FIELD OF THE INVENTION

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

dehydrogenase (Bed) and/or 3-hydroxybutyryl-CoA dehydrogenase (Hbd).

BACKGROUND OF THE INVENTION

Butanol is an advanced biofuel because it can be used in existing gasoline-powered vehicles and other liquid-fueled processes without technical modifications. Historically, the so-called acetone- butanol-ethanol (ABE) fermentation with strains from genus Clostridium was employed since 1920s for industrial butanol and acetone production. However, industrial ABE fermentation declined rapidly after the 1950s as a result of the cheaper petrochemical production routes. Given today's knowledge on economic and environmental issues of fossil oil resources and increasing environmental problems due to global warming, biotechnological routes for energy production become more and more important. Therefore, so-called 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.

The genus Clostridium represents a diverse group of strictly anaerobic Gram-positive bacteria with the ability to form heat-resistant endospores for long-term survival under unfavorable

environmental conditions. Besides several toxin-producing species such as Clostridium

perfringens, C. botulinum or C. tetani, a large number of terrestrial non-pathogenic species show biotechnologically interesting metabolic properties like the formation of ethanol, butanol, acetone, isopropanol or propanediol. Although many clostridial strains are capable of solvent production in various amounts and ratios, the major producers are C. acetobutylicum, C. beijerinckii,

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. Under certain growth conditions such as pH values > 5 and iron limitation, lactate can be the major fermentation product. Usually, C, acetobutylicum synthesizes acetate via phosphotransacetylase and acetate kinase reactions with the latter reaction providing ATP. For the biosynthesis of butyrate, two molecules of acetyl-CoA are condensed to acetoacetyl-CoA, followed by a reduction to butyryl-CoA, which is then converted to butyrate via phosphotransbutyrylase and butyrate kinase reactions with ATP generation. In the butyrate/butanol biosynthetic pathway, acetyl-CoA is first converted to butyryl-CoA via thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase and butyryl-CoA dehydrogenase reactions. Subsequently, butyryl-CoA is converted to butyrate in the acidogenic and to butanol in the solventogenic phase, the latter catalyzed by combined aldehyde/alcohol dehydrogenase activities.

Metabolic engineering efforts to improve butanol production have been of limited success

(reviewed by Liitke-Eversloh, Bahl 201 1 , Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production, Curr Opin Biotechnol 22:634-647; and Papoutsakis ET; 2008, Engineering solventogenic Clostridia; Curr Opin Biotechnol 19:420-429.)

The studies on overexpression of butanol biosynthesis pathway genes were to date mainly focused on heterologous overexpression in organisms other than Clostrida: E.coli (Atsumi S et al 2007, Metab Eng 10(6): 305-1 1 ), Lactobacillus brevis (RU2375451 ), Pseudomonas and Bacillus (Nielsen DR et al 2009 Metab Eng 1 1 (4-5):262-73). However the maximum obtained butanol concentrations were far below the wt Clostridium acetobutylicum level.

Metabolic engineering of Clostridium acetobutylicum strain M5 was done by overexpression of thiolase (Thl) combined with overexpression of alcohol dehydrogenase (Adh). The modification of the strain resulted in less side products as acetone but the maximal achievable concentration of the butanol remained at the wildtype level (Sillers R, Chow A, Tracy B, Papoutsakis ET. 2008.

Metabolic engineering of the non-sporulating, non-solventogenic Clostridium acetobutylicum strain M5 to produce butanol without acetone demonstrate the robustness of the acid-formation pathways and the importance of the electron balance. Metab Eng 10:321 -332.). In a similar study, Adh and Thl overexpression coupled with CoA transferase downregeulation lead to higher alcohol titers, but improved butanol titers were not achieved (Sillers R et al, 2009, Biotechnol Bioeng 102:38-49).

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.

Another problem of prior metabolic engineering approaches to improve clostridial butanol production was a concomitant increase of the acetate level which is not useable as fuel and thus undesirable for industrial production.

As a consequence of the limited success of rational metabolic engineering approaches to improve clostridial butanol production, Lutke-Eversloh, Bahl 201 1 , Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production. Curr Opin Biotechnol 22, 634-647, recommend an alternative approach to provide improved Clostridia strains, namely by selecting improved strains because of their phenotypic characteristics.

PROBLEM TO BE SOLVED

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.

SUMMARY OF THE INVENTION

The inventors have surprisingly found that the overexpression of specific enzymes involved in clostridial butanol production not only results in high butanol titer and faster kinetics, but also fails to lead to a significant increase in the acetate level. The inventors could also successfully show the scale-up of this approach to reactor scale.

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:

• a gene encoding crotonase {erf),

• a gene encoding butyryl-CoA dehydrogenase (bed),

• a gene encoding 3-hydroxybutyryl-CoA dehydrogenase (hbd).

The invention further provides a Clostridium cell or strain as defined above, a method for its production, including a vector for production of such cells.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows product concentrations of (Acetone-Butanol-Ethanol = ABE) and yields on glucose of recombinant C. acetobutylicum-slrains. Figure 1 legend: Acetone-butanol-ethanol (ABE) titers (a) and yields on glucose (b) of recombinant C. acetobutylicum strains. Cultivations were performed as described in Example 2, average data of three independent experiments are shown. 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.

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. Figure 2 legend: Characterization of C. acetobutylicum bed

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.

Figure 3 shows product concentrations during the batch cultivation of C. acetobutylicum

OverExpression-pT::crt and compares them to the wild type strains performance. Figure 3 legend: Characterization of C. acetobutylicum crt 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 crt

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.

DETAILED DESCRIPTION OF THE INVENTION

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:

• a gene encoding crotonase (erf),

• a gene encoding butyryl-CoA dehydrogenase (bed),

• a gene encoding 3-hydroxybutyryl-CoA dehydrogenase (hbd).

A cell with these characteristics is referred to herein as "genetically modified solventogenic Clostridia cell "genetically modified solventogenic Clostridium cell", and may also be referred to as "cell of the invention".

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

Clostridiales, more preferably a bacterial strain belonging to the taxonomic family Clostridiaceae, even more preferably a bacterial strain belonging to the taxonomic genus Clostridium. This includes the preferred embodiments of the bacterial strains belonging to one of the following species Clostridium aceticum, Clostridium acidisoli, Clostridium aciditolerans, Clostridium acidurici, Clostridium aerotolerans, Clostridium acetireducens, Clostridium massiliensis, Clostridium algidicarnis, Clostridium algidixylanolyticum, Clostridium aestuarii, Clostridium acetobutylicum, Clostridium timonensis, Clostridium akagii, Clostridium alcalibutyricum, Clostridium alcaliphilum, Clostridium aldenense, Clostridium aldenense, Clostridium aldrichii, Clostridium arbusti,

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 cochlearium, Clostridium colicanis , Clostridium colinum, Clostridium collagenovorans, Clostridium corinoforum, Clostridium bowmanii, Clostridium butyricum,

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 intestinale, Clostridium isatidis, Clostridium islandicum, Clostridium jejuense,

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

glycyrrhizinilyticum, Clostridium grantii, Clostridium haemolyticum, Clostridium taeniosporum, Clostridium tagluense, Clostridium tepidiprofundi, Clostridium termitidis, Clostridium tertium, Clostridium tetani, Clostridium tetanomorphum, Clostridium thermoalcaliphilum, Clostridium lacusfryxellense, Clostridium lavalense, Clostridium leptum, Clostridium disporicum, Clostridium ljungdahlii, Clostridium longisporum, Clostridium lundense, Clostridium magnum, Clostridium malenominatum, Clostridium maritimum, Clostridium mayombei, Clostridium metallolevans, Clostridium methoxybenzovorans, Clostridium methylpentosum, Clostridium neonatale, Clostridium neopropionicum, Clostridium frigoriphilum, Clostridium nitrophenolicum, Clostridium novyi, Clostridium oceanicum, Clostridium oroticum, Clostridium papyrosolvens, Clostridium

paraputrificum, 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. saccharolyticum, Clostridium saccharoperbutylacetonicum, Clostridium sardiniense, Clostridium sartagoforme, Clostridium scatologenes, Clostridium schirmacherense, Clostridium scindens, Clostridium septicu, Clostridium sphertoides , Clostridium sporosphaeroides, Clostridium straminisolvens, Clostridium thermobutyricum, Clostridium thermocellum, Clostridium

thermopalmarium, Clostridium thermophiius, Clostridium thermosuccinogenes, Clostridium thiosulfatireducens, Clostridium tunisiense, Clostridium limosum, Clostridium uliginosum,

Clostridium ultunense, Clostridium uzonii, Clostridium venationis, Clostridium vincentii, Clostridium viride , Clostridium xylanolyticum, Clostridium xylanovorans , Clostridium nexile, Clostridium psychrophilum, Clostridium puniceum, Clostridium tyrobutyricum, Clostridium bartlettii, Clostridium bolteae, Clostridium clostridioforme, Clostridium clostridioforme, Clostridium hathewayi, Clostridium leptum.

Among these, it is particularly preferred that the cell of the invention belongs to one of the following species: Clostridium propionicum, Clostridium cellulolyticum, Clostridium acetobutylicum,

Clostridium butyricum, Clostridium beijerinckii, Clostridium hydrogeniformans, Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium kluyveri, Clostridium lactatifermentans, Clostridium ljungdahlii, Clostridium methoxybenzovorans, Clostridium cf.

saccharolyticum, Clostridium thermobutyricum, Clostridium thermocellum, Clostridium

xylanolyticum, Clostridium xylanovorans, Clostridium tyrobutyricum. In even more preferred embodiments the bacterial cell belongs to one of the following species, Clostridium acetobutylicum Clostridium beijerinckii, Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum.

Thus, 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 sequence of the operon containing crt, bed and hbd (as well as etfA and etfB) is given under http://www.ncbi.nlm.nih.gov/nuccore/U171 10.1 (Accession is U171 10.1 ). These genes are chromosomal.

It is even more preferred that 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. Preferably, 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. Even more preferably, 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. Even more preferably 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. In this context, "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

C. acetobutylicum ATCC824, C, acetobutylicum DSM1731 , C. acetobutylicum ATCC4529, and C. acetobutylicum M5. In general, 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. It is also possible that the parent strain, from which the cell of the invention is derived or derivable, produces more than 1 mg/l, such as more than 10 mg/l, such as more than 100 mg/l of at least one of following organic compounds: acetone, butanol, ethanol. Such a parent strain is further improved by the methods described herein and/or by introducing at least one genetic modification described herein, whereby a strain of the invention is obtained. In some preferred embodiments, the strain of the invention produces larger quantities (i.e. larger when compared to its parent strain) of at least one of the following organic compounds: acetone, butanol, ethanol under identical culturing conditions (e.g. Example 6).

The term "produces more than x mg/l" (x being a number) of one or more organic compound(s) (such as one or more of acetone, butanol, ethanol) designates the production of said organic compound(s) when the Clostridium cell (parent cell or of the invention) is cultured under appropriate culturing conditions, wherein mg refers to the mg of said organic compound(s), and I refers to liters of culture broth. Culturing conditions for Clostridium cells are known in the art. The culture broth usually contains the cells and the culture medium. 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. At a certain process time (= hours after point t=0, Example 6) the production of said organic compound(s) is determined. 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).

The presence of a genetic modification that results in overexpression of one or more of the genes crt, bed, and hbd can be verified by the presence of genetic elements that cannot be found in wild- type strains and that have a positive impact on the relevant enzymatic activity. Such genetic elements are particularly additional copies of one or more of crt, bed and hbd. These additional copies have preferably 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 % or even 100 % identical to the amino acid sequence of SEQ ID No. 1 , having crotonase activity, 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 % or even 100 % identical to the amino acid sequence of SEQ ID No. 2, having butyryl-CoA dehydrogenase activity, 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 % or even 100 % identical to the amino acid sequence of identical to the amino acid sequence of SEQ ID No. 3, having 3- hydroxybutyryl-CoA dehydrogenase.

Comparison of sequence identity (alignments) is performed using the ClustalW Algorithm (Larkin .A., Blackshields G., Brown N.P., Chenna R., McGettigan P.A., McWilliam H., Valentin F., Wallace I.M., Wilm A., Lopez R., Thompson J.D., Gibson T.J. and Higgins D.G. (2007) ClustalW and ClustalX version 2. Bioinformatics 2007 23(21 ): 2947-2948).

These 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. n I m .nih.gov/nuccore/NC 003030; Accession is NC_003030). If the wild-type strain is available for comparison, 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. For example, 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. 2, 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 identical to the amino acid sequence of SEQ ID No. 3.

In the vector, 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 %.

In a particularly preferred embodiment, the vector is a shuttle vector for replication in E.coli and C. acetobutylicum.

Suitable promoters and vectors are described in Girbal et al., Applied and Environmental

Microbiology 2005, Vol. 71 , No. 5, p.2777-2781 and Mann et al. Biotechnol. Lett (2012) 34: 1643- 1649.

The cells of the present invention can be produced by a method comprising the steps of

(i) providing a wild-type Clostridium strain;

(ii) amplifying one or more of the following genes of said strain:

« a gene encoding crotonase (crt),

• a gene encoding butyryl-CoA dehydrogenase (bed),

• a gene encoding 3-hydroxybutyryl-CoA dehydrogenase (hbd),

(iii) cloning the amplified DNA sequences into plasmids;

(iv) transforming the wild-type Clostridium strain with the plasmids obtained in step (iii);

(v) selecting strains that show a higher activity of the enzyme encoded by the

amplified gene than the wild-type Clostridium strain.

It is particularly preferred that 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

Clostridium acetobutylicum strain by homologous overexpression of butyryl-CoA synthesis genes.

The present invention also provides a method for producing butanol, comprising the steps of fermenting a medium containing a sugar, or a precursor thereof, with a Clostridium cell of the present invention, wherein the Clostridium cell ferments sugar to butanol, and recovering butanol. The Clostridium cell also preferably ferments sugar to ethanol. Most preferably butanol and ethanol are generated in a molar ratio in the range of 1 :1 to 3:1 . More preferably, the maximum butanol concentration is at least 150 mM (e.g. in the range of 160 to 200 nM), and the maximum ethanol concentration is at least 60 mM (e.g. in the range of 100 to 150 nM). 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.

SEQUENCES

SEQ ID: No: 1: crotonase

MELNNVILEKEGKVAWTINRP ALNALNSDTLKEMDYVIGEIE

NDSEVLAVILTGAGEKSFVAGADISEMKEMNTIEGRKFGILGNKVFRRLELLEKPVI A AVNGFALGGGCEIAMSCDIRIASSNARFGQPEVGLGITPGFGGTQRLSRLVGMGMAKQ LIFTAQNIKADEALRIGLVNKWEPSELMNTAKEIANKIVSNAPVAVKLSKQAINRGM QCDIDTALAFESEAFGECFSTEDQ DAMTAFIEKRKIEGFKNR

SEQ ID No. 2: putative butyryl-CoA dehydrogenase

MDFNLTREQELVRQMVREFAENEVKPIAAEIDETERFPMENVKK

MGQYGMMGIPFSKEYGGAGGDVLSYIIAVEELSKVCGTTGVILSAHTSLCASLINEH G TEEQKQKYLVPLAKGEKIGAYGLTEPNAGTDSGAQQTVAVLEGDHYVINGSKIFITNG

GVADTFVIFAMTDRTKGTKGISAFIIE GFKGFSIGKVEQKLGIRASSTTELVFEDMI

VPVENMIGKEG GFPIAMKTLDGGRIGIAAQALGIAEGAFNEARAYMKERKQFGRSLD

KFQGIiAWMiyLADMDVAIESARYLVYKAAYLKQAGLPYTVDAARAKLH

VQLFGGYGYT DYPVERMMRDAKITEIYEGTSEVQKLVISGKIFR

SEQ ID No. 3 : 3 -hydroxybutyryl-CoA dehydrogenase

MKKVCVIGAGTMGSGIAQAFAAKGFEWLRDI DEFVDRGLDFI

NKNLS LVK G IEEAT VEILTRISGTVDLNMAADCDLVIEAAVERMDIKKQIFADL DNICKPETILASNTSSLSITEVASATKTNDKVIGMHFFNPAPVMKLVEVIRGIATSQE TFDAVKETSIAIG DPVEVAEAPGFVWRILIPMINEAVGILAEGIASVEDIDKAM L GANHPMGPLELGDFIGLDICLAIMDVLYSETGDSKYRPHTLLKKYVRAGWLGRKSGKG FYDYSK

SEQ ID: No: 4 : Thl prmoter 135 bp:

tcgactttttaacaaaatatattgataaaaataataatagtgggtataattaagttg ttagagaaaacgtataaattagggataaactatgga acttatgaaatagattgaaatggtttatctgttaccccgtag

EXAMPLES

Example 1

Cloning and homologous overexpression of BCS genes

In order to improve the metabolic fluxes through the butyryl-CoA synthesis (BCS) pathway, 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. To accomplish this, 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. Bacteriol, August 2006; 5469-5478) using the oligonucleotides listed in TABLE 1 as PCR primers. The DNA fragments were cloned via BamHI and KasI restriction sites into the plHydA plasmid as described (Girbal et al. 2005; Hillmann F, Doring C, Riebe O, Ehrenreich A, Fischer RJ and Bahl H, 2009. The role of PerR in 0 ₂ -affected gene expression of Clostridium acetobutylicum. J. Bacteriol. 191 :6082-6093.; Mann et al. 2012).

TABLE 1 : Oligonucleotides used for gene cloning. Genes were amplified from chromosomal DNA of C. acetobutylicum ATCC 824 and purified PCR products were cloned into the pT vector via BamHI and KasI restriction sites (underlined).

Gene (ORF) Primer Sequence (5'→ 3 ^* ) hbd_fw_BamHI AAAAAGGATCCATGAAAAAGGTATGTG hbd (CAC2708)

hbd_rv_Kasl AAAAAGGCGCCTTATTTTGAATAATCGTAG crt_fw_BamHI AAAAAGGATCCATGGAACTAAACAATG crt (CAC2712)

crt_rv_Kasl AAAAAGGCGCCCTATCTATTTTTGAAGCC bcd_etf AB_fw_Bam H I AAAAAGGATCCATGGATTTTAATTTAACAAG bcd/etfAB

(CAC2709-2711 )

bcd_etfAB_rv_Kasl AAAAAGGCGCCTTAATTATTAGCAGCTTTAAC

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. acetobutylicum ATCC 824 by electroporation and respective overexpression strains were selected on reinforced clostridial agar (RCA, Oxoid Deutschland GmbH, Wesel, Germany) containing 40 pg ml ^"1 erythromycin.

Example 2

Effect of BCS gene overexpression in C. acetobutylicum The recombinant strains of C. acetobutylicum were subjected to fermentation experiments at a 200 ml-scale in a defined synthetic medium to assess the phenotypic characteristics. The parental wildtype C. acetobutylicum ATCC 824 and the vector control strain C. acetobutylicum ATCC 824/pT (Mann et al. 2012) were used as references.

In general, 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 μΙ 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 & Krempel 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 χ 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 μg 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. Environ. Microbiol. 44, 1318-1324). glucose was used as carbon source at a concentrations of 60 g/l. Samples were regularly drawn to monitor growth and cell-free supernatant samples were stored at -20°C until quantification of glucose and fermentation products. Acetate, butyrate, acetone, ethanoi and butanol were determined as described previously on an Agilent 7890A gas chromatograph (Agilent Technologies, Boblingen, Germany) equipped with a Chromosorb 101 (80/100 mesh, 2.0 m x 3.0 mm x 1 .6 mm) glass column.

TABLE 2: Fermentation products of recombinant C. acetobutylicum-stra ^' s. Mean values and standard deviations after 88 h of cultivation of three independent replicates per strain are shown.

C. acetobutylicum strain

Products

Wildtype pT pj .hbd pT::crt pT:: bed

Acetate (mM) 5.9 ± 1.8 15.6 ± 5.2 8.6 ± 1 .2 14.2 + 0.6 18.1 ± 5.8 Butyrate (mM) Acetone (mM) 63.8 ± 4.1 82.0 ± 3.1 1 14.5 ± 1 1 .9 83.1 ± 2.3 81 .1 ± 1.1 Ethanoi (mM) 32.1 ± 2.1 21.7 ± 5.6 124.1 ± 1.0 140.8 ± 3.9 62.0 ± 2.4 Butanol (mM) 139.4 ± 3.4 139.1 ± 2.3 180.6 ± 1 .9 172.7 ± 2.4 166.4 + 4.4 Figure 1 shows product concentrations of (Acetone-Butanol-Ethanol = ABE) and yields on glucose of recombinant C. acefofet/iy//cum-strains.

Interestingly, all three novel overexpression strains exhibited a significantly improved solvent production, i. e. acetone, butanol and ethanol (ABE). The overall ABE titers were increased to 25.7 ± 0.9 g/l (hbd overexpression), 24.1 ± 0.5 g/l (crt overexpression) and 19.9 ± 0.5 g/l {bcd/etfAB overexpression), respectively, as compared to the vector control strain with 16.1 ± 0.6 g/l ABE (FIGURE 1 ). Whereas n-butanol was the main fermentation product in all recombinant C. acetobutylicum strains, hbd and crt overexpression also resulted in higher ethanol formation (TABLE 2). These results clearly indicate that increasing BCS pathway enzymes by gene overexpression constitutes a suitable metabolic engineering strategy to overcome natural limits in clostridial butanol production. Such approach was for first time demonstrated in this publication.

Example 3

Enzyme activities in recombinant C. acetobutylicum strains

To validate increased BCS enzyme dosages, the Hbd and Crt as well as the thiolase activities were measured in the recombinant C. acetobutylicum strains and compared to the wildtype and vector control strains.

For the determination of enzyme activities in crude extracts, cells of C. acetobutylicum grown in MS-MES were harvested in the late exponential growth, and cells were stored at -70°C when necessary. All following steps were done at 4°C or on ice, respectively. The cell pellets were resuspended in 100 mM Tris/HCI buffer, pH 7.6, and disrupted by sonication using a Hielscher UP200S ultrasonic processor equipped with the sonotrode S3 (Hielscher Ultrasonics GmbH, Teltow, Germany). The cell suspension was sonicated for 90 s at 80 % amplitude, followed by a break for 60 s, this cycle was repeated 8-12 times. When necessary, breaks of 4-5 min were included to prevent warming of the suspension, the process was controlled by light microscopy. The cell extract was centrifugated at 13,000 rpm for 30 min and the supernatant was used for enzyme assays as described below. The protein content was determined according to Bradford (1976).

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.

As summarized in TABLE 3, Hbd and Crt activities were significantly increased in C. acetobutylicum ^• plv.hbd and C. acetobutylicum pT::crt, respectively. Interestingly, activities of the thiolase, the first enzyme of the BCS pathway catalyzing the condensation of two molecules acetyl-CoA, was not significantly changed in any overexpression strain as compared to the wildtype and the vector control strain.

TABLE 3: Enzyme activities of recombinant C. acetobutylicum-strams. Average values and standard deviations of three independent measurements are shown. One unit of activity corresponds to the conversion of one millimole substrate per minute per milligram protein content of the crude extracts.

C. acetobutylicum 3-Hyd roxyb utyryl-CoA

Thiolase (U/mg) Crotonase (U/mg) strain dehydrogenase (U/mg)

Wildtype 31 .5 ± 1.2 10.3 ± 0.5 94.9 ± 4.5

PT 29.9 1 0.6 10.8 ± 0.6 97.8 ± 7.1 p Twhbd 29.7 ± 1 .3 19.0 ± 0.8 100.9 ± 2.5 pT::crt 31.9 ± 0.4 1 1 .5 ± 0.3 160.2 ± 4.4 plr.bcd 34.2 ± 2.0 9.9 ± 0.8 91.4 ± 2.2

Example 4

Clostridium preculture preparation

C. acetobutylicum ATCC 824, Clostridium acetobutylicum ύΕ- Tv.bcd, Clostridium acetobutylicum LIE-pT::crf), were cultivated anaerobically at 37°C. 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. After an initial growth phase of 16 h, the germed culture suspension was transferred to a modified mineral salt 2-(/V-morpholino) ethanesulfonic acid medium (MS-MES medium; 10% v/v). 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. Environ. Microbiol. 44, 1318- 1324). 18 mg L ^" ZnS0 · 7 H ₂ 0 were added, if declared. The exponentially growing microorganisms were transferred after 16 h into fresh MS-MES medium in anaerobic flasks. This procedure was repeated until sufficient cells were available for the inoculation of a stirred-tank reactor. Before inoculation the optical density (OD) was measured at 600 nm to adjust the inoculation volume to achieve an initial dry cell weight of 0.15 g L ^~1 in the stirred-tank bioreactors filled with sterile MS-MES medium.

Example 5

Cultivations on a milliliter scale

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 sterile gas cover of the parallelbioreactor system ensured identical gas flows into the headspaces of each parallel stirred-tank bioreactor. Afterwards the total gassing rate was reduced to 6 L h ^"1 (= 2.1 mL min ^-1 for each parallel bioreactor), the cultivation temperature was adjusted to 37°C, and headspace cooling was set to 2°C to reduce evaporation. This headspace cooling temperature resulted in a volume-specific evaporation rate identical to that on the liter scale (data not shown). For this purpose, evaporation experiments without

microorganisms were performed under these conditions. The volume-specific evaporation rates of butanol, acetone, and ethanol were 0.06, 0.14, and 0.01 g L ^~ h ^~ , respectively, on both scales. Evaporation of water was negligible. Because energy dissipation is a relevant scale criteria for the cultivation of C. acetobutylicum the stirrer speed at the milliliter scale was adjusted to 400 rpm, which resulted in a power supply of 0.2 W L ^"1 . Due to this relatively low rotational speed, no gas phase was sucked in by the gas-inducing stirrers for dispersion of gas bubbles into the

fermentation medium.

Example 6

Cultivations on a liter scale

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). After heat sterilization (121 °C, 20 min) of the reactor with MS-MES medium, the reactor was immediately sparged with sterile nitrogen gas (2 L min ^-1 , 5.0 Nitrogen; Air Liquide, Munich, Germany) while being cooled to establish and remain anaerobic conditions. At a temperature of 37°C gassing was reduced to 0. 7 L N ₂ min ^-1 . After inoculation, the initial pH was manually adjusted to pH 5.5 with KOH. The stirrer speed was increased from 50 rpm to 200 rpm after the pH dropped down to pH 5.4 (power consumption at 200 rpm for the liter-scale system: 0.2 W L ^"1 ; . The fermentation process time was initiated (t = 0 h) when the stirrer speed was increased to 200 rpm, because the initial lag phases varied extremely. The evaporation of water was negligible due to the low gassing rate.

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 process time axis of the cultivations on a mL scale was delayed for 10 hours. With this delay both scales show the same process performance for the recombinant strain.

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.

An earlier onset of butanol production and a significant increase in butanol production were evident. This resulted in higher butanol concentrations. At a process time of 14 hours butanol concentrations of 6 g/L were produced by the recombinant strain while the wild type strain produced only 2 g/L. At a process time of 20 hours a butanol concentration of 9 g/L were produced by the recombinant strain, while the wild type strain produced a maximal butanol concentration of 7 g/L after 31 hours.

The course of the acetone concentration was qualitatively comparable. The onset of the production of the recombinant strain occurred earlier and a maximum concentration of 4.4 g/L was reached at 20 h. The wild type strain reached a maximum acetone concentration of 4 g/L after 31 h. The decrease in cell dry mass immediately after reaching the maximum butanol concentration is noticeable with both strains.

The data clearly demonstrate that bed overexpression led to faster kinetics butanol production and to higher butanol yields. This is a first demonstration of butanol production improvement achieved by gene overexpression on reactor scale. Such improvement exceeds expectations and it is relevant for production level. Figure 3 shows product concentrations during the batch cultivation of C. acetobutylicum

OverExpression-pT::crt and compares them to the wild type strains performance.

In contrast to the recombinant OverExpression-pT::bcd strain, the C. acetobutylicum

OverExpression -pT::crt strain did not consume all glucose at a process time of 20 hours. 6 g/L glucose concentration remained in the medium at this process time but this is considerably less compared to the performance of the wild type strain (35 g/L glucose concentration at t = 20 h). 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.

The 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. At a process time of 20 hours the maximal solvent concentrations were obtained (9 g/L butanol concentration and 1 g/L ethanol concentration and 4,3 g/L acetone).

Again the data clearly demonstrate that bed overexpression led to faster kinetics of butanol production and to higher butanol yields. This is a first demonstration of butanol production improvement achieved by gene overexpression on reactor scale. Such improvement exceeds expectations and it is relevant for production level.