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  • 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
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/16—Butanols
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  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present Invention is related to the field of industrial microbiology and genetic engineering of organisms. More specifically the present invention relates to metabolic engineering of Clostridia such as Clostridium acetobutylicum. Even more specifically, the invention relates to overexpression of enzymes from the butanol metabolic pathway, namely crotonase (Crt), butyryl-CoA
• Butanol is an advanced biofuel because it can be used in existing gasoline-powered vehicles and other liquid-fueled processes without technical modifications.
• ABE acetone- butanol-ethanol
• strains from genus Clostridium was employed since 1920s for industrial butanol and acetone production.
• industrial ABE fermentation declined rapidly after the 1950s as a result of the cheaper petrochemical production routes.
• solventogenic Clostridia have regained much interest recently due to their unique metabolic capacity of n-butanol production from renewable resources.
• Solventogenic Clostridia are strains that produce organic chemical compounds (primary metabolites), including butanol, ethanol and acetone, particularly butanol. Solventogenic Clostridia are strictly anaerobic, endospore forming bacteria. Solventogenic Clostridia metabolize carbohydrates, including glucose, cellulose and hemicellulose. Solventogenic Clostridia are reviewed by Papoutsakis in Current Opinion in Biotechnology 2008, 19:420-429.
• C. saccharoperbutylacetonicum and C. saccharobutylicum with C. acetobutylicum being the model organism of solvent producing Clostridia for scientific research (Diirre P. 2011. Fermentative production of butanol - the academic perspective. Curr Opin Biotechnol 22:331 -336.)
• the fermentation of sugars by Clostridia typically causes three different growth phases: (i) exponential growth and formation of acids, (ii) transition to stationary growth phase with re- assimilation of acids and concomitant formation of solvents and (iii) formation of endospores.
• Sugars such as glucose or xylose are catabolized to pyruvate, and acetyl-CoA is primarily formed by the pyruvate:ferredoxin oxidoreductase.
• lactate can be the major fermentation product.
• WO2012045022 describes the overexpression of alcohol/aldehyde dehydrogenase and butanol dehydrogenase in a non-butanol producing strain of Clostridium tyrobutyricum. The production of butanol was demonstrated, however at a lower level than wt Clostridium acetobutylicum. A similar approach has been tried in application KR201 10033086. Comparable data are shown in (Sillers R et al, 2008 Metab Eng 10:321 -332.), where no significant increase in butanol production was achieved. A slightly different approach was described in CN10261979. In addition to Adh, overexpression tests with Acetoacetyl-acylCoA transferase were performed. However, the achieved increase of butanol level was not significant.
• the object of the present invention is thus to provide an improved process for the microbial production of butanol, and to provide microbial cells for such a process.
• the invention thus provides a method for producing butanol, comprising the steps of fermenting a medium containing a sugar, or a precursor thereof, with a Clostridium cell with the ability to produce butanol, wherein the cell comprises a genetic modification that results in overexpression of one or more of the following genes:
• Control C. acetobutylicum ATCC 824/pT (vector control); pJ::hbd, C. acetobutylicum ATCC 824/pT v.hbd; olv.crt, C. acetobutylicum ATCC 824/pT::crt; pT::bcd, C. acetobutylicum ATCC 824/pT v.bcd.
• overexpression strain Utilization of glucose, production of biomass, production of organic acids acetate, butyrate, and production of solvents acetone, ethanol and butanol is shown.
• White circles present performance of the bed overexpression strain in liter scale reactor and black circles present strain performance in milliliter scale reactor. Performance of maternal strain with wt genotype is presented with grey line. Error bars stay for standard deviation from 3 independent experiments.
• the cells for use in the method of the present invention are Clostridium cells with the ability to produce butanol, wherein the cell comprises a genetic modification that results in overexpression of one or more of the following genes:
• the genetically modified solventogenic Clostridia cell is a bacterial strain which belongs to the taxonomic class Clostridia, preferably a bacterial strain belonging to the taxonomic order
• Clostridium argentinense Clostridium asparagiforme, Clostridium alkalicellulosi, Clostridium autoethanogenum, Clostridium baratii, Clostridium beijerinckii, Clostridium bogorii, Clostridium boliviensis, Clostridium bolteae, Clostridium aminobutyricum, Clostridium aminophilum, Clostridium algoriphilum, Clostridium aminovorans, Clostridium amygdalinum, Clostridium amylolyticum, Clostridium celerecrescens, Clostridium cellobioparum, Clostridium cellulolyticum, Clostridium cellulosi, Clostridium cellulovorans, Clostridium chartatabidum, Clostridium aminovalericum, Clostridium chromoreductans, Clostridium citroniae, Clostridium clariflavum, Clostridium clostridioforme
• Clostridium cadaveris Clostridium aurantibutyricum, Clostridium botulinum, Clostridium bovipellis, Clostridium carboxidivorans, Clostridium carnis, Clostridium cavendishii, Clostridium celatum, Clostridium halophilum, Clostridium hatheway, Clostridium hathewayi, Clostridium hathewayi, Clostridium herbivorans, Clostridium histolyticum, Clostridium caliptrosporum, Clostridium caminithermale, Clostridium hveragerdense, Clostridium hydrogeniformans, Clostridium hydrogeniformans, Clostridium hydrolyticum, Clostridium hylemonae, Clostridium indolis,
• Clostridium josui Clostridium kluyveri, Clostridium lactatifermentans , Clostridium crotonatovorans, Clostridium cylindrosporum, Clostridium difficile, Clostridium diolis, Clostridium caenicola , Clostridium drakei, Clostridium elmenteitii, Clostridium estertheticum, Clostridium fallax, Clostridium favososporum, Clostridium felsineum, Clostridium filamentosum, Clostridium fimetarium,
• Clostridium formicaceticum Clostridium frigidicarnis, Clostridium chauvoei, Clostridium frigoris, Clostridium fusiformis, Clostridium ganghwense, Clostridium gasigenes , Clostridium
• Clostridium pascui Clostridium pasteurianum
• Clostridium peptidivorans Clostridium perfringens
• Clostridium phytofermentans Clostridium piliforme
• Clostridium polysaccharolyticum Clostridium populeti
• Clostridium propionicum Clostridium proteolyticum
• Clostridium proteolytics Clostridium homopropionicum
• Clostridium hungatei Clostridium purinilyticum, Clostridium putrefaciens, Clostridium quinii, Clostridium ragsdalei, Clostridium roseum, Clostridium ruminantium, Clostridium saccharobutylicum, Clostridium saccharogumia, Clostridium cf.
• thermopalmarium Clostridium thermophiius, Clostridium thermosuccinogenes, Clostridium thiosulfatireducens, Clostridium tunisiense, Clostridium limosum, Clostridium uliginosum,
• the cell of the invention belongs to one of the following species: Clostridium propionicum, Clostridium cellulolyticum, Clostridium acetobutylicum,
• the genetically modified Clostridium cell is preferably a cell or strain from C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum and C. saccharobutylicum, with C. acetobutylicum being preferred.
• Wild-type C. acetobutylicum is a strain that naturally produces the solvents acetone, butanol and ethanol in a ratio of 3:6:1.
• the cell of the invention is a cell which belongs to one of the following species Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum, and which produces more than 1 mg/l of at least one of following organic compounds: acetone, butanol, ethanol.
• said cell of the invention which belongs to one of said species produces more than 10 mg/l of at least one of following organic compounds: acetone, butanol, ethanol.
• said cell of the invention which belongs to one of said species produces more than 100 mg/l of at least one of following organic compounds: acetone, butanol, ethanol.
• said cell of the invention which produces more than 100 mg/l of at least one of following organic compounds: acetone, butanol, ethanol belongs to the species Clostridium acetobutylicum.
• "at least one” includes the integers one, two and three.
• Clostridium cells of this invention include derivatives of any parent strain.
• the cells of this invention may thus also be referred to as mutant cells.
• the parent strain can be any wild-type strain of any Clostrium species, particularly the above-specified Clostridium species and strains, including C. acetobutylicum ATCC824. Suitable wild-type strains for genetic modification include
• the derivative is obtainable by introducing at least one genetic modification into the parent strain, such as into the parent strain Clostridium acetobutylicum 824. Suitable genetic modifications are described in detail below. The presence of a genetic modification can be verified by comparing the genetic information of the parent strain and the derivative, e.g. as described below.
• the determination of said production of said organic compound(s) is determined, in a preferred embodiment, by preparing a Clostridium preculture as described in Example 4 (which renders the preculture suitable for inoculation of a batch culture), followed by inoculation of a batch culture prepared as described in Example 6 and cultured as described in Example 6.
• Suitable certain process times are 14 h, 20 h or 31 h, preferably 14 h or 20 h.
• the mg/l determined at said certain process times characterize a strain as producing a certain amount of said organic compound(s) (e.g. more than more than 1 mg/l, more than 10 mg/l, more than 100 mg/l) as defined in this document).
• Such additional copies may be present in the cell's chromosome, in episomes or in plasmids. Additionally, or alternatively, such genetic elements may be stronger native promoters (lie the thl promoter) that replace the wild-type promoters of the chromosomally located crt, bed, and hbd (which can be effected e.g. by homologous recombination). Furthermore, one may usually conclude from such an analysis of the genetic elements which (wild-type) strain was used for genetic modification. (For example, the entire wild-type Clostridium acetobutylicum ATCC824 genome is shown in http://www.ncbi.
• the genetic modification can also be verified by an increased enzymatic activity of one or more of Crt, Bed, and Hbd in comparison to the wild-type strain.
• the increased enzymatic activity can e.g. be shown by an increased butanol production.
• the genetic modification can be a genetic modification that results in overexpression of one of crt, bed and hbd, a genetic modification that results in overexpression of two of crt, bed and hbd, or a genetic modification that results in overexpression of all three of crt, bed and hbd.
• the overexpressed gene(s) is preferably homologous to the Clostridium cell. Alternatively, it may be heterologous to the Clostridium cell.
• the genes may originate from any other species belonging to the class of Actinobacteria.
• the genetic modification is preferably the result of a transformation with one or more vectors comprising one or more of the genes crt, bed and hbd.
• the vectors preferably contain a nucleic acid sequence encoding an amino acid sequence which is at least 70 % , more preferably at least 80 %, even more preferably at least 90 % such as at least 95 % identical to the amino acid sequence of SEQ ID No. 1 , and/or a sequence encoding an amino acid sequence which is at least 70 %, more preferably at least 80 %, even more preferably at least 90 % such as at least 95 % identical to the amino acid sequence of SEQ ID No.
• amino acid sequence which is at least 70 %, more preferably at least 80 %, even more preferably at least 90 % such as at least 95 % identical to the amino acid sequence of identical to the amino acid sequence of SEQ ID No. 3.
• the gene is operatively linked to a promoter.
• a preferred promoter is the promoter of C.acetobutylicum ATCC 824 thiolase gene having the sequence of SEQ ID No. 4., or a functional equivalent having a sequence identity of at least 90, more preferably 95 %.
• the vector is a shuttle vector for replication in E.coli and C. acetobutylicum.
• the cells of the present invention can be produced by a method comprising the steps of
• step (iv) transforming the wild-type Clostridium strain with the plasmids obtained in step (iii);
• the plasmids are transformed into E.coli for methylation prior to step (iv).
• the invention provides an improved process for producing butanol using Clostridia strains with increased metabolic fluxes through butyryl-CoA synthesis. The fluxes were increased in
• Acetate is preferably generated in a molar ratio butanol :acetate of at least 8:1 , preferably at least 12:1 , more preferably at least 20:1.
• the overall ABE titers are preferably in the range above 20 g/l ABE. These values can be determined e.g. after 20 hours of fermentation.
• the process is preferably carried out in batch mode.
• the reaction volume comprising the sugar, or precursor thereof and the bacteria is preferably 1 I or more, more preferably 10 I or more.
• the reaction time is preferably in the range of 15 to 25 hours.
• SEQ ID No. 2 putative butyryl-CoA dehydrogenase
• the BCS genes of Clostridium acetobutylicum were homologously overexpressed for increased dosages of 3-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonase (Crt) and butyryl-CoA dehydrogenase (Bed) complex.
• Hbd 3-hydroxybutyryl-CoA dehydrogenase
• Crt crotonase
• Bed butyryl-CoA dehydrogenase
• the genes hbd (CAC2708), crt (CAC2712) and bcd/etfAB (CAC2709-271 1 ) were amplified by PCR from chromosomal DNA of C. acetobutylicum ATCC 824 (Fischer et al. , J.
• E. coli DH5a The resulting plasmids pJ::hbd, pT: crt and pT/.bcd, respectively, were transformed into E. coli DH5a and validated by DNA sequencing (LGC Genomics GmbH, Berlin, Germany).
• E. coli strains were cultivated in LB medium comprising per liter 5 g yeast extract, 10 g tryptone and 10 g NaCI, ampicillin was added for plasmid maintenance at a concentration of 100 g/ml (Sambrock J and Russell DW, 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, NY, USA.). After in vivo methylation in E. coli ER2275 pAN2, the plasmids were transformed into C.
• recombinant C. acetobutylicum strains were cultivated anaerobically at 37°C without shaking in Hungate tubes or serum bottles, 40 pg/rnl erythromycin were added for plasmid maintenance.
• Resazurin (7-hydroxy-10-oxidophenoxazin-10-ium-3-one) was added as a redox indicator for anaerobiosis at a concentration of 1 mg/l and residual oxygen was removed by addition of 50-100 ⁇ titanium (III) nitrilotriacetic acid (NTA) solution (1.3 M NaOH, 0.16 M NTA, 0.27 M Na 2 C0 3 and 1 .3 % TiCI 3 ).
• Fermentation experiments were performed in 200 ml MS-MES medium in serum bottles (Muller & Krève AG, Bulach, Switzerland) with the following composition per liter: 0.55 g KH 2 P0 4 , 0.55 g K 2 HP0 4 , 0.22 g MgS0 4 ⁇ 7 H 2 0, 0.011 g FeS0 4 * 7 H 2 0 and 2.3 ml acetic acid; after the pH was adjusted to 6.6 with NH 4 OH, 40 ⁇ g p-aminobenzoic acid, 0.32 [ig biotin, 1 mg resazurin and 21 .3 g 2-(/V-morpholino) ethanesulfonic acid (MES) were added (modified from Monot, F et al, 1982, Appl.
• MES 2-(/V-morpholino) ethanesulfonic acid
• Thiolase activity was measured spectrophotometrically by acetoacetyl-CoA decrease at 303 nm.
• 3-Hydroxybutyryl-CoA dehydrogenase (Hbd) activity was determined by NADH decrease at 340 nm according to Madan et al. (1973). Crotonase activity was measured by crotonyl-CoA decrease at 263 nm as described previously.
• Precultures were obtained from spore suspensions inoculated to clostridial growth medium in anaerobic flasks with a liquid volume of 5 mL (glucose, 2.5 g L “ ; KH 2 P0 4 , 0.75 g L “1 ; K 2 HP0 4 , 0.75 g L “ ; MgS0 4 -7 H 2 0, 0.4 g L “1 ; MnS0 4 ⁇ H 2 0, 0.01 g L “1 ; FeS0 4 -7 H 2 0, 0.01 g L “1 ; NaCI, 1 g L ⁇ 1 ; (NH 4 ) 2 S0 4 , 2 g L ⁇ 1 ; yeast extract, 5 g L "1 ; asparagine, 2 g L ⁇ 1 ; pH 6.6 adjusted with NH 4 OH) and pasteurized at 80°C for 10 min.
• MS-MES medium 2-(/V-morpholino) ethanesulfonic acid medium
• MS-MES medium was prepared in anaerobic flasks (45 mL working volume) with glucose (60 g L ⁇ ), KH 2 P0 4 (0.55 g L " ), K 2 HP0 4 (0.42 g L ⁇ ), MgS0 4 ⁇ 7 H 2 0 (0.22 g L “ ), FeS0 4 ⁇ 7 H 2 0 (0.0 1 g L ⁇ 1 ), 0.08 mg L “1 biotin and 8 mg L "1 p-aminobenzoic acid, (NH 4 ) 2 S0 4 (5.496 g L "1 ) and acetic acid (2.3 g L ⁇ ) at pH 5.5 adjusted with KOH (modified from Monot, F et al, 1982, Appl.
• the parallel bioreactor system was applied with baffled single-use bioreactors made of polystyrene. To ensure initial anaerobic conditions, the system and all necessary components were stored in an anaerobic chamber overnight before each of the parallel bioreactors with a nominal volume of 20 mL was filled with 12 mL of an inoculated MS MES medium withO.1 mL L "1 polypropylene glycol as an antifoaming agent. The inoculated parallel bioreactor system was manually transferred to the control station outside the glove box. Immediately afterwards it was gassed with 48 L h " of N 2 for 1 h to ensure anaerobic conditions.
• the reference batch process was carried out at 37°C in a 2 L stirred tank reactor (Labfors 3, Infors, Switzerland) with 2 Rushton turbine impellers at a working volume of 1 L.
• the pH was monitored with the control software Iris NT Pro v 5.02 (Infors-HT, Bottmingen, Switzerland).
• the reactor was immediately sparged with sterile nitrogen gas (2 L min -1 , 5.0 Nitrogen; Air Liquide, Kunststoff, Germany) while being cooled to establish and remain anaerobic conditions.
• sterile nitrogen gas (2 L min -1 , 5.0 Nitrogen; Air Liquide, Kunststoff, Germany
• Figure 2 shows changes in product concentrations during the batch cultivation in stirred tank reactors of C. acetobutylicum OverExpression-pT::bcd are shown and compared to the wild type strains performance.
• the courses of all concentrations of the recombinant strain on both scales were comparable. Glucose was consumed completely at a process time of 20 hours. This resulted in a glucose consumption rate of 2.4 g/(Lh).
• the wild type strain showed a glucose consumption rate of 0.9 g/(Lh) at a process time of 20 hours and at a process time of 50 hours a rest glucose concentration of 10 g/L remained in the medium .
• the maximum cell dry weight of 4 g/L of C. acetobutylicum LIE- pT::bcd was 35 % higher and was obtained 10 h earlier compared to the wild type strain.
• the maximum acid concentrations were not determined, but the qualitative courses are lower compared to that of the wild types. This may be a proof for a better reassimilation of acids. Ethanol production was a bit higher than the wild type strains.
• the glucose consumption rates of C. acetobutylicum OverExpression-pT::crt were 250 % increased (2,3 g/L compared to 0,9 g/L wild type glucose consumtion rate).
• the maximum cell dry weight of 4 g/L of C. acetobutylicum OverExpression-pT::crt was 35 % higher and was obtained 10 h earlier compared to the wild type strain.
• butyrate concentrations were comparable to the one of the wild type strain but the acetate concentration was increased to 5 g/L compared to 3 g/L reached with the wild type strain before the onset of reassimilation.
• the solventogenic phase proceeded comparably to C. acetobutylicum OverExpression-pT::bcd.
• the maximal solvent concentrations were obtained (9 g/L butanol concentration and 1 g/L ethanol concentration and 4,3 g/L acetone).

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