne dans la production de polyhydroxyalkanoates chez pseudomonas putida kt2440 Download PDF

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WO2011086211A1
WO2011086211A1 PCT/ES2010/070858 ES2010070858W WO2011086211A1 WO 2011086211 A1 WO2011086211 A1 WO 2011086211A1 ES 2010070858 W ES2010070858 W ES 2010070858W WO 2011086211 A1 WO2011086211 A1 WO 2011086211A1
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strain
pseudomonas putida
lytic
pha
microorganism
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PCT/ES2010/070858
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Spanish (es)
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Virginia MARTÍNEZ LÓPEZ
Eduardo DÍAZ FERNÁNDEZ
Pedro GARCÍA GONZÁLEZ
José Luís GARCÍA LÓPEZ
Estrella DUQUE MARTÍN DE OLIVA
Juan Luís RAMOS MARTÍN
María Auxiliadora PRIETO JIMÉNEZ
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Consejo Superior De Investigaciones Científicas (Csic)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/78—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

  • the present invention relates to a bacterial strain of Pseudomonas putida KT2440, which comprises a lytic heterologous genetic system where said system in turn comprises the nucleotide sequence encoding the lytic cell wall enzyme endolysin Ejl, the nucleotide sequence encoding holina Ejh , and a nucleotide sequence encoding a gene regulation system, which in turn comprises a nucleotide sequence promoting gene expression, and a nucleotide sequence encoding and expressing a regulatory protein for said gene expression.
  • the strain is the microorganism with CECT deposit number 7659.
  • the present invention also relates to said strain where the Pseudomonas putida KT2440 microorganism is mutant in one or more of the tol-pal genes.
  • said strain is the microorganism with deposit number CECT 7658.
  • the present invention relates to the use of any of the strains described for the extraction of polyhydroxyalkanoate (PHA) synthesized by said bacterium as well as to a method for the extraction of said compound.
  • PHA polyhydroxyalkanoate
  • Bioprocess technology has undergone considerable progress in recent years trying to improve and adapt modern biotechnology to classical fermentation technologies. In this sense, recombinant DNA technology or, in a broader concept, molecular biology techniques, have been decisive so that a large number of organisms can be exploited and manipulated for the production of compounds of interest. To a large extent, this success has been possible thanks to the development of gene expression systems in heterologous organisms that are easier to manipulate and multiply.
  • PHAs commonly known as “bioplastics” are biodegradable polymers produced by certain bacteria, which accumulate inside the cell in the form of carbon source reserve granules when culture conditions are not optimal for growth (Madison and Huisman , 1999. Microbiol. Mol. Biol. Rev., 63: 21-53; Prieto et al. 2007. Synthesis and degradation of polyhydroxyalkanoates. In Pseudomonas: a Model System in Biology. Pseudomonas, vol. V, Eds, Ramos, JL and Filloux, A. Springer, 397-428).
  • biopolymers are biodegradable and the bacteria synthesize them from renewable sources such as glucose, fructose or fatty acids that are part of vegetable oils. Therefore, the term bioplastic can be defined as a biopolymer synthesized from renewable sources, which can be biodegraded under controlled conditions and that presents physicochemical characteristics similar to plastics derived from the petrochemical industry (Sarasa et al. 2009. Bioresour. Technol. 100: 3764-3768).
  • PHA granules are composed of a polyester (93-97% of the dry weight of the granule [PSG]) surrounded by phospholipids (1-6% of PSG) and granule-associated proteins (GAPs) (1 -2% of PSG), which form a thin layer on the surface of the granule (Steinbüchel et al. 1995. Can. J. Microbio /., 41: 94-105).
  • GAPs three classes of GAPs have been defined in bacteria: i) PHA synthases, involved in the polymerization of PHA, ii) PHA depolymerases, responsible for bioplastic degradation and iii) fasines, the most abundant GAPs, with a structural or regulatory function (Prieto et al. 1999a. Appl. Environ. Microbio!., 65: 3265-3271; Moldes et al., 2004. Appl. Environ. Microbio!., 70: 3205-3212).
  • PHAs are classified into two main types according to their chemical structure: short chain PHAs (scl-PHAs) obtained from monomers with 4 or 5 carbon atoms and medium chain (mcl-PHAs) monomers from monomers with 6 to 14 carbon atoms.
  • the different PHAs identified to date are linear polymers composed of fatty 3- hydroxy acids exclusively of the R (RHAs) configuration.
  • the molecular weight of these polymers varies between 50,000-1,000,000 and their diversity lies in the substitutions in the asymmetric carbon in position 3, which gives the polymer the chiral character (Prieto et al. (1999b) J. Bacterium !. 181 : 858-868).
  • composition of the polymer depends on the source of carbon present in the culture medium used during the fermentation of the producing bacteria (Durner, et al. 2001. Biotechnol. Bioeng., 72: 278-288, Jung et al. 2001. Biotechnol. Bioeng., 72: 19-24).
  • PHAs can also be useful for biomedical applications such as biomaterials (Zinn et al. 2001. Adv. Drug. Deliv. Rev., 53: 5-21).
  • PHA can be considered as a source of new chiral compounds (synthons) of great utility as precursors in the pharmaceutical industry, since they are difficult to achieve in pure state by conventional chemical processes.
  • bacteriophages or phage abbreviated
  • filamentous ones are lithic and break the wall of the host bacteria until their complete lysis.
  • phages use two proteins that act in a perfectly coordinated way: a holine and an endolysin.
  • the genes that encode these proteins are usually contiguous and their transcription and translation is late, starting only when the virions are in their final phase of packaging within the bacterial cytoplasm.
  • the holines constitute a group of phage proteins of very varied primary structure but which are grouped into three classes and functionally are very similar in their mode of action.
  • Endolysins are lytic enzymes encoded by phages, do not have a secretion signal peptide, accumulate in the cytoplasm during the vegetative cycle and are translocated to the cell wall through the pore formed by the oligomerization of holine.
  • the endolisins can be muramidases, glucosaminidases, transglycosylases, amidases or endopeptidases (Hermoso et al. (2007) Curr. Op. Microbiol .10: 461-472).
  • the Tol-Pal complex of bacteria is organized into two protein complexes: an inner membrane complex consisting of TolQ, TolA and ToIR proteins that interact with each other through transmembrane domains, and another membrane-associated complex external composed of TolB and Pal proteins, which also interacts with Lpp, OmpA and peptidoglycan (Lazzaroni et al. 1999. FEMS Microbiol. Lett., 177: 191-197). These proteins are located forming complexes in the cell envelope of Gram-negative bacteria, and are critical for maintaining the structure of the envelope. The alteration of the same leads to situations of weakening of the cell and increased sensitivity to detergents or chelating agents (Llamas et al. 2000. J. Bacterium!., 182: 4764-4772).
  • the present invention relates to a bacterial strain of Pseudomonas putida KT2440, which comprises a lytic heterologous genetic system where said system in turn comprises the nucleotide sequence encoding the lytic cell wall enzyme endolysin Ejl, the nucleotide sequence encoding holina Ejh , and a nucleotide sequence encoding a gene regulation system, which in turn comprises a nucleotide sequence promoting gene expression, and a nucleotide sequence encoding and expressing a regulatory protein for said gene expression.
  • the strain is the microorganism with deposit number CECT 7659.
  • the present invention also relates to said strain where the microorganism Pseudomonas putida KT2440 is mutant in one or more of the tol-pal genes.
  • said strain is the microorganism with deposit number CECT 7658.
  • the present invention relates to the use of any of the strains described for the extraction of polyhydroxyalkanoate (PHA) synthesized by said bacterium as well as to a method for the extraction of said compound.
  • PHA polyhydroxyalkanoate
  • a solution is offered to the extraction of compounds produced by the bacteria Pseudomonas putida KT2440, and particularly the extraction of polyhydroxylacanoates thereby decreasing the use of contaminating chemicals used to lyse the producing cells.
  • the results of the cell autolysis in two bacterial strains deposited in the Spanish Type Culture Collection (CECT) are shown in the examples of the present invention.
  • the two strains comprise a lytic heterologous genetic system that expresses genes involved in the degradation of the cell wall and in the permeability of the cell membrane, whose expression is controlled by a regulator system of expression dependent on an inducer.
  • the two strains described above and deposited in the CECT carry the lytic heterologous genetic system in monocopy.
  • the recombinant strain CECT 7659 that carried the lytic heterologous genetic system in monocopy was more stable than the native strain transformed with the plasmid carrying the lithic system in multicopy.
  • mutations in the tol-pal genes cause instability of the outer cell membrane in the Pseudomonas putida KT2440 bacteria and allows the periplasmic proteins to be secreted and the bacteria lysed in the presence of some detergents and EDTA.
  • this mutation could be useful for inducing lysis by detergent and EDTA in PHA producing cells.
  • the presence of PHA instead of destabilizing the mutant cell in the tol-pal genes made the cell more stable and insensitive to lysis by the detergent and the EDTA chelating agent. Therefore, this mutation was unusable for the proposed purpose.
  • the mutant Pseudomonas putida KT2440 bacteria in the tol-pal genes was not individually useful for the proposed purpose, it was analyzed whether the combination of this lysis system of the mutant strain in the tol-pal genes with the lytic heterologous genetic system in monocopy of the present invention, generated some unexpected synergistic effect that would propitiate cell lysis under the desired conditions.
  • a synergic effect of the threshold type is generated, a priori unpredictable, in such a way that until all elements are not combined in a precise way, the threshold of de-structuring of the cell envelope necessary for lysis and therefore the cell is not exceeded. It remains perfectly viable. Under these conditions, the release of the compounds contained in the cell cytoplasm occurs with high efficiency in the presence of a detergent and a chelating agent only when the lytic heterologous genetic system is induced in monocopy, thus becoming the new recombinant strain created (CECT 7658 ) in a new body very useful for the proposed objective.
  • one aspect of the present invention relates to a bacterial strain of Pseudomonas putida KT2440, which comprises a lytic heterologous genetic system where said system in turn comprises the nucleotide sequence encoding a lytic cell wall enzyme, the nucleotide sequence that encodes a holine, and a regulatory system of gene expression.
  • the lytic cell wall enzyme is a protein capable of breaking the cell wall or the substances that make up that wall.
  • Said lithic enzyme can come from bacteria, fungi, yeasts, plants, animals or bacteriophages.
  • the lytic cell wall enzyme is selected from the list comprising, but not limited to, Exl of the bacteriophage EJ-1 of Streptococcus pneumoniae, Cpl-1 of the bacteriophage Cp-1 of Streptococcus pneumoniae, transglicosidase R of the bacteriophage ⁇ of Escherichia coli, lysozymes of bacteriophage P22 from Salmonella and bacteriophage T4 from Escherichia coli, protein E from bacteriophage 0X174 from E. coli, Sgl transglicosidase and AmiA amidase from Escherichia coli (Young, R. 1992. Microbiol. Rev. 56: 430-481).
  • Holin is a phageal protein with transmembrane domains that is located in the cytoplasmic membrane forming a pore that allows the passage of the lytic enzyme responsible for breaking the bacterial wall and causing lysis.
  • Holines have a very varied primary structure but are grouped into three classes and functionally they are very similar in their mode of action. Said holina comes from a bacteriophage.
  • said holina is selected from the list comprising, but not limited to, Exh of bacteriophage EJ-1 of Streptococcus pneumoniae, Cph-1 of bacteriophage Cp-1 of Streptococcus pneumoniae, holina of bacteriophage 06 of Pseudomonas phaseolicola, holina S of bacteriophage ⁇ of Escherichia coli, holina from bacteriophage P22 from Salmonella (Young, R. 1992. Microbiol. Rev. 56: 430-481).
  • the scientific classification of the bacteria of the present invention is: Kingdom: Bacteria I Division: Proteobacteria I Order: Pseudomonadales I Family: Pseudomonadaceae Genus: Pseudomonas Species: Pseudomonas putida.
  • the complete genome sequence of Pseudomonas putida KT2440 has been disclosed by Nelson et al., (2002) (Nelson et al., 2002. Environ Microbiol., 4 (12): 799-808).
  • the access number of the complete sequence of Pseudomonas putida KT2440 is AE015451 .1 and its taxonomic identification (Taxonomy ID) is 160488.
  • lytic heterologous genetic system refers to a genetic construct that comprises nucleotide sequences that correspond to one or more different organisms of Pseudomonas putida.
  • the lytic cell wall enzyme and holine may be the nucleotide sequences encoding the amino acid sequence of the proteins that have such activity, from phage of Streptococcus pneumoniae, Lactobacillus casei, Lactobacillus rhamnosus, Bacillus subtilis or Bacillus megaterium.
  • said sequences come from phage EJ-1 of Streptococcus pneumoniae.
  • gene expression regulation system refers to a set of nucleotide sequences that regulate the expression of the "lytic heterologous genetic system” and can be heterologous or not, that is, these sequences may or may not come from Pseudomonas putida. That is, said term refers to a set of nucleotide sequences that have an effect on the functionality of a nucleotide sequence with respect to the beginning of transcription or the start of translation of an RNA sequence or other sequences.
  • genes of gene expression are those that are called inducible regulatory systems that are constituted by one or more nucleotide sequences called promoters / operators and by one or more nucleotide sequences that encode and express one or more proteins.
  • regulators that activate or repress gene expression in the presence of one or more compounds called inducers.
  • An example of gene expression activator is the Pseudomonas putida XyIS protein that belongs to a family of transcriptional regulators called XyIS / AraC, involved in stimulating the transcription of at least 90 different proteins involved in different cellular processes such as the metabolism of carbon, pathogenesis and the response to alkylating agents in bacteria.
  • the gene expression regulatory protein is selected from the list comprising, but not limited to, any protein that can act as a regulator of gene expression in Pseudomonas putida.
  • a preferred embodiment relates to the bacterial strain of Pseudomonas putida KT2440, which comprises a lytic heterologous genetic system, where the lytic cell wall enzyme is the endolysin Ejl, the holine is the Ejh protein, and the gene expression regulatory system is xyl / Pm.
  • Nucleotide sequences encoding Ejl endolysin and Ejh holine may, for example, but not be limited to, from phage of Streptococcus pneumoniae, Bacillus subtilis or Bacillus megaterium. Preferably said sequences come from phage EJ-1 of Streptococcus pneumoniae. Said nucleotide sequences may be part of a cassette in which the ejl sequence precedes the ejh sequence, in the 5 ' to 3 ' direction .
  • the gene expression regulatory sequence, xyl / Pm consists of a nucleotide sequence that encodes and expresses the XyIS protein, a transcription regulator of the TOL plasmid promoter that regulates the expression of the Pm promoter of the target degradation pathway.
  • the XyIS protein belongs to the family of XyIS / AraC transcription regulators.
  • Pm has two motifs called A and B, whose sequences are TGCA and GGNTA, respectively. These motifs are repeated in the Pm promoter and as a consequence the XyIS protein monomers bind to said repetitions, activating transcription.
  • an activator such as, but not limited to, 3- methylbenzoic acid (hereinafter 3-MB).
  • the lytic heterologous genetic system described in previous paragraphs may also comprise a resistance marker that allows the selection of the bacteria that contain said system.
  • antibiotic resistance genes are an important tool in genetic engineering in general. Antibiotic resistance genes have the ability to deactivate selectively certain antibiotics and, consequently, protect the cells against those antibiotics. An antibiotic resistance gene, therefore, can be used to betray the existence of the lytic heterologous genetic system of the present invention.
  • the antibiotic gene is linked to said system before being transformed into the recipient bacterial cell. These cells are then incubated in the presence of the antibiotic. Only the cells that reproduce under these conditions are the ones that will have incorporated the antibiotic resistance gene along with that genetic system.
  • the marker confers resistance to the cells against the antibiotic kanamycin.
  • the marker can confer resistance against chemicals or against the presence of unusual nutrients.
  • a preferred embodiment of the present invention relates to any bacterial strain derived from Pseudomonas putida KT2440, described in previous paragraphs, where said strain is the microorganism with CECT deposit number 7659. Said microorganism has been deposited in the Spanish Type Culture Collection ( CECT) on December 16, 2009, the deposit number CECT 7659 corresponded to it. The address of said International Deposit Authority is: University of Valencia / Research building / Campus of Burjassot / 46100 Burjassot (Valencia).
  • the strain CECT 7659 comprises a lytic heterologous genetic system.
  • This strain has the ejh and ejl genes that encode a holine (Ejh) and a lytic enzyme (Ejl) of the bacteriophage EJ-1, respectively, under the transcriptional control of the Pm promoter (ejh-ejl cassette, xyIS regulatory construction) / Pm and a kanamycin resistance gene).
  • the nucleotide sequence that codes for holina Ejh is SEQ ID NO: 1 and the sequence that codes for endolysin Ejl is SEQ ID NO: 2 (get ejh-ejl; Accession number NC_005294.1), and the nucleotide sequence of components of the xyl / Pm gene expression regulatory system is SEQ ID NO: 5 for Xyl (Accession number NP_542858) and SEQ ID NO: 6 for Pm (Accession number X01 103).
  • KTHL can be used interchangeably.
  • strain CECT 7659 The culture and maintenance conditions of strain CECT 7659, communicated to the International Deposit Authority, are:
  • Another preferred embodiment refers to any strain derived from the strain Pseudomonas putida KT2440 comprising said lithic heterologous system, wherein said bacterial strain of Pseudomonas putida KT2440, described in previous paragraphs, is mutant in one or more of the tol-pal genes.
  • the term "mutant" as used in the present invention refers to the sequence resulting from the deletion of the tol-pa genes.
  • the Pseudomonas putida KT2440 cell mutant in at least one or more of the tolpalar genes is selected from the lysia comprising Pseudomonas putida KT2440 AX (tolA :: xylE which generates a TolA protein shortened to 94 amino acids), Pseudomonas putida KT2440 BX (tolB :: xylE that generates a TolB protein shortened to 29 amino acids), Pseudomonas putida KT2440 QX ⁇ tolQr.xylE that generates a TolQ protein shortened to 17 amino acids) or Pseudomonas putida KT2440 RX (tolR :: xylE that does not generate a ToIR protein because the toIR gene is deleted).
  • a more preferred embodiment refers to the strain described in the previous paragraph, mutant in one or more of the tol-pal genes, wherein said strain is the microorganism with deposit number CECT 7658.
  • the strain CECT 7658 corresponds to the strain of Pseudomonas putida KT2440 BX (tolB :: xylE that generates a TolB protein shortened to 29 amino acids).
  • This strain has mutated the gene that encodes the TolB protein of the Tol-Pal system (P. putida KT2440 tolB :: xylE, with TolB shortened to 29 amino acids).
  • the promoter Pm cassette ejh-ejl, regulatory construction xylS / Pm and a kanamycin resistance gene.
  • strain CECT 7658 in addition to presenting the mutation of the tol-pal genes comprises the lytic heterologous genetic system where the nucleotide sequence that codes for holina Ejh is SEQ ID NO: 1, the sequence that codes for endolysin Ejl ( marry ejh-ejl) is SEQ ID NO: 2, and the nucleotide sequence encoding the regulatory sequence of gene expression xyl / Pm is SEQ ID NO: 5 for Xyl and SEQ ID NO: 6 for Pm.
  • BXHL can be used interchangeably.
  • strain CECT 7658 The culture and maintenance conditions of strain CECT 7658, communicated to the International Deposit Authority, are:
  • the lytic heterologous genetic system can be introduced into cells by transforming vectors that contain said genetic system.
  • the transformation may result in a transient expression of the genes of interest or a stable expression.
  • the stable expression allows that, after successive divisions of the cell, the incorporated sequence continues to be expressed.
  • Another preferred embodiment refers to any strain described in previous paragraphs, where the lytic heterologous genetic system is inserted into its chromosomal DNA.
  • a more preferred embodiment refers to said strain where the lytic heterologous genetic system inserted in its chromosomal DNA is in a single copy.
  • Another aspect of the present invention is a microbial population comprising any strain described in previous paragraphs.
  • microorganism of the present invention or "microorganism of the invention” can be used.
  • Another aspect of the present invention relates to the use of the microorganism of the invention for the extraction of at least one compound synthesized by said bacterium. Since the present invention focuses on the insertion of a heterologous genetic system capable of inducing, in an induced manner, the lysis of the cells that contain it, the microorganism of the invention can be used for the extraction of any compound synthesized by said bacterium. .
  • the fact that the amount of PHA synthesized by said bacterium has been measured in the present invention does not limit the type of compound synthesized by it.
  • a preferred embodiment relates to the use of the microorganism of the invention for the extraction of at least one compound synthesized by said bacterium, wherein the synthesized compound is polyhydroxyalkanoate (PHA).
  • PHA polyhydroxyalkanoate
  • Another aspect of the present invention relates to a method for the extraction of PHA synthesized by the microorganism of the invention, comprising: a) culturing the microorganism of the invention in a minimal culture medium,
  • step (a) adding an effector compound of the expression of the lytic heterologous genetic system to the culture of step (a), and
  • minimum medium refers to a culture medium with a defined composition of mineral salts and a compound that the Pseudomonas putida bacteria can use as a carbon source.
  • a preferred embodiment refers to the use of the medium designated 0.1 N M63, composed of 13.6 g KH 2 PO 4 ; 0.2 g (NH 4 ) 2 SO 4 ; 0.5 mg SO 4 Fe ⁇ 7 h O per liter, buffered to pH 7, and supplemented with 1 mM MgSO 4 and a trace element solution (Moldes et al., 2004. Appl. Environ. Microbio!., 70: 3205 -3212).
  • the carbon source can be any compound that the Pseudomonas putida bacteria can use as a carbon source.
  • a carbon source Preferably, but not limited to, any molecule capable of being transformed into hydroxyalkanoyl-CoA by bacterial metabolism.
  • Said medium is appropriate for the cells to produce the compound of interest.
  • effector compound refers to a compound capable of inducing the expression of said genetic system by means of the gene expression regulatory system.
  • the expression of the cassette with the two lithic genes is induced by the addition to the culture medium of said effector compound. In this way the joint action of the lytic enzyme and holine leads to spontaneous rupture of the bacterial cell envelope and the release of the compounds of interest contained in the cell cytoplasm.
  • the isolation of the compound synthesized by said bacterium, according to step (c) of the method is carried out by techniques that are part of the common general knowledge and are therefore available to a person skilled in the art.
  • the isolation or recovery of the enzymatic product is carried out, for example, but not limited, by precipitation of said product with saline fractionation, heat precipitation, isoelectric precipitation, with organic solvents or with polymers, with cold acetone or by chromatography .
  • a preferred embodiment refers to the method for the extraction of at least one compound synthesized by the microorganism of the invention, where it also comprises a step prior to step (a) where the strain or bacterial population is grown in a rich culture medium.
  • the term "rich medium” as used in the present invention refers to a medium containing yeast extract or other substances of complex and poorly defined composition that can serve as a source of nutrients and energy for the growth of the microorganism.
  • the rich medium is Luria-Bertani (LB) (Sambrook and Rusell, 2001. Molecular Cloning. A Laboratory Manual. CSHL Press, Cold Spring Harbor, New York).
  • Said rich medium is appropriate for the cells to divide but not produce the compound of interest. That is, the method described in this preferred embodiment consists of the following sequential steps:
  • the microorganism of the invention can be isolated or not.
  • the microorganism of the invention is isolated by techniques known to those skilled in the art and transferred to said minimum culture medium, - adding an effector compound of the expression of the lytic heterologous genetic system to the culture of the previous step,
  • Another preferred embodiment relates to the method for the extraction of at least one compound synthesized by the microorganism of the invention, where the effector compound of the expression of the lytic heterologous genetic system is 3-MB acid.
  • Another preferred embodiment relates to the method for the extraction of at least one compound synthesized by the microorganism of the invention, where in addition, after cultivation of the microorganism of the invention, one or more chemical compounds are added to the minimum culture medium, with detergent and chelating properties, in small amounts that accelerate cell lysis mediated by the lytic heterologous genetic system, but which in these quantities are not capable of producing on their own the lysis of a native bacterium unmodified by the lytic heterologous genetic system.
  • the amounts of chemical compounds used as lysis accelerators are much lower than the amounts that would be needed to lyse the bacteria if it were not genetically modified, and therefore are less polluting.
  • a PHA precursor is added to the minimum culture medium.
  • the PHA precursor is a fatty acid or any molecule capable of being transformed into hydroxyalkanoyl-CoA by bacterial metabolism.
  • FIG. 1 It shows a scheme of the cloning of the ejh-ejl cassette on the Pseudomonas putida KT2440 chromosome under the transcriptional control of the Pm promoter.
  • FIG. 2. Shows the distribution profiles obtained by ultracentrifugation with sucrose gradient.
  • Tube 1 wild strain Pseudomonas putida KT2440 without inductor
  • tube 2 wild strain Pseudomonas putida KT2440 with inductor
  • tube 3 recombinant strain Pseudomonas putida KTHL without inducer
  • tube 4 recombinant strain Pseudomonas putida KTHL with inductor.
  • the white band located at the gradient interface of this last tube corresponds to the PHA granules released to the extracellular medium.
  • FIG. 3. It shows the count of viable cells (cfu / ml) at 0, 6 and 24 h of the Pseudomonas putida KTHL and Pseudomonas putida BXHL strains in the presence and absence of 3-MB.
  • FIG. 4. Shows the distribution profiles obtained by ultracentrifugation with sucrose gradient.
  • Tube 1 Pseudomonas wild strain. putida KT2440 incubated with 10 mM EDTA and 0.1% SDS; tube 2: recombinant strain Pseudomonas putida BXHL with 3-MB incubated with 10 mM EDTA and 0.1% SDS.
  • FIG. 5 Shows the intracellular PHA content of the Pseudomonas putida BXHL strain in the presence of 3-MB and in the presence of 3-MB, 10 mM EDTA and 0.1% SDS.
  • the invention will now be illustrated by illustrative and non-limiting tests, carried out by the inventors describing the use of the strain of the invention for the extraction of any compound synthesized by the strains of Pseudomonas putida KTHL and Pseudomonas putida BXHL, mutant in the Tol-pal system, carriers of the lithic system of the KTHL strain.
  • EXAMPLE 1 Construction of the strain of Pseudomonas putida KTHL and extraction of compounds (PHA) synthesized by them.
  • Pseudomonas putida KTHL strain carrying the ejh-ejl lithic cassette in monocopy A cell autolysis system in Pseudomonas putida KT2440 has been designed by constructing a carrier strain on the chromosome of the genes encoding a holine (Ejh) and an endolysin (Ejl). The resulting strain has been called Pseudomonas putida KTHL. To control the expression of the lytic system in Pseudomonas putida KT2440, a monocopy expression system of the ejh-ejl cassette was constructed in order to introduce it into the genome of the strain by triparental conjugation.
  • 1,209 bp fragment was amplified by PCR using plasmid pEDF12 as a template (Table 4) and V08 and V09 as oligonucleotides (Table 5). Said fragment was digested with Xba ⁇ and BamH ⁇ and after cloning it into plasmid pUC18Not it was cloned into pCNB1 causing plasmid pCNBHL (FIG. 1).
  • the Pseudomonas putida KTHL cells were centrifuged at 31,000 * g for 1 h in a Sorvall centrifuge and the pellets were resuspended in pH 8 phosphate buffer to preserve cell integrity. 50% of each sample was lyophilized and analyzed by GC-MS to quantify the total PHA content. The rest of the sample was subjected to fractionation in a gradient of preformed sucrose with a first volume of 20% sucrose (11 ml) and a second volume of 15% sucrose (11 ml). The samples were placed on top of the gradient and ultracentrifuged at 126,000 ⁇ g for 20 h in an XL-90 ultracentrifuge (Beckman).
  • the growth of the different strains in plastic production conditions in a single fermentative phase allows to grow from a single source of carbon (octanoic acid) and achieve a high biomass / PHA yield.
  • Table 1 Shows the optical density value (at 6 oo nm); the intracellular content of PHA; the viable cell count (cfu / ml) and the biomass (g / l) corresponding to cells grown in a fermentative phase at 23h of culture of the wild strain Pseudomonas putida KT2440 and of the recombinant strain Pseudomonas putida KTHL in the presence and absence of 3-MB.
  • the growth in two fermentative phases consists of a new fermentation / production system that allows an improvement in the production of PHA in Pseudomonas putida KTHL and in the functioning of the lysis system.
  • the procedure consists in culturing the cells in a rich medium (LB medium) and then transferring them to a specific PHA production medium, where they will be lysed after activation of the lytic system.
  • LB medium rich medium
  • Pseudomonas putida KTHL is able to grow and accumulate PHA in a manner similar to the wild strain in terms of ⁇ levels reached by both strains (Table 2).
  • Table 2 Shows the optical density value ( ⁇ 6 ⁇ nm); the intracellular content of PHA; viable cell count (cfu / ml) and biomass (g / l) corresponding to cells grown in two fermentative phases at 23h of culture of the wild strain Pseudomonas putida KT2440 and of the recombinant strain Pseudomonas putida KTHL in the presence and absence of 3-MB.
  • the degrees of sensitivity of the strain Pseudomonas putida KTHL against the chemical agents sodium dodecyl sulfate (SDS), deoxycholate (DOC) or ethylenediaminetetraacetic acid (EDTA) were analyzed in order to improve the performance of the bioplastic extraction process.
  • the cells were incubated for 18 h at 30 ° C in LB medium and in LB medium supplemented with 0.2 mM EDTA, 0.1% DOC -0.05% (w / v) or 0.01% SDS (w / v ).
  • the minimum concentrations of EDTA, DOC and SDS that do not alter the growth of Pseudomonas putida KTHL induced with 3-MB were determined, these being the following: EDTA 0.05-01 mM, DOC 0.01% ( p / v) and SDS 0.005% (p / v).
  • the concentrations of EDTA and DOC that alter the growth of the wild Pseudomonas strain were also determined. putida KT2440, these being the following: EDTA 0.4 mM and DOC 0.1-0.2% (w / v).
  • the sensitivity levels of the recombinant strain Pseudomonas putida KTHL against EDTA, DOC and SDS in plastic production medium were analyzed. After the fermentation in two phases, the cells were incubated for 7 h at room temperature with 10 mM EDTA and 0.1% SDS simultaneously. At these concentrations, the recombinant strain Pseudomonas putida KTHL induced with 3-MB presented a similar sensitivity that in the absence of inducer and that the wild strain. The fact of not achieving the lysis of any of the strains despite the high concentration of the chemical agents used, suggests that the presence of the PHA granule inside the cell confers resistance to the bacteria.
  • EXAMPLE 2 Construction of mutant Pseudomonas putida strains in the Tol-pal system, carriers of the lytic system of the KTHL strain, and extraction of compounds (PHA) synthesized by them. 2.1. Study of the expression of the cell autolysis system in the mutant strains in the Tol-pal, Pseudomonas putida AX, BX, QX and RX system.
  • mutant strains were tested in the Tol-pal complex, which have the unstructured cell envelope.
  • the intracellular PHA content of the described strains grown in two fermentative phases was quantified, this being less than 0.2 g / l in the Pseudomonas putida AXHL, Pseudomonas putida RXHL and Pseudomonas putida QXHL strains.
  • the PHA content of Pseudomonas putida BXHL (0.88 g / l) is similar, and even greater, to that of the strain Pseudomonas putida KTHL (0.78 g / l).
  • mutant strain was selected in the TolB protein carrying the autolysis system, Pseudomonas putida BXHL.
  • the PHA content present in the cell sediments resulting from the sucrose gradient ultracentrifugation after inducing cell lysis was compared.
  • the quantification of the PHA present in the sediments resulting from the ultracentrifugation allows us to conclude that the percentage of rupture obtained after induction of the lytic system in the strain Pseudomonas putida BXHL is significantly lower than that obtained with the French press, where the cell rupture was practically total (results not shown).
  • Pseudomonas Pseudomonas putida mt-2 cured from plasmid 2002. Environ putida KT2440 TOL, hsdR Microbe !. 4: 782-786.
  • Oligonucleotides used the amplification of products by PCR.
  • the underlined sequences correspond to the restriction targets created.
  • the rich medium used to grow E. coli and Pseudomonas putida cells was Luria-Bertani (LB) (Sambrook and Rusell, 2001. Molecular Cloning. A Laboratory Manual. CSHL Press, Cold Spring Harbor, New York).
  • the minimum medium used to grow the cells was the medium named 0.1 N M63 (13.6 g KH 2 P0 4 ; 0.2 g (NH 4 ) 2 S0 4 ; 0.5 mg S0 4 Fe ⁇ 7 H 2 0 per liter, pH 7) supplemented with 1 mM MgS0 4 and a trace element solution (Moldes et al., 2004. Appl. Environ. Microbiol., 70: 3205-3212).
  • the strains were stored at 4 ° C in LB plates or minimum medium.
  • the bacteria were frozen in the corresponding culture medium with 15% (v / v) glycerol and kept at -80 ° C.
  • the Pseudomonas putida KT2440 cells were cultured for 24 h in M63 0.1 N medium whose composition is similar to that of M63 but with 0.2 g / l of (NH 4 ) 2 S0 instead of 2 g / l, using 15 mM octanoate as the sole source of carbon (Moldes et al., 2004. Appl. Environ. Microbio /., 70: 3205-3212).
  • E. coli cells were genetically modified by transformation after making them competent by the RbCI method (Sambrook and Rusell, 2001. Molecular Cloning. A Laboratory Manual. CSHL Press, Cold Spring Harbor, New York.), Or by electroporation ( Wirth et al., 1989 Mol Gen Genet 216: 175-177).
  • Pseudomonas putida cells were genetically modified by electroporation transformation.
  • To electroporate the Pseudomonas putida cells cells were collected in liquid culture or cell mass from agar plates and five washes were performed with sterile water at 4 ° C.
  • the conditions of the Gene Pulser / Pulse Controller electroporation equipment (Bio-Rad) were 2.5 kV, 25 ⁇ and 200 ⁇ .
  • the plasmids were mobilized to Pseudomonas putida by bi- or tri-parental conjugation following the method described by de Lorenzo and Timmis, (1994) Methods Enzymol 235: 386-405) and using E. coli strain HB101 (pRK600) As auxiliary strain.
  • the transconjugants of Pseudomonas putida were selected in LB medium plates with the corresponding antibiotics or in minimum medium plates with 0.2% citrate and the corresponding antibiotic.
  • Plasmid DNA extraction was carried out using the High Puré Plasmid Purification Kit (Roche) system, according to the manufacturer's protocol.
  • Genomic DNA extraction was carried out using the GenomicPrepTM Cells and Tissue DNA Isolation Kit (GE Healthcare) according to the manufacturer's instructions. DNA amplification was performed on a Mastercycler Gradient device from Eppendorf. The reaction mixtures contained 1.5 mM MgC ⁇ , 0.2 mM dNTPs, 10% dimethylsulfoxide, 0.5 units of DNA polymerase, 100 ng of template DNA and oligonucleotides at a final concentration of 0.5 ⁇ . The DNA fragments were purified using agarose gels, using the GeneClean kit (BIO 101) or the "High Puré TM PCR Product Purification Kit" (Boehringer Mannheim).
  • the different oligonucleotides used in the PCR reactions were acquired in Sigma-Genosys and are indicated in Table 5.
  • Quantitative analysis was carried out by calculating the response factors of the monomers with respect to 3-MB. For the calculation of the factors, mixtures of known concentrations of PHA were used, obtaining a response factor for each of the quantified monomers whose coefficient of variation did not exceed 5%.
  • the strains were inoculated in LB medium at DO600 0.5. After 6 h of incubation under orbital agitation different concentrations of the chemical agents mentioned above were added for 18 h. The growth of these cultures was carried out in 96-well plates at 30 ° C with 2 min of strong orbital agitation every 15 min (Multiskan Ascent, Thermo). The growth values shown are the average of 3 replicates.
  • SDS sodium dodecyl sulfate
  • DOC deoxycholate
  • EDTA ethylenediaminetetraacetic acid

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Abstract

La présente invention concerne une souche bactérienne de Pseudomonas putida KT2440, qui comprend un système génétique hétérologue lytique, ledit système comprenant lui-même une séquence nucléotidique qui code pour l'enzyme lytique de paroi cellulaire endolisine Ejl, la séquence nucléotidique qui code pour la holine Ejh, et une séquence nucléotidique qui code pour un système de régulation génique, lequel comprend une séquence nucléotidique promotrice de l'expression génique, et une séquence nucléotidique qui code pour et exprime une protéine régulatrice de ladite expression génique. De préférence, la souche est le micro-organisme portant le numéro d'entrée CECT 7659. L'invention concerne également ladite souche dans laquelle le micro-organisme Pseudomonas putida KT2440 est mutant dans un ou plusieurs des gènes tol-pal. De préférence, ladite souche est le micro-organisme portant le numéro d'entrée CECT 7658. L'invention concerne en outre l'utilisation de l'une quelconque des souches décrites pour l'extraction de polyhydroxyalkanoate (PHA) fritté par ladite bactérie ainsi qu'un procédé pour l'extraction dudit composé.
PCT/ES2010/070858 2009-12-23 2010-12-21 Système d'autolyse cellulaire pour le traitement de la biomasse bactérienne dans la production de polyhydroxyalkanoates chez pseudomonas putida kt2440 WO2011086211A1 (fr)

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WO2014096276A1 (fr) 2012-12-21 2014-06-26 Carbios Préparation de poly(hydroxyacides gras) à longue chaîne
EP3819380A1 (fr) 2019-11-05 2021-05-12 Consejo Superior De Investigaciones Científicas (CSIC) Souches de pseudomonas putida recombinant pour la production de polyhydroxyalcanoate
RU2785601C1 (ru) * 2022-10-07 2022-12-09 Федеральное государственное бюджетное учреждение "Государственный научный центр "Институт иммунологии" Федерального медико-биологического агентства (ФГБУ "ГНЦ Институт иммунологии" ФМБА России) Штамм Pseudomonas putida для биодеградации гептила, штамм Rhodococcus erythropolis для биодеградации авиационного керосина и способ биоремедиации почвы, загрязненной компонентами ракетных топлив

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ES2458465B1 (es) * 2012-10-01 2015-02-11 Consejo Superior De Investigaciones Cientificas (Csic) Procedimiento de fermentación para la producción de polihidroxialcanoatos y derivados que comprende la utilizacion de predadores bacterianos

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014096276A1 (fr) 2012-12-21 2014-06-26 Carbios Préparation de poly(hydroxyacides gras) à longue chaîne
EP3819380A1 (fr) 2019-11-05 2021-05-12 Consejo Superior De Investigaciones Científicas (CSIC) Souches de pseudomonas putida recombinant pour la production de polyhydroxyalcanoate
WO2021089636A1 (fr) 2019-11-05 2021-05-14 Consejo Superior De Investigaciones Cientificas (Csic) Souches recombinantes de pseudomonas putida pour la production de polyhydroxyalcanoate
RU2785601C1 (ru) * 2022-10-07 2022-12-09 Федеральное государственное бюджетное учреждение "Государственный научный центр "Институт иммунологии" Федерального медико-биологического агентства (ФГБУ "ГНЦ Институт иммунологии" ФМБА России) Штамм Pseudomonas putida для биодеградации гептила, штамм Rhodococcus erythropolis для биодеградации авиационного керосина и способ биоремедиации почвы, загрязненной компонентами ракетных топлив

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