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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • 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
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/145—Clostridium

Definitions

• the present invention concerns a new modified microorganism for the production of 1 ,3- propanediol.
• This microorganism is adapted for growth and production of 1 ,3-propanediol from a culture medium with high glycerine content and specifically with a high concentration of industrial glycerine.
• the invention also concerns culture conditions of said adapted microorganisms and process for the production of 1 ,3-propanediol.
• the invention concerns, finally, 1 ,3-propanediol produced by the modified microorganism and its applications.
• PDO trimethylene glycol or propylene glycol
• trimethylene glycol or propylene glycol is one of the oldest know fermentation products. It was originally identified as early as 1881 by August Freund in a glycerine fermented culture containing Clostridium pasteurianum. PDO is a typical product of glycerine fermentation and has been found in anaerobic conversions of other organic substrates. Only very few organisms, all of them bacteria, are able to form it. They include enterobacteria of the genera Klebsiella (K. pneumoniae), Enterobacter (E. agglomerans) and Citrobacter (C. freunddi), Lactobacilli (L brevis and L buchneri) and Clostridia of the C. butyricum and the C. pasteurianum group.
• PDO as a bifunctional organic compound, may potentially be used for many synthesis reactions, in particular as a monomer for polycondensations to produce polyesters, polyethers, polyurethanes, and in particular, polytrimethylene terephtalate (PTT).
• PTT polytrimethylene terephtalate
• PDO can be produced by different chemical routes but they generate waste stream containing extremely polluting substances and the cost of production is high.
• chemically produced PDO can not compete with the petrochemically available diols like 1 ,2-ethanediol, 1 ,2-propanediol and 1 ,4-butanediol.
• DuPont started a research program for the biological conversion of glucose to PDO. Although this process is environmentally friendly it has the disadvantage to i) use vitamin B12 a very expensive cofactor and ii) be a discontinuous process due to the instability of the producing strain.
• C. butyricum was previously described as being able to grow and produce PDO from industrial glycerine in batch and two-stage continuous fermentation (Papanikolaou et al., 2000).
• the maximal PDO titer obtained was 48.1 g.L “1 at a dilution rate of 0.02 h "1 , meaning a productivity of 0.9 g.L “1 .h “1 .
• the cultures were conducted with a maximum glycerine concentration in the fed medium of 90g.L "1 and in the presence of yeast extract, a costly compound containing organic nitrogen that is known by the man skilled in the art to help increase bacterial biomass production.
• WO2006/128381 discloses the use of this glycerine for the production of PDO with batch and fed-batch cultures using natural PDO producing organisms such as Klebsiella pneumoniae, C. butyricum or C. pasteuricum. Furthermore, the medium used in WO2006/128381 also contains yeast extract. As described in this patent application, the maximal productivity reached was comprised between 0.8 and 1.1g.l “1 .h "1 .
• C. acetobutylicum DG1 pSPD5 The performance of a C. acetobutylicum strain modified to contain the vitamin B12- independent glycerine-dehydratase and the PDO-dehydrogenase from C. butyricum, called "C. acetobutylicum DG1 pSPD5" has been described in Gonzalez-Pajuelo et al., 2005. This strain originally grows and produces PDO in a fed medium containing up to 120 g. 1 of pure glycerine. In addition, analyses in a fed medium containing a maximum of 60 g. 1 of pure or industrial glycerine did not point out to any differences. These results have been obtained in presence of yeast extract.
• the present invention concerns a population of Clostridium acetobutylicum useful for the production of 1 ,3-propanediol (PDO), wherein said population comprises at least one strain of a Clostridium acetobutylicum sp. comprising mutations selected among the mutations identified in Table 1 , wherein relative percentages of said mutations are selected among the following gene families:
• the population of the invention comprises at least one strain of Clostridium acetobutylicum selected among the group consisting of :
• CNCM means "Collection Nationale de Cultures de Microorganismes” at the Pasteur Institute, Paris.
• the population comprises the above strains further mutated with at least one of the following point mutations: C is replaced with T at locus CA_C0175, position 198989 in the Clostridium acetobutylicum genome, coding for a predicted sugar phosphate isomerase, homolog of an eukaryotic glucokinase regulator (carbohydrate metabolism) G is replaced with A at locus CA_C1300, position 1444099 in the Clostridium acetobutylicum genome, coding for an RNA polymerase sigma factor RPOD ( transcription and translation regulation)
• C is replaced with T at locus CA_C1610, position 1752341 in the Clostridium acetobutylicum genome, coding for a branched-chain amino acid permease (transporter).
• the present invention also concerns a method for the production of 1 ,3- propanediol, comprising culturing a population of Clostridium acetobutylicum useful for the production of 1 ,3-propanediol (PDO) according to the invention in a culture medium comprising glycerine as sole source of carbon and recovering the 1 ,3-propanediol produced from the culture medium.
• PDO Clostridium acetobutylicum useful for the production of 1 ,3-propanediol
• a population of Clostridium acetobutylicum useful for the production of 1 ,3- propanediol means one or more strains of Clostridium acetobutylicum genetically modified for the production of 1 ,3-propanediol from glycerine as sole source of carbon. Such strains are known in the art and disclosed, particularly, in applications WO200104324 and WO2008052595.
• the population according to the invention may be a combination of several strains, the majority of which comprising the mutations according to the invention, as well as a single strain, and particularly strain DG1 pSPD5 PD0001VE05c01 , DG1 pSPD5 PD0001VE05c05 or DG1 pSPD5 PD0001VE05c07 deposited at CNCM under accession numbers I-4378, I-4379, I-4380 respectively, or strain DG 1 pSPD5 PD0001VE05c08.
• Mutations are changes of nucleotides in the strain genome, more particularly SNPs ("Single Nucleotide Polymorphisms”), identified when compared to the mother strain DG1 pSPD5 PD0001VT.
• SNPs Single Nucleotide Polymorphisms
• Said strain is disclosed in WO200104324 and is derived from strain ATCC824 which genome sequence has been published (Nolling et al., 2001).
• Mutations can occur in coding or non-coding sequences. These mutations can be synonymous wherein there is not modification of the corresponding amino acid or non- synonymous wherein the corresponding amino acid is altered. Synonymous mutations do not have any impact on the function of translated proteins, but may have an impact on the regulation of the corresponding genes or even of other genes, if the mutated sequence is located in a binding site for a regulator factor. Non-synonymous mutations may have an impact on the function of the translated protein as well as on regulation depending the nature of the mutated sequence.
• Clostridium acetobutylicum useful for the production of 1 ,3- propanediol may preferably comprise one of those deposited strains comprising additional modifications, at least one of the following modifications:
• It may preferably comprise any combinations of these mutations, comprising 1 , 2, 3, 4 or 5 of these mutations.
• the population of strains of the invention is capable of growing on a medium comprising up to 120 g.L "1 of glycerine and more particularly of industrial glycerine.
• strains of the population of the invention may be obtained using standard techniques of mutagenesis and/or gene replacement in Clostridium, such as disclosed in application WO2008040387 which contents are incorporated herein by reference.
• the population of the invention comprises strain DG1 pSPD5 PD0001VE05c08, which mutations are identified in Table 1.
• the person skilled in the art knows how to introduce the mutations into a Clostridium strain to generate a strain similar to strain DG1 pSPD5 PD0001VE05c08, starting from one of strains DG1 pSPD5 PD0001VE05c01 , DG1 pSPD5 PD0001VE05c05 or DG1 pSPD5 PD0001VE05c07, deposited at CNCM under accession numbers I-4378, I-4379, I-4380 respectively and using standard gene replacement and recombination techniques.
• An "appropriate culture medium” or a “culture medium” refers to a culture medium optimized for the growth and the diol-production of the Clostridium strains or population.
• the fermentation process is generally conducted in reactors with a synthetic, particularly inorganic, culture medium of known defined composition adapted to the Clostridium species used and containing glycerine.
• synthetic medium means a culture medium comprising a chemically defined composition on which organisms are grown.
• glycerine is advantageously the single source of carbon.
• glycol and 'glycerol are synonymous and used interchangeably in this invention to refer to the same molecule.
• glycerine is added to the medium in the form of glycerine composition comprising at least 50% of glycerine, preferably at least 85% of glycerine.
• the glycerine used in the culture medium of the invention is industrial glycerine.
• “Industrial glycerine” means a glycerine product obtained from an industrial process without substantial purification. I ndustrial glycerine can also be designated as "raw glycerine”.
• Industrial glycerine comprises more than 70% of glycerine, preferably more than 80%, water and impurities such as mineral salts and fatty acids. The maximum content of glycerine in industrial glycerine is generally 90%, more generally about 85%.
• Industrial processes form which industrial glycerine is obtained are, inter alia, manufacturing methods where fats and oils, particularly fats and oils of plant origin, are processed into industrial products such as detergent or lubricants. In such manufacturing methods, industrial glycerine is considered as a by-product.
• the industrial glycerine is a by-product from biodiesel production and comprises known impurities of glycerine obtained from biodiesel production, comprising about 80 to 85% of glycerine with salts, water and some other organic compounds such as fatty acids.
• Industrial glycerine obtained from biodiesel production has not been subjected to further purification steps.
• the culture medium comprises high concentrations of glycerine.
• high glycerine content or "high concentration of glycerine” means more than 90 g.L “1 of glycerine in the culture medium . I n preferred embodiments, the concentration is comprised between 90 and 200 g.L “1 of glycerine, more particularly between 90 and 140 g/L of glycerine, preferably about 120 g.L "1 of glycerine.
• the culture medium is a synthetic medium without addition of organic nitrogen.
• the production is advantageously done in a batch, fed-batch or continuous process.
• Culturing microorganisms at industrial scale for the production of 1 ,3-propanediol i s kn own i n th e a rt, pa rti cu l arl y d i s cl osed i n PCT/EP2010/056078 filed on 05/20172010 and PCT/EP2010/064825 filed on 5/10/2010, which content are incorporated herein by reference.
• 1 ,3-propanediol may be isolated by distillation. In most embodiments, 1 ,3-propanediol is distilled from the fermentation medium with a by-product, such as acetate, and then further purified by known methods.
• Clone isolation was performed on agar plates starting from a growing flask culture of the population strain Clostridium acetobutylicum DG1 pSPD5 PD0001 VE05.
• the synthetic media used for flask culture contained per liter of deionized water : glycerine, 30g; KH 2 P0 4 , 0.5g; K 2 HPO 4 , 0.5g; MgS0 4 , 7H 2 0, 0.2g; CoCI 2 6H 2 0, 0.01g; acetic acid, 99.8%, 2.2ml; NH 4 CI, 1.65g; MOPS, 23.03g, biotin, 0.16mg; p-amino benzoic acid, 32mg; FeS0 4 , 7H 2 0, 0.028g; resazurin, 1 mg and cysteine, 0.5g.
• the pH of the medium was adjusted to 6.5 with NH 4 OH 6N.
• agar medium which contains per liter of deionized water : commercial or raw glycerine, 30g; yeast extract, 5g; KH 2 P0 4 , 0.75; K 2 HP0 4 , 0.75g; MgS0 4 , 7H 2 0, 0.4g; asparagine, 2g; (NH 4 ) 2 S0 4 , 2g; NaCI, 1 g; MnS0 4 , H20, 10mg; FeS0 4 , 7H 2 0, 10mg; MOPS, 23.03g; resasurin, 1 mg and cysteine, 15g.
• the pH of the medium was adjusted to 6.6 with NH 4 OH 3N.
• Isolated clones were considered pure after three subsequent subcultures on agar plates. Pure clones were then transferred into liquid rich medium which contained either commercial or raw glycerine (Table 2). Subsequently, growing liquid cultures were conserved on glycerine 20% at -80°C until further characterization.
• Measurement of viability after conservation evaluation of growth rate of cells on synthetic medium ;
• the synthetic media used for Clostridia batch cultivations contained per liter of deionized water: glycerine, 30g; KH 2 P0 4 , 0.5g ; K 2 HP0 4 , 0.5g ; MgS0 4 , 7H 2 0, 0.2g ; CoCI 2 6H 2 0, 0.01g ; H 2 S0 4 , 0.1ml ; NH 4 CI, 1.5g ; biotin, 0.16mg ; p-amino benzoic acid, 32mg and FeS0 4 , 7H 2 0, 0.028g.
• the pH of the medium was adjusted to 6.3 with NH 4 OH 3N.
• Commercial glycerine purchased from Sigma (purity 99.5%) was used for batch cultivation.
• the feed medium for continuous cultures contained per liter of tap water : raw glycerine, 105g ; KH 2 P0 4 , 0.5g ; K 2 HP0 4 , 0.5g ; MgS0 4 , 7H 2 0, 0.2g ; CoCI 2 6H 2 0, 0.026g ; NH 4 CI, 1.5g ; biotin, 0.16mg ; p-amino benzoic acid, 32mg ; FeS0 4 , 7H 2 0, 0.04g ; anti- foam, 0,05ml ; ZnS0 4 , 7H 2 0, 8mg ; CuCI 2 , 2H 2 0, 4mg ; MnS0 4 , H 2 0, 40mg ; H 3 B0 3 , 2mg ; Na 2 Mo0 4 , 2H 2 0, 0.8mg.
• Medium pH was not adjusted in this case.
• the bioreactor gas outlet was protected from oxygen by a pyrogallol arrangement (Vasconcelos et al, 1994). After sterilisation the feed medium was also flushed with sterile 0 2 -free nitrogen until room temperature was attained and maintained under nitrogen at 200 mbar to avoid 0 2 entry. Batch and continuous cultures process:
• a culture growing in a 100ml flask on synthetic medium (the same as described above for batch culture but with addition of acetic acid, 2.2 g.L “1 and MOPS, 23.03g.L “1 ) taken at the end of exponential growth phase was used as inoculum (5% v/v).
• Cell concentration was measured turbidimetrically at 620nm and correlated with cell dry weight determined directly.
• Glycerine, 1 ,3-propanediol, ethanol, butanol, acetic and butyric acids concentrations were determined by HPLC analysis. Separation was performed on a Biorad Aminex HPX-87H column and detection was achieved by refractive index. Operating conditions were as follows: mobile phase sulphuric acid 0.5mM; flow rate 0.5ml/min, temperature, 25°C.
• Table 3 performances of the C. acetobutylicum DG1 pSPD5 population PD0001VE05 (mean data from 4 chemostats), of clone c08 PD0001VE05c08.
• the feed medium contained 105g.L “1 of raw glycerine, dilution rate was 0.060h “1 and 0.025h “1 . Values correspond to the average of samples analyzed after at least 3 residences times at dilution rate of 0.060h "1 .
• Nl no information.
• the PD0001VT strain can not grow in a medium lacking yeast extract.
• Genomic DNA from strains PD0001VT, PD0001VE05, PD0001 VE05c01 , PD0001VE05c05, PD0001 VE05c07 and PD0001VE05c08 was extracted using Qiagen Genomic kit 500G (Qiagen, Inc., Valencia, CA). Briefly, cells were grown anaerobically respectively in rich or synthetic glycerine medium (as described in example 1 and 2) in penicillin vials (70 ml_) to late exponential phase (A 6 2o 1 .5 to 2.0). Strictly anaerobic conditions were maintained throughout cell lysis.
• Cells were collected and washed twice in SET buffer (25% sucrose, 0.05 M Tris-HCI, 0.05 M EDTA). Cell pellets were suspended in 1 1 ml_ B1 kit buffer with 44 ⁇ _ RNase, 30 mg/mL lysozyme and 100 ⁇ g/mL proteinase K. The mixtures were incubated at 37°C for 45 min, centrifuged and supernatants were used for DNA extraction according to the Qiagen DNA purification kit instructions. The DNAs were then suspended in 50 ⁇ _ of 10 mM Tris-HCI (pH8.0). Sequencing analysis
• Genomes of the native DG1 pSPD5 PD0001VT strain and the evolved population DG1 pSPD5 PD0001 VE05 were sequenced using the Roche GS FLX technology.
• the sequencing project was performed by Eurofins Genomics MWG/ Operon (ZA de Courtabeauf-9 Avenue de la Laponie, 91978 Les Ulis Cedex) with for each strain 1 Long- Tag paired end libraries (8 Kb), generation of sequence and scaffolding of the contigs with GS FLX Titanium series chemistry using a half run (max. 600 000 reads, max 180 000- 300 000 true paired end reads).
• Isolated clones from the evolved population were sequenced using the comparative genomic sequencing (CGS) method developed by NimbleGen (Roche NimbleGen Inc. 500 S. Rosa Rd. Madison Wl 53719).
• CGS comparative genomic sequencing
• NimbleGen Roche NimbleGen Inc. 500 S. Rosa Rd. Madison Wl 53719.
• the CGS analysis was performed in two phases: in phase 1 , regions of genomic difference were identified by a comparative hybridization of DNA of the native strain and the evolved clones. In phase 2, only the identified regions of genomic differences were sequenced so as to produce a set of fully characterized single nucleotide polymorphisms (SNPs).
• SNPs single nucleotide polymorphisms
• Bioinformatics and SNP analysis of the evolved population were performed by Eurofins Genomics MWG / Operon.
• the read sets of both strains were separately mapped to the Genbank reference sequence (Clostridium acetobutylicum ATCC 824 http://www.ncbi.nlm.nih.gov/nuccore/AE001437) using the software gsMapper (Roche 454, V2.3) .
• Three SN Ps files were delivered comparing DG 1 pSPD5 PD0001VT to ATCC824, DG1 pSPD5 PD0001VE05 to ATCC824 and DG1 pSPD5 PD0001VT to DG1 pSPD5 PD0001VE05.
• Unique SNPs between the native and the evolved strains are presented below. Low coverage ( ⁇ 25) and low variant frequency ( ⁇ 85%) were removed resulting in 160 unique SNPs distributed in 17 families according to the KEGG database used for the family group annotations.
• SNP analysis of the isolated clones was performed by NimbleGen (Roche).
• the SNP files we re d e l i ve red co m pa ri n g n ati ve D G 1 pS P D 5 P D0001 VT to DG 1 pS P D5 PD0001VE05C01 , DG1 pSPD5 PD0001VE05c05, DG1 pSPD5 PD0001VE05c07 or DG1 pSPD5 PD0001VE05c08 using Genbank reference sequence (Clostridium acetobutylicum ATCC 824 http://www.ncbi.nlm.nih.gov/nuccore/AE001437).
• AA change categorizes coding SNPs base on whether or not they change the amino acid sequence of a protein.
• S indicates synonymous SNPs (no amino acid change).
• N indicates nonsynonymous SNPs (altered amino acid).
• FC Fre-Change indicates a modification in protein translation because of insertion or deletion of a nucleotide
• Table 1 Mutations between native and evolved strains. Mutations were first identified in the adapted population and then presence of each mutat was verified in isolated clones (four last columns: Y for presence and N for absence of mutation).

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