# Patent

## Title
Recombinant microorganisms and methods of use thereof

## Abstract

The invention relates to methods for the production of chemical compounds, particularly but not exclusively ethanol, by microbial fermentation. Also described are genetically modified micro-organisms capable of using carbon monoxide to produce one or more products, particularly but not exclusively ethanol as a main product, and producing a reduced amount or substantially no 2,3-butanediol and/or a precursor thereof.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. patent application Ser. No. 13/876,563 filed Mar. 28, 2013, which is a national phase of International Patent Application PCT/NZ2013/000012 filed Jan. 30, 2013, which claims priority to U.S. Provisional Patent Application 61/593,269 filed Jan. 31, 2012, the entirety of which are incorporated herein by reference.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with Government support under contract no W911NF-11C-0090 awarded by Defense Advanced Research Projects Agency (DARPA). The Government may have certain rights in this invention.


FIELD
The present invention relates to methods for the production of chemical compounds, particularly but not exclusively ethanol, by microbial fermentation and genetically modified micro-organisms of use in such methods.
BACKGROUND
Acetogenic microorganisms are known to be useful for the production of fuels (for example, ethanol or butanol) and other chemicals by fermentation of substrates including carbon monoxide, carbon dioxide, hydrogen and methanol, for example. Many of these microorganisms naturally produce at least two, if not more, products. However, where micoorganisms are being used to produce products, particularly on a commercial scale, it is not always desirable for the microorganisms to produce multiple products. For example, production of multiple products can come at the expense of production efficiency and yield of a product of particular value, as by-products can divert carbon away from the pathways involved in producing the main desired product. In addition, by-products may be toxic to the microorganism, the production of multiple products can make recovery and separation of desired products difficult and, it can be difficult to control fermentation conditions to favour production of one product over another. By-products may also be a potential source of contamination in a fermenter as they may be substrates for undesirable organisms.
In the case of ethanol production by microbial fermentation of substrates comprising carbon monoxide, 2,3-butanediol is typically produced as a by-product. This may reduce the ethanol production efficiency and yield, as well as cause other problems, as noted above.
It is an object of the invention to overcome one or more of the disadvantages of the prior art, or to at least to provide the public with a useful choice.
SUMMARY OF INVENTION
The invention relates, inter alia, to novel genetically modified microorganisms capable of using carbon monoxide to produce one or more product and producing a reduced amount of 2,3 butanediol and/or a precursor thereof compared to a parental microorganism. In one embodiment, the genetically modified microorganism produces substantially no 2,3 butanediol and/or a precursor thereof compared to a parental microorganism. In one particular embodiment the microorganism produces ethanol as the main product.
In a first aspect, the invention provides a carboxydotrophic acetogenic microorganism which is adapted to produce one or more product and a reduced amount or substantially no 2,3 butanediol and/or a precursor thereof upon fermentation of a substrate comprising carbon monoxide, the microorganism comprising one or more genetic modification which disrupts the 2,3-butanediol biosynthesis pathway compared to a parental microorganism.
In one particular embodiment, the invention provides a carboxydotrophic acetogenic microorganism which is adapted to produce ethanol as the main product and a reduced amount or substantially no 2,3 butanediol and/or a precursor thereof upon fermentation of a substrate comprising carbon monoxide, the microorganism comprising one or more genetic modification which disrupts the 2,3-butanediol biosynthesis pathway compared to a parental microorganism.
In one embodiment, the microorganism is adapted to further produce one or more of formate, lactate, pyruvate, succinate, valine, leucine, isoleucine, acetolactate, malate, fumerate, 2-oxogluterate, citrate.
In one embodiment, the microorganism is adapted to produce an increased amount of one or more of ethanol, formate, lactate, pyruvate, succinate, valine, leucine, isoleucine, acetolactate, malate, fumerate, 2-oxogluterate, citrate compared to a parental microorganism.
In one embodiment, the microorganism comprises at least one genetic modification which disrupts the expression and/or activity of one or more enzyme capable of converting pyruvate to acetolactate.
In one embodiment, the one or more enzyme capable of converting pyruvate to acetolactate is an acetolactate synthase (alsS).
In one embodiment, the microorganism comprises at least one genetic modification which disrupts the expression and/or activity of one or more capable of converting acetolactate to acetoin.
In one embodiment, the one or more enzyme capable of converting acetolactate to acetoin is an acetolactate decarboxylase (budA).
In one embodiment, the microorganism comprises at least one genetic modification which disrupts the expression and/or activity of one or more enzyme capable of converting acetoin to 2,3-butanediol.
In one embodiment, the one or more enzyme capable of converting acetoin to 2,3-butanediol is an enzyme chosen from 2,3-butanediol dehydrogenase (2,3bdh), an acetoin reductase, a primary:secondary alcohol dehydrogenase.
In one embodiment, the microorganism comprises at least one genetic modification which disrupts the expression and/or activity of a combination of two or more of the enzymes capable of converting pyruvate to acetolactate, acetolactate to acetoin, and/or acetoin to 2,3-butanediol.
In one embodiment, the genetic modification disrupts the expression and/or activity of one or more:

    
    
        Acetolactate synthase (alsS);
        Acetolactate decarboxylase (BudA);
        2,3-Butanediol dehydrogenase (2,3 bdh);
        Acetoin reductase; and,
        Primary :secondary alcohol dehydrogenase.
    
    


In one embodiment, the genetic modification disrupts the expression and/or activity of one or more:

    
    
        Acetolactate synthase (alsS);
        Acetolactate decarboxylase (BudA); and,
        2,3-Butanediol dehydrogenase (2,3 bdh).
    
    


In one embodiment, the one or more genetic modification disrupts one or more of the genes encoding one or more of the above enzymes. In one embodiment, the one or more genetic modification disrupts the activity of a compound required for the expression or activity of one or more of the above enzymes. In one embodiment, the one or more genetic modification increases the expression or activity of one or more compounds which inhibit the expression or activity of one or more of the above enzymes.
In one particular embodiment, the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei and related isolates. In another embodiment, the group also comprises Clostridium coskatii. 
In one particular embodiment, the microorganism is Clostridium autoethanogenum DSM23693.
In a second aspect, the invention provides a method for the production of a carboxydotrophic acetogenic microorganism which is adapted to produce one or more product and a reduced amount or substantially no 2,3 butanediol and/or a precursor thereof upon fermentation of a substrate comprising carbon monoxide, the method comprising genetically modifying a carboxydotrophic acetogenic parental microorganism to disrupt the 2,3-butanediol biosynthesis pathway.
In one embodiment, the method results in increased production of the one or more product compared to a parental microorganism.
In one particular embodiment, the invention provides a method for the production of a carboxydotrophic acetogenic microorganism which is adapted to produce ethanol as the main product and a reduced amount or substantially no 2,3 butanediol and/or a precursor thereof upon fermentation of a substrate comprising carbon monoxide, the method comprising genetically modifying a carboxydotrophic acetogenic parental microorganism to disrupt the 2,3-butanediol biosynthesis pathway.
The invention also provides microorganisms made by a method of the second aspect.
In one embodiment, the method comprises introducing to the parental microorganism one or more genetic modifications which disrupt one or more genes encoding one or more enzymes capable of converting pyruvate to acetolactate. In one embodiment, the one or more enzymes capable of converting pyruvate to acetolactate is an acetolactate synthase (alsS).
In one embodiment, the method comprises introducing to the parental microorganism one or more genetic modifications which disrupt one or more genes encoding one or more enzymes capable of converting acetolactate to acetoin. In one embodiment, the one or more enzymes capable of converting acetolactate to acetoin is an acetolactate decarboxylase (budA).
In one embodiment, the method comprises introducing to the parental microorganism one or more genetic modifications which disrupt one or more genes encoding one or more enzymes capable of converting acetoin to 2,3-butanediol. In one embodiment, the one or more enzymes capable of converting acetoin to 2,3-butanediol is chosen from a 2,3-butanediol dehydrogenase (2,3bdh), an acetoin reductase, a primary:secondary alcohol dehydrogenase.
In one embodiment, the method comprises introducing to the parental microorganism one or more genetic modifications which disrupt a combination of two or more of the genes encoding an an enzyme capable of converting pyruvate to acetolactate, acetolactate to acetoin, and/or acetoin to 2,3-butanediol.
In one embodiment, the method comprises introducing to the parental microorganism one or more genetic modifications which disrupts one or more of the genes encoding one or more acetolactate synthase (alsS), acetolactate decarboxylase (BudA) and 2,3-Butanediol dehydrogenase (2,3 bdh).
In one embodiment, the method comprises introducing a genetic modification which disrupts the activity of a compound required for the expression or activity of one or more of the above enzymes.
In one embodiment, the method comprises introducing a genetic modification which increases the expression or activity of one or more compounds which inhibit the expression or activity of one or more of the above enzymes.
In a third aspect, the invention provides a method for the production of one or more product. In one embodiment, the method is for the production of one or more of ethanol, formate, lactate, pyruvate, succinate, valine, leucine, isoleucine, acetolactate, malate, fumerate, 2-oxogluterate, citrate.
In one particular embodiment, the invention provides a method for the production of one or more products (in one embodiment including ethanol and one or more other products) by microbial fermentation comprising fermenting a substrate comprising CO using one or more microorganism of the first aspect of the invention and/or made by the method of the second aspect of the invention. In one embodiment, the one or more other products are chosen from the group consisting succinate, lactate, formate, valine, leucine, pyruvate, isoleucine, acetolactate, malate, fumerate, 2-oxogluterate, citrate.
The invention also provides a method for reducing the total atmospheric carbon emissions from an industrial process.
In one embodiment the method comprises the steps of:

    (a) providing a substrate comprising CO to a bioreactor containing a culture of one or more microorganism of the first aspect of the invention and/or made by a method of the second aspect of the invention; and
    (b) anaerobically fermenting the culture in the bioreactor to produce one or more of the abovementioned products, preferably including ethanol.


In another embodiment the method comprises the steps of:

    capturing CO-containing gas produced as a result of the industrial process, before the gas is released into the atmosphere;
    the anaerobic fermentation of the CO-containing gas to produce one or more of the above mentioned products, preferably including ethanol, by a culture containing one or more microorganism of the first aspect of the invention and/or made by the method of the second aspect of the invention.


In particular embodiments of the method aspects, the microorganism is maintained in an aqueous culture medium.
In particular embodiments of the method aspects, the fermentation of the substrate takes place in a bioreactor.
Preferably, the substrate comprising CO is a gaseous substrate comprising CO. In one embodiment, the substrate comprises an industrial waste gas. In certain embodiments, the gas is steel mill waste gas or syngas.
In one embodiment, the substrate will typically contain a major proportion of CO, such as at least about 20% to about 100% CO by volume, from 20% to 70% CO by volume, from 30% to 60% CO by volume, and from 40% to 55% CO by volume. In particular embodiments, the substrate comprises about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO, or about 55% CO, or about 60% CO by volume.
While it is not necessary for the substrate to contain any hydrogen, the presence of H2 should not be detrimental to product formation in accordance with methods of the invention. In particular embodiments, the presence of hydrogen results in an improved overall efficiency of alcohol production. For example, in particular embodiments, the substrate may comprise an approx 2:1, or 1:1, or 1:2 ratio of 117:CO. In one embodiment the substrate comprises about 30% or less H2 by volume, 20% or less H2 by volume, about 15% or less H2 by volume or about 10% or less II2 by volume. In other embodiments, the substrate stream comprises low concentrations of H2, for example, less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or is substantially hydrogen free. The substrate may also contain some CO, for example, such as about 1% to about 80% CO2 by volume, or 1% to about 30% CO2 by volume.
In certain embodiments the methods further comprise the step of recovering the one or more products from the fermentation broth. In one embodiment, ethanol is recovered from the fermentation broth. In one embodiment, one or more other products are recovered from the fermentation broth including formate, lactate, pyruvate, succinate, valine, leucine, isoleucine, acetolactate, malate, fumerate, citrate and 2-oxogluterate.
In a fourth aspect, the invention provides one or more product when produced by a method of the third aspect. In one embodiment, the one or more products are chosen from the group consisting of ethanol, formate, lactate, pyruvate, succinate, valine, leucine, isoleucine, acetolactate, malate, fumerate, citrate and 2-oxogluterate. In one particular embodiment, the one or more products at least comprises ethanol.
In a fifth aspect, the invention provides a carboxydotrophic acetogenic microorganism in which one or more non-essential gene has been disrupted compared to a parental microorganism.
In a sixth aspect, the invention provides a method of producing a carboxydotrophic acetogenic microorganism in which one or more non-essential gene has been disrupted, the method comprising genetically modifying one or more non-essential gene in a parental microorganism.
The invention also provides microorganisms made by the methods of the sixth aspect.
In one embodiment, the one or more non-essential gene is a gene encoding an enzyme that converts acetolactate to acetoin and/or encoding an enzyme that converts acetoin to 2,3 Butanediol. In one embodiment, the enzymes are as herein described.
In certain embodiments the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium coskatii Butyribacterium limosum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii, and Thermoanaerobacter kiuvi. 
In one particular embodiment, the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei. In another embodiment, the group also comprises Clostridium coskatii. 
In one particular embodiment, the microorganism is Clostridium autoethanogenum DSM23693.
In a seventh aspect, the invention provides a method for the production of one or more product by microbial fermentation using one or more microorganism of the fifth aspect and/or made by a method of the sixth aspect.
In one particular embodiment, the invention provides a method for the production of ethanol and one or more other products by microbial fermentation comprising fermenting a substrate comprising CO using one or more microorganism of the fifth aspect and/or made by a method of the sixth aspect.
In one embodiment the method comprises the steps of:

    (a) providing a substrate comprising CO to a bioreactor containing a culture of one or more microorganism of the fifth aspect and/or made by a method of the sixth aspect; and
    (b) anaerobically fermenting the culture in the bioreactor to produce one or more products.


In another embodiment the method comprises the steps of:

    (a) capturing CO-containing gas produced as a result of the industrial process, before the gas is released into the atmosphere;
    b) the anaerobic fermentation of the CO-containing gas to produce one or more products by a culture containing one or more microorganism of the fifth aspect and/or made by a method of the sixth aspect.


In one embodiment, the one or more product is as herein described.
In one embodiment, the substrate comprising CO is as herein described.
The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.



BRIEF DESCRIPTION OF THE FIGURES
These and other aspects of the present invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only, with reference to the accompanying figures, in which:
FIG. 1a shows the metabolic pathway from CO in 2,3 butanediol-producing carboxydotrophic aceotgens (for example C. autoethanogenum DSM23693).
FIG. 1b illustrates the effects of knocking out the 2,3 butanediol biosynthesis pathway in 2,3-butanediol producing carboxydotrophic actogens with redistribution of carbon flux towards ethanol and shows production of new products, for example, succinate, 2-oxogluterate, formate, valine, leucine from CO.
FIG. 2 shows the budA gene and its 5′ and 3′ flanking regions on C. autoethanogenum DSM23693 genome. Also indicated are the primers used for PCR amplification and subsequent cloning of the flanking fragments in pMTL85141 plasmid.
FIG. 3 shows an exemplanary pMTL85141-budA-ko plasmid harbouring the 5′ and 3′ budA gene flanking DNA fragments separated by a lacZ gene for budA gene knockout in C. autoethanogenum DSM23693.
FIG. 4 shows an exemplary methylation plasmid of use in the invention
FIG. 5 shows a graphical presentation of genomic region of C. autoethanogenum DSM23693 following budA gene knockout and also indicates the position of primers used for screening C. autoethanogenum DSM23693 budA gene knockouts and the expected size of PCR products from wildtype C. autoethanogenum DSM23693 and its corresponding budA gene knockout. FIG. 5 also shows an agarose gel electrophoresis image of PCR screening of C. autoethanogenum DSM23693 budA gene knockouts. Lane 1 and 9 show GeneRulerTM 1 kb Plus DNA Ladder. Lane 2-6 shows PCR amplication of budA target region from genomic DNA isolated from wildtype C. autoethanogenum DSM23693 (+ve, 2.7 kb) and six potential C. autoethanogenum DSM23693 budA gene knockouts (1-6, 2.2 kb) with primers Og09 and Og12r. Lane 10-16 shows PCR with genomic DNA isolated from wildtype (+ve) C. autoethanogenum DSM23693 and six potential C. autoethanogenum DSM23693 budA gene knockouts with primers Og44f and Og45r specific to 273 bp internal region of budA gene (*).
FIG. 6: PCR confirmation of RAM insertion in C. autoethanogenum DSM23693 budA and 2,3bdh genes using primers Og44f/Og45r and Og42f/Og43r.
FIG. 7 shows the rate of conversion of acetoin to butanediol by C. autoethanogenum DSM23693 and Δ2,3bdh ClosTron mutant in fermentation.



BRIEF DESCRIPTION OF SEQUENCE LISTING
This specification is accompanied by a sequence listing in which the following sequences are listed.

    Seq. ID 1: Nucleotide sequence of the nucleotide sequence of C. autoethanogenum DSM23693 budA gene.
    Seq. ID 2: Amino acid sequence of C. autoethanogenum DSM23693 budA protein.
    Seq. ID 3: Nucleotide sequence of the 5′ flanking region of C. autoethanogenum DSM23693 budA gene.
    Seq. ID 4: Nucleotide sequence of 3′ flanking sequence of budA gene
    Seq ID 5 to 8 and 10 and 11: Are described in table 1 herein after.
    Seq. ID 9: Nucleotide sequence of E. coli-Clostridium shuttle vector—plasmid pMTL85141
    Seq. ID. 12: Nucleotide sequencing results of pMTL85141-budA-ko which demonstrates that the flanking DNA fragments found on the plasmid were free of mutations.
    Seq ID 13: 16s rRNA gene of C. autoethanogenum (Y18178, GI:7271109)
    Seq ID 14: 16s rRNA gene of colony 1 of potential budA knockout transformant of C. autoethanogenum DSM23693: (93%) identity
    Seq. ID 15: 16s rRNA gene of colony 2 of potential budA knockout transformant of C. autoethanogenum DSM23693: (94%)
    Seq. ID 16: 16s rRNA gene of colony 3 of potential budA knockout transformant of C. autoethanogenum DSM23693: (95%)
    Seq. ID 17: 16s rRNA gene of colony 4 of potential budA knockout transformant of C. autoethanogenum DSM23693: (93%).
    Seq. ID 18: 16s rRNA gene of colony 5 of potential budA knockout transformant of C. autoethanogenum DSM23693: (94%).
    Seq. ID 19: 16s rRNA gene of colony 6 of potential budA knockout transformant of C. autoethanogenum DSM23693: (92%).
    Seq ID 20. Nucleotide sequencing result of Colony 1 PCR product of potential budA knockout transformant of C. autoethanogenum DSM23693 with primer Og09f. (92%)
    Seq ID 21. Nucleotide sequencing result of Colony 1 PCR product of potential budA knockout transformant of C. autoethanogenum DSM23693 with primer Og12r. (92%)
    Seq ID 22. Nucleotide sequencing result of Colony 3 PCR product of potential budA knockout transfbrmant of C. autoethanogenum DSM23693 with primer Og12r. (92%)
    Seq ID 23. Nucleotide sequencing result of Colony 4 PCR product of potential budA knockout transformant of C. autoethanogenum DSM23693 with primer Og12r. (92%)
    Seq ID 24. Nucleotide sequencing result of Colony 5 PCR product of potential budA knockout transformant of C. autoethanogenum DSM23693 with primer Og12r.
    Seq ID 25. Nucleotide sequencing result of Colony 6 PCR product of potential budA knockout transformant of C. autoethanogenum DSM23693 with primer Og09f.
    Seq ID 26. Nucleotide sequencing result of C. autoethanogenum DSM23693 budA target region from clone 6 with primer Og12r.
    Seq ID 27 and 28: are described in table 4 herein after
    Seq 29 and 30: are described in table 4 herein after
    SEQ ID 31: nucleotide sequence of novel methyltransferase gene fused with an inducible lac promoter.
    SEQ ID 32: protein sequence of a novel methyltransferase.
    SEQ ID 33: nucleotide sequence of plasmid pGS20.
    SEQ_ID NO 34: Amino acid sequence of a novel alcohol dehydrogenase from C. autoethanogeum, C. ljungdahlii and C. ragsdalei. 
    SEQ_ID NO 35: Nucleic acid sequence of novel alcohol dehydrogenase gene from C. autoethanogeum. 
    SEQ_ID NO 36: Nucleic acid sequence of novel alcohol dehydrogenase gene from C. ljungdahlii. 
    SEQ ID NO 37: Nucleic acid sequence of novel alcohol dehydrogenase gene from C. ragsdalei. 
    Seq. ID. 38: Nucleotide sequence of Malic enzyme 1 of C. autoethanogenum 
    Seq. ID. 39: Amino acid sequence of Malic enzyme 1 of C. autoethanogenum: 
    Seq. ID. 40: Nucleotide sequence of Malic enzyme 2 of C. autoethanogenum 
    Seq. ID. 41: Amino acid sequence of Malic enzyme 2 of C. autoethanogenum 
    Seq. ID. 42: Nucleotide sequence of Malate dehydrogenase of C. autoethanogenum 
    Seq. ID. 43: Amino acid sequence of Malate dehydrogenase of C. autoethanogenum. 
    Seq. ID. 44: Nucleotide sequence of Pyruvate phosphate dikinase of C. autoethanogenum. 
    Seq. ID. 45: Amino acid sequence of Pyruvate phosphate dikinase of C. autoethanogenum. 
    Seq. ID. 46: Nucleotide sequence of Pyruvate carboxylase of C. autoethanogenum. 
    Seq. ID. 47: Amino acid sequence of Pyruvate carboxylase of C. autoethanogenum 
    Seq. ID. 48: Nucleotide sequence of PEP carboxykinase of C. autoethanogenum. 
    Seq. ID. 49: Amino acid sequence of PEP carboxykinase of C. autoethanogenum 
    Seq. ID. 50: Nucleotide sequence of Fumarate hydratase subunit A of C. autoethanogenum 
    Seq. ID. 51: Amino acid sequence of Fumarate hydratase subunit A of C. autoethanogenum. 
    Seq. ID. 52: Nucleotide sequence of Fumarate hydratase subunit B of C. autoethanogenum 
    Seq. ID. 53: Amino acid sequence of Fumarate hydratase subunit B of C. autoethanogenum. 
    Seq. ID. 54: Nucleotide sequence of Fumarate reductase 1 of C. autoethanogenum 
    Seq. ID. 55: Amino acid sequence of Fumarate reductase 1 of C. autoethanogenum. 
    Seq. ID. 56: Nucleotide sequence of Fumarate reductase 2 of C. autoethanogenum. 
    Seq. ID. 57: Amino acid sequence of Fumarate reductase 2 of C. autoethanogenum 
    Seq. ID. 58: Nucleotide sequence of Fumarate reductase 3 of C. autoethanogenum. 
    Seq. ID. 59: Amino acid sequence of Fumarate reductase 3 of C. autoethanogenum 
    Seq. ID. 60: Nucleotide sequence of Malic enzyme 1 of C. ragsdalei. 
    Seq. ID. 61: Amino acid sequence of Malic enzyme 1 of C. ragsdaiei. 
    Seq. ID. 62: Nucleotide sequence of Malate dehydrogenase of C. ragsdalei 
    Seq. ID. 63: Amino acid sequence of Malate dehydrogenase of C. ragsdalei. 
    Seq. ID. 64: Nucleotide sequence of Pyruvate phosphate dikinase of C. ragsdalei. 
    Seq. ID. 65: Amino acid sequence of Pyruvate phosphate dikinase of C. ragsdalei. 
    Seq. ID. 66: Nucleotide sequence of Pyruvate carboxylase of C. ragsdalei. 
    Seq. ID. 67: Amino acid sequence of Pyruvate carboxylase of C. ragsdalei 
    Seq. ID. 68: Nucleotide sequence of PEP carboxykinase of C. ragsdalei. 
    Seq. ID. 69: Amino acid sequence of PEP carboxykinase of C. ragsdalei 
    Seq. ID. 70: Nucleotide sequence of Fumarate hydratase subunit A of C. ragsdalei 
    Seq. ID. 71: Amino acid sequence of Fumarate hydratase subunit A of C. ragsdaiei 
    Seq. ID. 72: Nucleotide sequence of Fumarate hydratase subunit B of C. ragsdalei. 
    Seq. ID. 73: Amino acid sequence of Fumarate hydratase subunit B of C ragsdalei 
    Seq. ID. 74: Nucleotide sequence of Fumarate reductase 1 of C. ragsdalei. 
    Seq. ID. 75: Amino acid sequence of :Fumarate reductase 1 of C. ragsdalei 
    Seq. ID. 76: Nucleotide sequence of Fumarate reductase 2 of C. ragsdalei 
    Seq. ID. 77: Amino acid sequence of Fumarate reductase 2 of C. ragsdalei 
    Seq. ID 78: 5′ upstream sequence or homology arm of Clostridium ljungdahlii budA gene.
    Seq. ID 79: 3′ downstream sequence or homology arm of Clostridium ljungdahlii budA gene
    Seq. ID 80: 5′ upstream sequence or homology arm of Clostridium ragsdalei budA gene
    Seq. ID 81: 3′ downstream sequence or homology arm of Clostridium ragsdalei budA gene
    Seq ID 82: nucleotide sequence of ClosTron targeting region in C. autoethanogenum DSM23693 budA
    Seq ID 83 nucleotide sequence of ClosTron targeting region in C. autoethanogenum DSM23693 2.3bdh.
    Seq ID 84: oligonucleotide Og42f used for screening Δ2,3bdh ClosTron mutants.
    Seq ID 85: oligonucleotide Og43r used for screening Δ12,3bdh ClosTron mutants.
    Seq. ID 86: Nucleotide sequence of the 16s rRNA PCR product amplified from C. autoethanogenum DSM23693 Δ2, 3bdh ClosTron clone 2 obtained using primer fD1.
    Seq ID 87: Nucleotide sequence of the 16s rRNA PCR product amplified from C. autoethanogenum DSM23693 Δ2, 3bdh ClosTron clone 2 obtained using primer rP2.
    Seq. ID 88: Nucleotide sequence of the 16s rRNA PCR product of C. autoethanogenum DSM23693 Δ2,3bdh ClosTron clone 4 obtained using primer fD1
    Seq ID 89: Nucleotide sequence of the 16s rRNA PCR product of C. autoethanogenum DSM23693 Δ2,3bdh ClosTron clone 4 obtained using primer rP2
    Seq. ID 90: Nucleotide sequence of the 16s rRNA PCR product of C. autoethanogenum DSM23693 ΔbudA ClosTron clone 1 obtained using primer fD1.
    Seq ID 91: Nucleotide sequence of the 16s rRNA PCR product of C. autoethanogenum DSM23693 ΔbudA ClosTron clone 1 obtained using primer rP2.
    Seq. ID 92: Nucleotide sequence of the 16s rRNA PCR product of C. autoethanogenum DSM23693 ΔbudA ClosTron clone 3 obtained using primer fD1.
    Seq ID and 93: Nucleotide sequence of the 16s rRNA PCR product of C. autoethanogenum DSM23693 ΔbudA ClosTron clone 3 obtained using primer rP2.
    Seq ID 94 nucleotide sequence of 5′ homology arm of C. autoethanogenum DSM23693 2,3bdh gene.
    Seq ID 95: nucleotide sequence of 3′ homology arm of C. autoethanogenum DSM23693 2,3bdh gene.
    Seq. ID 96 and 97: the primers used to amplify 5′ homology arm of C. autoethanogenum DSM23693 2,3bdh gene.
    Seq. ID 98 and 99: the primers used to amplify 3′ homology arm of C. autoethanogenum DSM23693 2,3bdh gene.
    Seq. ID 100 and 101: flanking primers that can be used to confirm knockout of C. autoethanogenum DSM23693 2,3bdh gene.
    Seq ID 102: nucleic acid sequence of 5′ homology arm of C. autoethanogenum D5N423693 SecAdh gene
    Seq ID 103: nucleic acid sequence of 3′ homology arm of C. autoethanogenum DSM23693 SecAdh gene.
    Seq. ID 104 and 105: primers used to amplify 5′ homology arm of C. autoethanogenum DSM23693 2,3bdh gene.
    Seq. ID 106 and 107 primers used to amplify 3′ homology arm of C. autoethanogenum DSM23693 2,3bdh gene.
    Seq. ID 108 and 109: primers that can be used to confirm knockout of C. autoethanogenum DSM23693 SecAdh gene.
    Seq ID 110: nucleotide sequence of group II intron targeting cassette for C. autoethanogenum DSM23693 SecAdh gene.
    Seq. ID 111 and 112: flanking primers that can be used to confirm insertional inactivation of C. autoethanogenum DSM23693 SecAdh gene.
    Seq ID 113: nucleotide sequence of 5′ homology arm of C. autoethanogenum DSM23693 alsS gene.
    Seq ID 114: nucleotide sequence of 3′ homology arm of C. autoethanogenum DSM23693 alsS gene.
    Seq. ID 115 and 116: sequences of primers used to amplify 5′ homology arm of C. autoethanogenum DSM23693 alsS gene.
    Seq. ID 117 and 118: sequences of primers used to amplify 3′ homology arm of C. autoethanogenum DSM23693 alsS gene.
    Seq. ID 119 and 120: sequences of flanking primers that can be used to confirm knockout of C. autoethanogenum DSM23693 alsS gene.
    Seq ID 120: nucleotide sequence of 5′ homology arm of C. autoethanogenum DSM23693 ilvC gene.
    Seq ID 121: nucleic acid sequence of 3′ homology arm of C. autoethanogenum DSM23693 ilvC gene.
    Seq. ID 123 and 124: sequences of primers used to amplify 5′ homology arm of C. autoethanogenum DSM23693 ilvC gene.
    Seq. ID 125 and 126: sequences of primers used to amplify 3′ homology arm of C. autoethanogenum DSM23693 ilvC gene.
    Seq. ID 127 and 128: sequences of flanking primers that can be used to confirm knockout of C. autoethanogenum DSM23693 ilvC gene.
    Seq ID 129: nucleotide sequence of 5′ homology arm of C. autoethanogenum DSM23693 ilvl gene.
    Seq ID 130: nucleotide sequence of 3′ (Seq. ID 130) homology arm of C. autoethanogenum DSM23693 ilvl gene.
    Seq. ID 131 and 132: sequences of primers used to amplify 5′ homology arm of C. autoethanogenum DSM23693 ilvl gene.
    Seq. ID 133 and 134: sequences of primers used to amplify 3′ homology arm of C. autoethanogenum DSM23693 ilvl gene.
    Seq. ID 135 and 136: sequences of flanking primers that can be used to confirm knockout of C. autoethanogenum DSM23693 ilvl gene.
    Seq ID 137 nucleotide sequence of 5′ homology arm of C. autoethanogenum DS14123693 ilvB gene.
    Seq ID 138: nucleotide sequence of 3′ homology arm of C. autoethanogenum DSM23693 ilvB gene.
    Seq. ID 139 and 140: sequences of primers used to amplify 5′ homology arm of C. autoethanogenum DSM23693 gene.
    Seq. ID 141 and 142: sequences of primers used to amplify 3′ homology arm of C. autoethanogenum DSM23693 ilvB gene.
    Seq. ID 143 and 144: sequences of flanking primers that can be used to confirm knockout of C. autoethanogenum DSM23693 livB gene.
    Seq ID 145: example ClosTron intron targeting nucleotide sequence of alsS
    Seq ID 146: example ClosTron intron targeting nucleotide sequence of ilvC
    Seq ID 147: example ClosTron intron targeting nucleotide sequence of ilvl
    Seq ID 148: example ClosTron intron targeting nucleotide sequence of ilvB
    Seq ID 149 and 150: oligonucleotides that can be used to screen alsS ClosTron mutants
    Seq ID 151 and 152: oligonucleotides that can be used to screen ilvC ClosTron mutants
    Seq ID 153 and 154: oligonucleotides that can be used to screen ilvl ClosTron mutants
    Seq ID 155 and 156: oligonucleotides that can be used to screen ilvB ClosTron mutants.


Standard IUPAC abbreviations are used for all sequences, see http://en.m.wikipedia.org/wiki/Nucleic_acid_notation#section_1. By way of example:

    
    
        A Adenosine
        C Cytidine
        G Guanosine
        T Thymidine
        W A or T
        S C or G
        M A or C
        K G or T
        R A or G
        Y C or T
        B C, G or T
        D A, G or T
        H A, C or T
        V A, C or G
        N or—any base (not a gap), A, C, G, T
    
    


DETAILED DESCRIPTION OF THE INVENTION
The following is a description of the present invention, including preferred embodiments thereof, given in general terms. The invention is further elucidated from the disclosure given under the heading “Examples” herein below, which provides experimental data supporting the invention, specific examples of various aspects of the invention, and means of performing the invention.
The invention provides microorganisms capable of producing one or more products by fermentation of a substrate comprising CO. In one particular embodiment, the invention provides microorganisms capable of producing ethanol or, ethanol and one or more other products, by fermentation of a substrate comprising CO. The recombinant microorganism produces at least a reduced amount of 2,3 butanediol and/or a precursor thereof compared to a parental microorganism. In one embodiment the microorganism produces substantially no 2,3 butanediol or a precursor thereof compared to a parental microorganism.
Through various gene knockout studies, the inventors have surprisingly identified that if the 2,3-butanediol biosynthesis pathway is disrupted in a carboxydotrophic acetogenic microorganism, the microorganism is able to produce increased levels of thrmate, lactate, succinate, 2-oxogluterate, valine, leucine, isoleucine and ethanol, as compared to a parental microorganism. The inventors also believe that the microorganisms produce increased levels of pyruvate and TCA cycle intermediate compounds acetolactate, malate, fumarate, citrate as these are precursors of succinate, 2-oxogluterate and valine, leucine and isoleucine production. This has a number of significant advantages. One primary advantage is an increase in the efficiency of ethanol production including higher levels of ethanol produced. Without wanting to be bound by any particular theoty, the inventors believe that the increased levels of valine, leucine, formate, lactate and pyruvate, result in more of these chemicals being available to the microorganisms to feed ethanol production. In addition, fermentation broths must often be supplemented with amino acids and other chemicals to ensure the viability and production efficiency of the microorganisms during fermentation. The production of valine, leucine, formate, lactate and pyruvate by a recombinant microorganism of the invention obviates the need to supplement the fermentation broth with these chemicals, which can result in cost savings. Further, the reduction or removal of 2,3-butanediol production in the microorganisms of the invention has advantages. 2,3-butanediol can be toxic to microorganisms and thus may have a negative effect on fermentation and growth. Reducing or removing 2,3-butanediol from the fermentation broth also allows for easier recovery of ethanol from the broth; typically both ethanol and 2,3-butanediol must be recovered together and then separated in a subsequent step. 2,3-butanediol is also a source for potential microbial contamination in a fermenter as it is a substrate for many undesirable organisms. In addition, succinate, 2-oxogluterate, formate, lactate, pyruvate, leucine and isoleucine have independent economic value as they may be used in a number of commercial processes and as intermediate compounds in the production of downstream chemical products.
The inventor's have for the first time demonstrated the disruption or knock out of a non-essential gene in a carboxydotrophic acetogenic microorganism. Accordingly, in another aspect, the invention also provides carboxydotrophic acetogenic microorganisms in which one or more non-essential gene has been disrupted compared to a parental microorganism, along with methods of producing such microorganisms and methods of using these microorganisms. A “non-essential” gene is one which encodes a protein which is not necessary for the survival of a microorganism, such that the microorganism can survive without supplementation of the protein. Examples of non-essential genes include those encoding acetolactate decarboxylase and 2,3 butanediol dehydrogenase. Skilled persons will be able to identify non-essential genes using standard techniques in the art, including recombinant techniques to disrupt genes (as described herein) along with standard assays to test whether such genetic modifications have an effect on the survival of the microorganisms.
While the description of the invention herein after focuses on disruption of the 2,3-butanediol biosynthesis pathway by genetic modification, it should be appreciated that microorganisms of the invention may also include one or more additional genetic modifications if desired (including disruption of one or more non-essential gene not associated with the 2,3-butanediol biosynthesis pathway). In the case of the aspect of the invention relating to disruption of non-essential genes it should be appreciated that genetic modifications in genes encoding enzymes other than in the 2,3-butanediol pathway is encompassed.
In addition, while the description hereinafter may focus on the production and recovery of ethanol as a main product, it should be appreciated that the invention may be used to increase the level of production of one or more product other than ethanol or in addition to ethanol.
Definitions
As referred to herein, a “fermentation broth” is a culture medium comprising at least a nutrient media and bacterial cells.
As referred to herein, a shuttle microorganism is a microorganism in which a methyltransferase enzyme is expressed and is distinct from the destination microorganism.
As referred to herein, a destination microorganism is a microorganism in which the genes included on an expression construct/vector are expressed and is distinct from the shuttle microorganism.
The term “main fermentation product” is intended to mean the one fermentation product which is produced in the highest concentration and/or yield.
The terms “increasing the efficiency”, “increased efficiency” and the like, when used in relation to a fermentation process, include, but are not limited to, increasing one or more of the rate of growth of microorganisms catalysing the fermentation, the growth and/or product production rate, the volume of desired product (such as alcohols) produced per volume of substrate consumed, the rate of production or level of production of the desired product, and the relative proportion of the desired product rodt ced compared with other by-products of the fermentation.
The phrase “substrate comprising carbon monoxide” and like terms should be understood to include any substrate in which carbon monoxide is available to one or more strains of bacteria for growth and/or fermentation, for example.
The phrase “gaseous substrate comprising carbon monoxide” and like phrases and terms includes any gas which contains a level of carbon monoxide. In certain embodiments the substrate contains at least about 20% to about 100% CO by volume, from 20% to 70% CO by volume, from 30% to 60% CO by volume, and from 40% to 55% CO by volume. In particular embodiments, the substrate comprises about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO, or about 55% CO, or about 60% CO by volume.
While it is not necessary for the substrate to contain any hydrogen, the presence of H2 should not be detrimental to product formation in accordance with methods of the invention. In particular embodiments, the presence of hydrogen results in an improved overall efficiency of alcohol production. For example, in particular embodiments, the substrate may comprise an approx 2:1, or 1:1, or 1:2 ratio of H2:CO. In one embodiment the substrate comprises about 30% or less H2 by volume, 20% or less H2 by volume, about 15% or less H2 by volume or about 10% or less H2 by volume. In other embodiments, the substrate stream comprises low concentrations of H2, for example, less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or is substantially hydrogen free. The substrate may also contain some CO2 for example, such as about 1% to about 80% CO2 by volume, or 1% to about 30% CO2 by volume. In one embodiment the substrate comprises less than or equal to about 20% CO2 by volume. In particular embodiments the substrate comprises less than or equal to about 15% CO2 by volume, less than or equal to about 10% CO2 by volume, less than or equal to about 5% CO2 by volume or substantially no CO2.
In the description which follows, embodiments of the invention are described in terms of delivering and fermenting a “gaseous substrate containing CO”. However, it should be appreciated that the gaseous substrate may be provided in alternative forms. For example, the gaseous substrate containing CO may be provided dissolved in a liquid. Essentially, a liquid is saturated with a carbon monoxide containing gas and then that liquid is added to the bioreactor. This may be achieved using standard methodology. By way of example, a microbubble dispersion generator (Hensirisak et. al. Scale-up of microbubble dispersion generator fur aerobic fermentation; Applied Biochemistry and Biotechnology Volume 101, Number 3/October, 2002) could be used. By way of further example, the gaseous substrate containing CO may be adsorbed onto a solid support. Such alternative methods are encompassed by use of the term “substrate containing CO” and the like.
In particular embodiments of the invention, the CO-containing gaseous substrate is an industrial off or waste gas. “Industrial waste or off gases” should be taken broadly to include any gases comprising CO produced by an industrial process and include gases produced as a result of ferrous metal products manufacturing, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, gasification of biomass, electric power production, carbon black production, and coke manufacturing. Further examples may be provided elsewhere herein.
Unless the context requires otherwise, the phrases “fermenting”, “fermentation process” or “fermentation reaction” and the like, as used herein, are intended to encompass both the growth phase and product biosynthesis phase of the process. As will be described further herein, in sonic embodiments the bioreactor may comprise a first growth reactor and a second fermentation reactor. As such, the addition of metals or compositions to a fermentation reaction should be understood to include addition to either or both of these reactors.
The term “bioreactor” includes a fermentation device consisting of one or more vessels and/or towers or piping arrangement, which includes the Continuous Stirred Tank Reactor (CSTR), immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, Static Mixer, or other vessel or other device suitable for gas-liquid contact. As is described herein after, in some embodiments the bioreactor may comprise a first growth reactor and a second fermentation reactor. As such, when referring to the addition of substrate to the bioreactor or fermentation reaction it should be understood to include addition to either or both of these reactors where appropriate.
When used in relation to the products of fermentation in accordance with the invention “one or more products” and like phrases is intended to include ethanol, succinate, pyruvate, lactate, valine, formate, isoleucine, and leucine, for example. In one embodiment, “one or more products” may also include one or more of acetolactate, malate, fumarate, citrate, and 2-oxogluterate. It should be appreciated that the methods of the invention are applicable to methods intended for the production and recovery of ethanol (alone or in combination with other products) or the production and recovery of products other than ethanol.
The term “acetate” includes both acetate salt alone and a mixture of molecular or free acetic acid and acetate salt, such as the mixture of acetate salt and free acetic acid present in a fermentation broth as described herein, The ratio of molecular acetic acid to acetate in the fermentation broth is dependent upon the pH of the system. The terms succinate, pyruvate, lactate, formate, acetolactate, malate, fumarate, citrate and 2-oxogluterate should be construed similarly.
Unless the context requires otherwise, reference to any compound herein which may exist in one or more isomeric forms (for example, D, L, meso, S, R, cis or trans form) should be taken generally to include refrence to any one or more such isomers of the compound. For example, reference to “acetoin” should be taken to include reference to either or both the D and L isomers thereof.
“Exogenous nucleic acids” are nucleic acids which originate outside of the microorganism to which they are introduced. Exogenous nucleic acids may be derived from any appropriate source, including, but not limited to, the microorganism to which they are to be introduced, strains or species of organisms which differ from the organism to which they are to be introduced, or they may be artificially or recombinantly created. The exogenous nucleic acid may be adapted to integrate into the genome of the microorganism to which it is to be introduced or to remain in an extra-chromosomal state.
The “2,3-butanediol biosynthesis pathway” is a pathway of reactions including the conversion of pyruvate into acetolactate, acetotatate into acetoin, and acetoin into 2,3-butanediol.
As used herein, “disrupt the 2,3-butanediol biosynthesis pathway” and like phrases, is intended to mean that the production of 2,3-butanediol is reduced, or in one embodiment substantially eliminated.
A “precursor of 2,3-butanediol” is intended to encompass acetoin and acetolactate.
An enzyme is “capable of converting” a first compound or substrate into a second compound or product, if in its active form it can catalyse a reaction in which at least a portion of the first compound is converted into the second compound.
Reference to “alcohol dehydrogenases” should be taken to include alcohol dehydrogenases which are capable of catalysing the conversion of ketones (such as acetoin) to secondary alcohols (such as 2,3-butanediol), or vice versa. Such alcohol dehydrogenases include secondary alcohol dehydrogenases and primary alcohol dehydrogenases. A “secondary alcohol dehydrogenase” is one which can convert ketones (such as acetoin) to secondary alcohols (such as 2,3-butanediol), or vice versa. A “primary alcohol dehydrogenase” is one which can convert aldehydes to primary alcohols, or vice versa; however, a number of primary alcohol dehydrogenases are also capable of catalysing the conversion of ketones to secondary alcohols, or vice versa. These alcohol dehydrogenases may also be referred to as “primary-secondary alcohol dehydrogenases”. Accordingly, in certain embodiments of the invention, reference to “2,3-butanediol dehydrogenase” should be taken to include reference to 2,3-butanediol dehydrogenases which may be categorised as primary, secondary or primary-secondary alcohol dehydrogenases.
A “genetic modification which disrupts” the 2,3-butanediol biosynthesis pathway or the expression or activity of one or more enzyme in accordance with the invention should be taken broadly to include any genetic modification which at least reduces the biosynthesis of 2,3-butanediol, the expression or activity of one or more enzymes or in one embodiment substantially blocks the expression or activity of one or more enzymes or substantially prevents the production of 2,3-butanediol. The phrase should be taken to include, for example: modification to a gene encoding one or more of the enzymes, including a modification to a genetic regulatory element involved in the expression of a gene; introduction of a nucleic acid which produces a protein which reduces or inhibits the activity of one or more of the enzymes, or which reduces or prevents expression of one or more of the enzymes; introduction of a nucleic acid which expresses a nucleic acid which is adapted to block expression of a gene (for example, antisense RNA, siRNA (small interfering RNA), CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)); reducing or inhibiting a protein which is required for expression or activity of one or more of the enzymes by introducing a modification to a gene encoding the protein. It should be appreciated that a protein which is required for expression or activity of one or more of the enzymes may act directly on a gene or one or more enzymes, or may act indirectly via another compound. Similarly, a protein which reduces or inhibits the activity or expression of the one or more enzymes may act directly on the gene or the one or more enzymes, or may act indirectly via another compound.
A “genetic modification” should be taken broadly and is intended to include, for example, introducing one or more exogenous nucleic acids to a microorganism, introducing a mutation to a genetic site, adding to or removing from the genome one or more nucleotides, substitution of one or more nucleotides with different nucleotides, substitution of a gene, removal of a gene, addition of a gene and the like.
A “parental microorganism” is a microorganism used to generate a recombinant microorganism of the invention. In one embodiment, the parental microorganism may be one that occurs in nature (ie a wild type microorganism) or one which has been previously modified (a genetically modified or recombinant microorganism). In embodiments of the invention relating to microorganisms which produce a reduced amount or substantially no 2,3-butanediol, the parental microorganism is one which includes a functional 2,3-butanediol pathway (including those that occur in nature or those that have been previously modified). Examples of parental microorganisms that include a functional 2,3-butanediol biosynthesis pathway include Clostridium autoethanogenum, Clostridium ljungdahiii, Clostridium ragsdaiei, Clostridium coskatii and related isolates.
A “functional” 2,3-butanediol biosynthesis pathway is one in which the microorganism can convert pyruvate to 2,3-butanediol. In one particular embodiment, the pathway includes conversion of pyruvate to acetolactate, acteolactate to aceotin, and acetoin to 2,3-butanediol. In one particular embodiment, conversion of pyruvate to acetolactate is catalysed by an acetolactate synthase, conversion of acteolactate to aceotin is catalysed by a aceotiatate decarboxylase, and conversion of acetoin to 2,3-butanediol is catalysed by a 2,3-butanediol dehydrogenase or an acetoin reductase.
The terms nucleic acid “constructs” or “vectors” and like terms should be taken broadly to include any nucleic acid (including DNA and RNA) suitable for use as a vehicle to transfer genetic material into a cell. The terms should be taken to include plasmids, viruses (including bacteriophage), cosmids and artificial chromosomes. Constructs or vectors may include one or more regulatory elements, an origin of replication, a multicloning site and/or a selectable marker, among other elements, sites and markers. In one particular embodiment, the constructs or vectors are adapted to allow for the disruption of a gene native to a parental microorganism. In another embodiment, the constructs or vectors are adapted to allow expression of one or more genes encoded by the construct or vector. Nucleic acid constructs or vectors include naked nucleic acids as well as nucleic acids formulated with one or more agents to facilitate delivery to a cell (for example, liposome-conjugated nucleic acid, an organism in which the nucleic acid is contained).
Throughout this specification exemplary sequence information is provided for enzymes applicable to the invention (for example, acetolactate synthase, aceotlactate decarboxylase, 2,3-butanediol dehydrogenase, acetoin reductase). This information is provided to identify exemplary enzymes applicable to the invention and to allow a skilled person to practise specific embodiments of the invention without undue experimentation. It should be appreciated that nucleic acid and amino acid sequences for the enzymes may differ from one microorganism to another. Accordingly, the invention should not be construed as being limited to these specific embodiments but rather to extend to the disruption of enzymes having different sequences but which are capable of catalysing the conversion of pyruvate to acetolactate, the conversion of acteolactate to aceotin, and/or the conversion of acetoin to 2,3-butanediol, Typically, such enzymes will have at least approximately 75% amino acid sequence identify to an enzyme exemplified herein. In particular embodiments, such enzymes will have at least approximately 80%, 85%, 90%, 95% or 99% sequence identify to an enzyme exemplified herein. At the nucleic acid level, genes encoding such variant enzymes will have at least approximately 75% sequence homology to a nucleic acid encoding an enzyme exemplified herein. In particular embodiments, such nucleic acidsw will have at least approximately 80%, 85%, 90%, 95% or 99% sequence homology to a nucleic acid encoding an enzyme exemplified herein.
It should also be appreciated that the variant enzyme need not have the same level of activity as an enzyme specifically exemplified herein. All that is required is that it has some level of activity in catalysing the conversion of interest. Skilled persons will readily appreciate other such enzymes, particularly in light of the information contained herein. Enzyme assays of use in assessing activities of enzymes for the 2,3-butanediol pathway include fore example the assay Voges-Proskauer testare described by Speckman and Collins (Specificity of the Westerfeld Adaptation of the Voges-Proskauer Test, 1982, Appl. Environ. Microbiol. 44: 40-43) or Dulieu and Poncelet (Spectrophotometric assay of a-acetolactate decarboxylase, 1999, Enzy and Microbiol Technol, 25, 537-42).
Microorganisms
As discussed herein before, the invention provides a recombinant microorganism capable of using carbon monoxide to produce one or more products (in one particular embodiment, ethanol as the main product) and producing a reduced amount or substantially no 2,3 butanediol and/or a precursor thereof compared to a parental microorganism. The microorganism comprises one or more genetic modifications (compared to a parental microorganism) which disrupts the 2,3-butanediol biosynthesis pathway.
As noted above, in one embodiment the microorganism produces ethanol as the main product. In one embodiment, the microorganism also produces one or more of formate, lactate, pyruvate, succinate, valine, leucine, isoleucine. In one particular embodiment, the microorganism is adapted to produce an increased amount of one or more of ethanol, formate, lactate, pyruvate, succinate, valine. leucine, isoleucine compared to a parental microorganism. In certain embodiments, the microorganism produces one or more of acetolactate, malate, citrate, fumerate, 2-oxogluterate. In one particular embodiment, the microorganism is adapted to produce an increased amount of one or more of acetolactate, malate, fumerate, 2-oxogluterate.
The one or more genetic modifications preferably disrupts the expression and/or activity of one or more enzymes capable of converting pyruvate to acetolactate, acteolactate to aceotin, acetoin to 2,3-butanediol. In certain embodiments, the one or more genetic modification disrupts the conversion of pyruvate to acetolactate only, the conversion of acetolactate to acetoin only, or the conversion of acetoin to 2,3-butanediol only. In other embodiments, the one or more genetic modifications disrupts two or three of these conversions.
In one embodiment, the one or more enzymes capable of converting pyruvate to aceotlacta e is an acetolactate synthase (alsS).
Acetolactate synthase activity is capable of converting pyruvate to acetolactate and is essential for branched-chain amino acid (including valine, leucine, isoleucine) production FIGS. 1a and 1b. One or more enzyme having acetolactate synthase activity may be expressed. in a parental microorganism. Exemplary amino acid sequence from C. autoethanogenum (AEI90719.1, AEI90730.1, .AE190731.1, AE190713.1, AEI90714.1), C. ljungdahlii (ADK15104.1, ADK115104.1, ADK15105.1 ADK15400.1, ADK15400.1), and C. ragsdalei (AEI90734.1, AEI90734.1, AEI90735.1, AEI90727.1, AEI90727.1) and respective nucleic acid sequences from C. autoethanogenum (HQ876013.1, HQ876023.1, HQ876021.1), C. ljungdahlii (CP001666.1—CLJU_c38920, CLJU_c32420, CLJU_c20420-30), and C. ragsdalei (HQ876014.1, HQ876024.1, HQ876022.1) can be obtained from GenBank. However, as noted herein before, the sequence of the gene encoding such enzymes and the amino acid sequence of the enzymes may vary from one microorganism to another.
In certain embodiments, a parental microorganism may contain more than one enzyme which is capable of converting pyruvate to acetolactate. Where a parental microorganism contains more than one enzyme which is capable of converting pyruvate to acetolactate, one or more genetic modification may be introduced such that expression and/or activity of two or more of the enzymes is disrupted. Where more than one enzyme is present in a parental microorganism, disrupting more than one such enzyme may have the effect of increasing the production of succinate, one or more TCA cycle intermediates and/or ethanol above the level that may be achieved if only a single enzyme is disrupted. Production levels may be further increased with the disruption of each additional enzyme present in the parental microorganism. While disrupting expression and/or activity of all such enzymes activity may provide some advantage in terms of production of desired products, the inventors do not contemplate it to be necessary to disrupt expression and/or activity of all such enzymes in order to gain the benefits of the invention.
In one embodiment, at least two, three, four or five enzymes capable of converting pyruvate to acetolactate are disrupted.
In embodiments of the invention where the conversion of pyruvate to acetolactate is substantially or completely blocked, growth of and fermentation by the microorganism may require supplementation with one or more amino acids, including, for example, valine, leucine and isoleucine. This can be achieved by any means which makes the amino acid(s) available to the microorganism. By way of example, one or more amino acid may be added to a culture, growth or fermentation media, to a culture of the microorganisms, and/or to a fermentation broth. In certain embodiments, the amino acid(s) may be added directly to the media or broth or added in the form of an extract, for example yeast extract.
In one embodiment, the one or more enzymes capable of converting acetolactate to acetoin is an acetolactate decarboxylase (budA).
Acetolactate, decarboxylase activity is capable of converting acetolactate, to acetoin FIGS. 1a and 1b. One or more enzyme having acetolactate decarboxylase activity may be expressed in a parental microorganism. Exemplary amino acid (AEI90717.1, ADK.13906.1, AEI90718.1) and nucleic acid (HQ876011.1, CP001666.1-CLJU_c08380, HQ876012.1) sequence information for acetolactate decarboxylase from C. autoethanogenum, C. ljungdahlii and C. ragsdalei can be obtained from GenBank. However, as noted herein before, the sequence of the gene encoding such enzymes and the amino acid sequence of the enzymes may vary from one microorganism to another.
In certain embodiments, a parental microorganism may contain more than one enzyme which is capable of converting acetolactate to acetoin. Where a parental microorganism contains more than one such enzyme, one or more genetic modification may be introduced such that expression and/or activity of two or more of the enzymes is disrupted. Were more than one such enzyme is present in a parental microorganism, disrupting more than one enzyme may have the effect of increasing the production of valine, leucine, isoleucine, ethanol, lactate, formate and succinate, andlor one or more TCA cycle intermediates above the level that may be achieved if only a single enzyme is disrupted. Production levels may be further increased with the disruption of each additional enzyme present in the parental microorganism. While disrupting expression and/or activity of all such enzymes may provide some advantage in terms of production of desired products, the inventors do not contemplate it to be necessary to disrupt expression and/or activity of all such enzymes in order to gain the benefits of the invention.
In one embodiment, the one or more enzyme capable of converting acetoin to 2,3-butanediol is chosen from the group comprising a 2,3-Butanediol dehydrogenase (2,3bdh) and an acetoin reductase.
2,3-butanediol dehydrogenase activity is capable of converting acetoin to 2,3-butanediol FIGS. 1a and 1b. Exemplary amino acid (AEI90715.1, ADK15380.1, AEI90716.1) and nucleic acid sequence (HQ876009.1, CP001666.1-CLJU_c23220, HQ876010.1) information for 2,3-butanediol dehydrogenase from C. autoethanogenum, C. ljungdahlii and C. ragsdalei can be obtained from GenBank. One or more enzyme having acetolactate synthase activity may be expressed in a parental microorganism. By way of example, the inventors have identified that C. autoethanogenum, C. ragsdalei and C. ljungdahiii include an additional primary-secondary alcohol dehydrogenase capable of converting acetoin to 2,3-butanediol. Exemplary sequence information for this enzyme is provided in SEQ ID nos 34, 35, 36, and 37. However, as noted herein before, the sequence of the gene encoding such enzymes and the amino acid sequence of the enzymes may vary from one microorganism to another.
In certain embodiments, a parental microorganism may contain more than one enzyme which is capable of converting acetoin to 2,3-butanediol. Where a parental microorganism contains more than one such enzyme, one or more genetic modification may be introduced such that expression and/or activity of two or more of the enzymes is disrupted. Where more than one such enzyme is present in a parental microorganism, disrupting more than one such enzyme may have the effect of increasing the production of leucine, isoleucine, ethanol, lactate, formate and succinate, and/or one or more TCA cycle intermediates above the level that may be achieved if only a single enzyme is disrupted. Production levels may be further increased with the disruption of each additional enzyme present in the parental microorganism. While disrupting expression and/or activity of all such enzymes may provide some advantage in terms of production of desired products, the inventors do not contemplate it to be necessary to disrupt expression and/or activity of all such enzymes in order to gain the benefits of the invention.
In one embodiment, at least two or three enzymes capable of converting acetoin to 2,3-butanediol are disrupted.
In one embodiment, the microorganism is selected from the group of acetogenic carboxydotrophic organisms comprising the species Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Acetobacterium woodii, Alkalibaculum bacchii, Moorella thermoacetica, Sporomusa ovate, Butyribacterium methylotrophicum, Blautia producta, Eubacterium limosum, Thermoanaerobacter kiuvi. 
These carboxydotrophic acetogens are defined by their ability to utilize and grow chemoautotrophically on gaseous one-carbon (C1) sources such as carbon monoxide (CO) and carbon dioxide (CO2) with carbon monoxide (CO) and/or hydrogen (H2) as energy source under anaerobic conditions forming acetyl-CoA, acetate and other products. They share the same mode of fermentation, the Wood-Ljungdahl or reductive acetyl-CoA pathway, and are defined by the presence of the enzyme set consisting of Carbon monoxide dehydrogenase (CODH), Hydrogenase, Formate dehydrogenase, Formyl-tetrahydrofolate synthetase, Methylene-tetrahydrofolate dehydrogenase, Formyl-tetrahydrofolate cyclohydrolase, Methylene-tetrahydrofotate reductase, and Carbon monoxide dehydrogenase/Acetyl-CoA synthase (CODH/ACS), which combination is characteristic and unique to this type of bacteria (Drake, Küset, Matthies, Wood, & Ljungdahl, 2006). In contrast to chemoheterotrophic growth of sugar-fermenting bacteria that convert the substrate into biomass, secondary metabolites and pyruvate from which products are formed (either via acetyl-CoA or directly), in acetogens the substrate is channelled directly into acetyl-CoA, from which products, biomass, and secondary metabolites are formed.
In a one embodiment, the microorganism is selected from a cluster of carboxydotrophic Clostridia comprising the species C. autoethanogenum, C. ljungdahlii, and “C. ragsdalei” and related isolates. These include but are not limited to strains C. autoethanogenum JAI-1T (DSM10061) (Abrini, Naveau, & Nyns, 1994), C. autoethanogenum LBS1560 (DSM19630) (WO/2009/064200), C. autoethanogenum LBS1561 (DSM23693), C. ljungdahlii PETCT (DSM13528=ATCC 55383) (Tanner, Miller, & Yang, 1993), C. ljungdahlii ERI-2 (ATCC 55380) (U.S. Pat. No. 5,593,886), C. ljungdahlii C-01 (ATCC 55988) (U.S. Pat. No. 6,368,819), C. ljungdahlii O-52 (ATCC 55989) (U.S. Pat. No. 6,368,819), or “C. ragsdalei P11T” (ATCC BAA-622) (WO 2008/028055), and related isolates such as “C. coskatii” (US patent 2011/0229947), and mutant strains thereof such as C. ljungdahlii OTA-1 (Tirado-Acevedo O. Production of Bioethanol from Synthesis Gas Using Clostridium ljungdahlii. PhD thesis, North Carolina State University, 2010).
These strains form a suhcluster within the Clostridial rRNA cluster I (Collins et al., 1994), having at least 99% identity on 16S rRNA gene level, although being distinct species as determined by DNA-DNA reassociation and DNA fingerprinting experiments (WO 2008/028055, US patent 2011/0229947).
The strains of this cluster are defined by common characteristics, having both a similar genotype and phenotype, and they all share the same mode of energy conservation and fermentative metabolism. The strains of this cluster lack cytochromes and conserve energy via an Rnf complex.
All strains of this cluster have a genome size of around 4.2 MBp (Köpke et al., 2010) and a CC composition of around 32 % mol (Abrini et al., 1994; Köpke et al., 2010; Tanner et al., 1993) (WO 2008/028055; US patent 2011/0229947), and conserved essential key gene operons encoding for enzymes of Wood-Ljungdahl pathway (Carbon monoxide dehydrogenase, Formyl-tetrahydrofolate synthetase, Methylene-tetrahydrofolate dehydrogenase, Formyl-tetrahydrofolate cyclohydrolase, Methylene-tetrahydrofolate reductase, and Carbon monoxide dehydrogenase/Acetyl-CoA synthase), hydrogenase, formate dehydrogenase, Rnf complex (rnfCDGEAB), pyruvate:ferredoxin oxidoreductase, aldehyde:ferredoxin oxidoreductase (Köpke et al., 2010, 2011). The organization and number of Wood-Ljungdahl pathway genes, responsible for gas uptake, has been found to be the same in all species, despite differences in nucleic and amino acid sequences (Köpke et al., 2011).
The strains all have a similar morphology and size (logarithmic growing cells are between 0.5-0.7×3-5 μm), are mesophilic (optimal growth temperature between 30-37° C.) and strictly anaerobe (Abrini et al., 1994; Tanner et al., 1993)(WO 2008/028055). Moreover, they all share the same major phylogenetic traits, such as same pH range (pH 4-7.5, with an optimal initial pH of 5.5-6), strong autotrophic growth on CO containing gases with similar growth rates, and a metabolic profile with ethanol and acetic acid as main fermentation end product, with small amounts of 2,3-butanediol and lactic acid formed under certain conditions (Abrini et at, 1994; Köpke et al., 2011; Tanner et at, 1993)(WO 2008/028055). Indole production has been observed with all species. However, the species differentiate in substrate utilization of various sugars (e.g. rhamnose, arabinose), acids (e.g. gluconate, citrate), amino acids (e.g. arginine, histidine), or other substrates (e.g. betaine, butanol). Some of the species were found to be auxotroph to certain vitamins (e.g. thiamine, biotin) while others were not. Reduction of carboxylic acids into their corresponding alcohols has been shown in a range of these organisms (Perez, Richter, Loftus, & Angenent, 2012).
The traits described are therefore not specific to one organism like C. autoethanogenum or C. ljungdahlii, but rather general traits for carboxydotrophic, ethanol-synthesizing Clostridia. Thus, the invention can be anticipated to work across these strains, although there may be differences in performance.
In certain embodiments, the parental microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei. In one embodiment, the group also comprises Clostridium coskatii. In one particular embodiment, the parental microorganism is Clostridium autoethanogenum DSM23693.
Parental microorganisms may be modified to arrive at the microorganisms of the invention using any number of known transformation and recombinant nucleic acid techniques. Such techniques are described for example in Sambrook et al, (Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). By way of further example, the methodology described in the examples section hereinafter can be used.
By way of general example, in the case of introducing a mutation into a gene, or otherwise disrupting or knocking out a gene, an appropriate nucleic acid construct or vector can be designed to integrate into the genome of the parental microorganism to disrupt the gene. Such constructs will typically include nucleic acid sequences (arms) homologous to a region within or flanking the gene to be disrupted, which allow for homologous recombination to occur, and the introduction of a mutation, the excision of a region of nucleic acid from the gene, or the substitution of a region of the gene with a nucleic acid on the contrast, to occur. While it is preferred that the arms on the constructs have 100% complementarity to the region in the genome which they are targeted to, this is not necessary, provided that the sequence is sufficiently complementary to allow for targeted recombination with the genetic region of interest. Typically, the arms will have a level of homology which would allow for hybridisation to a target region under stringent conditions, as defined in Sambrook et al 1989.
Skilled persons will appreciate nucleic acid sequences sufficient to allow for targeted homologous recombination and integration of an exogenous nucleic acid into the genome of a parental microorganism having regard to the available sequence information for the enzymes involved in the 2,3-hutanediol biosynthesis pathway. However, by way of example, in the case of budA, the flanking homology arms described herein may be used (for example, Seq ID 3, 4 and 78-81), or in the case of C. ljungdahlii, designed from the nucleic acid sequence information on Genbank (CP001666,1). “By way of further example, the flanking sequences of genes encoding enzymes to be disrupted in accordance with the invention may he determined from genomic sequence information from relevant microorganisms. By way of particular example, flanking sequences in C. ljundahlii can be determined from the information on GenBank CP001666.1
By way of further general example, where a nucleic acid is introduced into a parental microorganism to express a protein or nucleic acid which inhibits the expression or activity of an enzyme in the 2,3-butanediol biosynthesis pathway, or to express a protein which increases the expression of a compound which inhibits the expression or activity of an enzyme in the 2,3-butanediol biosynthesis pathway, the construct will be designed to allow for expression of the protein in the microorganism. Typically it will include appropriate regulatory elements, including a promoter. Constitutive or inducible promoters may be used.
Where the invention employs the direct disruption of a gene by introducing a mutation or the like, the construct or vector used to transfbrm the parental microorganism will be adapted to integrate into the genome of microorganism, as mentioned above. in the case of expression of a protein or nucleic acid that is adapted to disrupt the expression or activity of an enzyme in the 2,3-butanediol biosynthesis pathway, or increase the expression or activity of an inhibitor of an enzyme involved in the pathway, the constructs may remain extra-chromosomal upon transformation of a parental microorganism or may be adapted for intergration into the genome of the microorganism. Accordingly, constructs of use in the invention may include nucleotide sequences adapted to assist integration (for example, a region which allows for homologous recombination and targeted integration into the host genome) or expression and replication of an extrachromosomal construct (for example, origin of replication, promoter and other regulatory sequences).
Nucleic acid constructs of use in the invention may be constructed using any number of techniques standard in the art. For example, chemical synthesis or recombinant techniques may be used. Such techniques are described, for example, in Sambrook et at (Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Further exemplary techniques are described in the Examples section herein after. Essentially, the individual genes, regulatory elements, homology arms and the like will be operably linked to one another so that they can perform their desired function. Suitable vectors for use in the invention will be appreciated by those of ordinary skill in the art. However, by way of example, the following vectors may be suitable: pMTL, pIMP, pJIR and the plasmids exemplified in the Examples section herein after.
It should be appreciated that nucleic acids of use in generating the microorganisms of the invention may be in any appropriate form, including RNA, DNA, or cDNA, including double-stranded and single-stranded nucleic acids.
The one or more exogenous nucleic acids may be delivered to a parental microorganism as naked nucleic acids or may be formulated with one or more agents to facilitate the tranformation process (for example, liposome-conjugated nucleic acid, an organism in which the nucleic acid is contained). The one or more nucleic acids may be DNA, RNA, or combinations thereof, as is appropriate.
The microorganisms of the invention may be prepared from a parental microorganism and one or more exogenous nucleic acids using any number of techniques known in the art for producing recombinant microorganisms. By way of example only, transformation (including transduction or transfection) may be achieved by electroporation, conjugation, prophage induction, or chemical and natural competence. Suitable transformation techniques are described for example in Sambrook J, Fritsch E F, Maniatis T: Molecular Cloning: A laboratory Manual, Cold Spring Harbour Labrotary Press, Cold Spring Harbour, 1989.
By way of further example, the electroporation techniques described in: Koepke et al. 2010, Poc. Nat. Acad. Sci. U.S.A. 107: 13087-92; PCT/NZ2011/000203; WO2012/053905; Straetz et al., 1994, Appl. Environ. Microbiol. 60:1033-37; Mermelstein et al., 1992, Biotechnology, 10, 190-195; Jennert et al., 2000, Microbiology, 146: 3071-3080; Tyurin et al., 2004, Appl. Environ. Microbiol. 70: 883-890; may be used. By way of further example, prophage induction techniques as described in Prasanna Tamarapu Parthasarathy, 2010, Development of a Genetic Modification System in Clostridium scatologenes ATCC 25775 for Generation of Mutants, Masters Project Western Kentucky University, could be used. By way of further example, the conjugation methods described in Herbert et al., 2003, FEMS Microhiol. Lett. 229: 103-110 or Williams et al., 1990, J Gen. Microbiol. 136: 819-826 could be employed.
In certain embodiments, due to the restriction systems which are active in the microorganism to be transformed, it is necessary to methylate the nucleic acid to be introduced into the microorganism. This can be done using a variety of techniques, including those described below, and further exemplified in the Examples section herein after.
By way of example, in one embodiment, a recombinant microorganism of the invention is produced by a method comprises the following steps:
introduction into a shuttle microorganism of (i) a construct/vector to be introduced to the parental microorganism as described herein and (ii) a methylation construct/vector comprising a methyltransferase gene;
expression of the methyltransferase gene;
isolation of one or more constructs/vectors from the shuttle microorganism; and,
introduction of the one or more construct/vector into a destination microorganism.
In one embodiment, the methyltransferase gene of step B is expressed consitutively. In another embodiment, expression of the methyltransferase gene of step B is induced.
The shuttle microorganism is a microorganism, preferably a restriction negative microorganism, that facilitates the methylation of the nucleic acid sequences that make up the expression constructivector. In a particular embodiment, the shuttle microorganism is a restriction negative E. coli, Bacillus subiillis, or Lactococcus lactis. 
The methylation construct/vector comprises a nucleic acid sequence encoding a methyltransferase.
Once the expression construct/vector and the methylation construct/vector are introduced into the shuttle microorganism, the methyltransferase gene present on the methylation construct/vector is induced. Induction may be by any suitable promoter system although in one particular embodiment of the invention, the methylation construct/vector comprises an inducible lac promoter (for example, as in SEQ_ID NO 31) and is induced by addition of lactose or an analogue thereof, more preferably isopropyl-β-D-thio-galactoside (IPTG). Other suitable promoters include the ara, tet, or T7 system. In a further embodiment of the invention, the methylation constructivector promoter is a constitutive promoter.
In a particular embodiment, the methylation constructivector has an origin of replication specific to the identity of the shuttle microorganism so that any genes present on the methylation construct/vector are expressed in the shuttle microorganism. Preferably, the construct/vector to be introduced into the parental microorganism has an origin of replication specific to the identity of the microorganism.
Expression of the methyltransferase enzyme results in methylation of the genes present on the construct/vector to be introduced to a parental microorganism. The constmctivector may then be isolated from the shuttle microorganism according to any one of a number of known methods. By way of example only, the methodology described in the Examples section described hereinafter may be used to isolate the construct/vector.
In one particular embodiment, both constructivector are concurrently isolated.
The construct/vector destined for the parental microorganism may be introduced into the microorganism using any number of known methods. However, by way of example, the methodology described in the Examples section hereinafter may be used.
It is envisaged that a methyltransferase gene may be introduced into a shuttle microorganism and over-expressed. Thus, in one embodiment, the resulting methyltransferase enzyme may be collected using known methods and used in vitro to methylate the construct to be introduced into the parental microorganism. The construct/vector may then be introduced into the destination (parental) microorganism. In another embodiment, the methyltransferase gene is introduced into the genome of the shuttle microorganism followed by introduction of the construct destined for the parental microorganism into the shuttle microorganism, isolation of one or more constructs/vectors from the shuttle microorganism and then introduction of the construct/vector into the destination arental) microorganism.
It is envisaged that the construct/vector destined for the parental microorganism and the methylation construct/vector as defined above may be combined to provide a composition of matter. Such a composition has particular utility in circumventing restriction barrier mechanisms to produce the recombinant microorganisms of the invention.
In one particular embodiment, the construct/vectors described herein before are plasmids.
Skilled person will appreciate a number of suitable methyltransferases of use in producing the microorganisms of the invention. However, by way of example the Bacillus subtilis phage ΦT1 methyltransferase and the methyltransferase described in the Examples herein after may be used. Nucleic acids encoding suitable methyltransferases will be readily appreciated having regard to the sequence of the desired methyltransferase and the genetic code. In one embodiment, the nucleic acid encoding a methyltransferase is described in the Examples herein after (for example the nucleic acid of SEQ_ID NO. 31).
Any number of constructs/vectors adapted to allow expression of a methyltransferase gene may be used to generate the methylation construct/vector. However, by way of example, the plasmid described in the Examples section hereinafter may be used.
From the information contained herein, it will be appreciated that one may tailor the genetic modification of a parental microorganism to favour the production of one or more products over one or more other products. For example, disrupting the conversion of pyruvate to acetolactate favours the production of lactate, formate, malate, fumarate, citrate, succinate and 2-oxogluterate over the production of valine, leucine and isoleucine.
Production Method
The invention provides a method for producing one or more product by microbial fermentation comprising fermenting a substrate comprising CO using a microorganism of the invention. In one particular embodiment, the method is for producing ethanol or one or more other products by microbial fermentation comprising fermenting a substrate comprising CO using a microorganism of the invention. The methods of the invention may be used to reduce the total atmospheric carbon emissions from an industrial process.
Preferably, the fermentation comprises the steps of anaerobically fermenting a substrate in a bioreactor to produce the one or more products (in one particular embodiment, ethanol, or ethanol and one or more other products) using a recombinant microorganism of the invention.
In one embodiment the method comprises the steps of:

    
    
        (a) providing a substrate comprising CO to a bioreactor containing a culture of one or more microorganism of the first aspect of the invention; and
        (b) anaerobically fermenting the culture in the bioreactor to produce one or more products (in one embodiment including ethanol).
    
    


In one embodiment the method comprises the steps of:

    i. capturing CO-containing gas produced as a result of the industrial process, before the gas is released into the atmosphere;
    ii. the anaerobic fermentation of the CO-containing gas to produce one or more products (in one embodiment including ethano)1 by a culture containing one or more microorganism of the first aspect of the invention.


In an embodiment of the invention, the gaseous substrate fermented by the microorganism is a gaseous substrate containing CO. The gaseous substrate may be a CO-containing waste gas obtained as a by-product of an industrial process, or from some other source such as from automobile exhaust fumes. In certain embodiments, the industrial process is selected from the group consisting of ferrous metal products manufacturing, such as a steel mill, non-ferrous products manufacturing, petroleum refining processes, gasification of coal., electric power production, carbon black production, ammonia production, natural gas refining, methanol production and coke manufacturing. In these embodiments, the CO-containing gas may be captured from the industrial process before it is emitted into the atmosphere, using any convenient method. The CO may be a component of syngas (gas comprising carbon monoxide and hydrogen). The CO produced from industrial processes is normally flared off to produce CO2 and therefore the invention has particular utility in reducing CO2 greenhouse gas emissions and producing butanol for use as a biofuel. Depending on the composition of the gaseous CO-containing substrate, it may also be desirable to treat it to remove any undesired impurities, such as dust particles before introducing it to the fermentation. For example, the gaseous substrate may be filtered or scrubbed using known methods.
It will be appreciated that for growth of the bacteria and CO-to-ethanol (and/or other product(s)) to occur, in addition to the CO-containing substrate gas, a suitable liquid nutrient medium will need to be fed to the bioreactor. The substrate and media may be fed to the bioreactor in a continuous, batch or batch fed fashion. A nutrient medium will contain vitamins and minerals sufficient to permit growth of the micro-organism used. Anaerobic media suitable for fermentation to produce ethanol (and optionally one or more other products) using CO are known in the art. For example, suitable media are described in Biebel (Journal of Industrial Microbiology & Biotechnology (2001) 27, 18-26). The substrate and media may be fed to the bioreactor in a continuous, hatch or batch fed fashion. In one embodiment of the invention the media is as described in the Examples section herein after.
The fermentation should desirably be carried out under appropriate conditions for the CO-to-ethanol (and/or other product(s)) fermentation to occur. Reaction conditions that should be considered include pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum gas substrate concentrations to ensure that CO in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition.
In addition, it is often desirable to increase the CO concentration of a substrate stream (or CO partial pressure in a gaseous substrate) and thus increase the efficiency of fermentation reactions where CO is a substrate. Operating at increased pressures allows a significant increase in the rate of CO transfer from the gas phase to the liquid phase where it can be taken up by the micro-organism as a carbon source for the production of ethanol (and/or other product(s)). This in turn means that the retention time (defined as the liquid volume in the bioreactor divided by the input gas flow rate) can be reduced when bioreactors are maintained at elevated pressure rather than atmospheric pressure. The optimum reaction conditions will depend partly on the particular micro-organism of the invention used. However, in general, it is preferred that the fermentation be performed at pressure higher than ambient pressure. Also since a given CO-to-ethanol (and/or other product(s)) conversion rate is in part a function of the substrate retention time, and achieving a desired retention time in turn dictates the required volume of a bioreactor, the use of pressurized systems can greatly reduce the volume of the bioreactor required, and consequently the capital cost of the fermentation equipment. According to examples given in U.S. Pat. No. 5,593,886, reactor volume can be reduced in linear proportion to increases in reactor operating pressure, i.e. bioreactors operated at 10 atmospheres of pressure need only be one tenth the volume of those operated at 1 atmosphere of pressure.
The benefits of conducting a gas-to-ethanol fermentation at elevated pressures has been described elsewhere. For example, WO 02/08438 describes gas-to-ethanol fermentations performed under pressures of 30 psig and 75 psig, giving ethanol productivities of 150 g/l/day and 369 g/l/day respectively. However, example fermentations performed using similar media and input gas compositions at atmospheric pressure were found to produce between 10 and 20 times less ethanol per litre per day.
It is also desirable that the rate of introduction of the CO-containing gaseous substrate is such as to ensure that the concentration of CO in the liquid phase does not become limiting. This is because a consequence of CO-limited conditions may be that the ethanol product is consumed by the culture.
The composition of gas streams used to feed a fimmentation reaction can have a significant impact on the efficiency and/or costs of that reaction. For example, O2 may reduce the efficiency of an anaerobic fermentation process. Processing of unwanted or unnecessary gases in stages of a fermentation process before or after fermentation can increase the burden on such stages (e.g. where the gas stream is compressed before entering a bioreactor, unnecessary energy may be used to compress gases that are not needed in the fermentation). Accordingly, it may be desirable to treat substrate streams, particularly substrate streams derived from industrial sources, to remove unwanted components and increase the concentration of desirable components.
In certain embodiments a culture of a bacterium of the invention is maintained in an aqueous culture medium. Preferably the aqueous culture medium is a minimal anaerobic microbial growth medium. Suitable media are known in the art and described for example in U.S. Pat. Nos. 5,173,429 and 5,593,886 and WO 02/08438, and as described in the Examples section herein after.
The one or more products produced by a method of the invention (in one embodiment ethanol, or a mixed alcohol stream containing ethanol and/or one or more other products) may be recovered from the fermentation broth by methods known in the art, such as fractional distillation or evaporation, pervaporation, and extractive fermentation, including for example, liquid-liquid extraction. By-products such as acids including acetate may also be recovered from the fermentation broth using methods known in the art. For example, an adsorption system involving an activated charcoal filter or electrodialysis may be used. Alternatively, continuous gas stripping may also be used.
In certain preferred embodiments of the invention, ethanol and/or one or more other products are recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration), and recovering one or more products from the broth. Alcohols may conveniently be recovered for example by distillation, and acids may be recovered for example by adsorption on activated charcoal. The separated microbial cells are preferably returned to the fermentation bioreactor. The cell free permeate remaining after any alcohol(s) and acid(s) have been removed is also preferably returned to the fermentation bioreactor. Additional nutrients (such as B vitamins) may be added to the cell free permeate to replenish the nutrient medium before it is returned to the bioreactor.
Also, if the pH of the broth was adjusted as described above to enhance adsorption of acetic acid to the activated charcoal, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.
Succinate can be recovered from the fermentation broth using a number of techniques such as acidification, electrodialysis coupled with ion-exchange chromatography (Song and Lee, 2006, Enzyme Microb Technol 39, 352-361), precipitation with Ca(OH) coupled with filtration and addition of sulfuric acid (Lee et al 2008, Appl Microbiol Biotechnol 79, 11-22) or reactive extraction with amine-based extractants such as tri-n-octylamine (Huhet al, 2006, Proc Biochem 41, 1461-1465). For all methods it is crucial to have the free acid form, and not the salt. Most biotechnological production processes for succinic acid however operate at neutral or slightly acidic range of pH 6-7. Given the pKa of succinic acid (pKa=4.16 and 5.61), the majority is present as salt and not as free acid under these conditions. C. autoethanogenum and carboxydotrophic acetogenas however are known to tolerate and grow at a desirable low pH range of pH 4-6.
Branched-chain amino acids valine, leucine, and isoleucine can be relatively easily recovered from the fermentation broth by concentration (e.g. reverse osmosis) and crystallization or removal of the biomass (e.g. ultrafiltration or centrifugation) and ion exchange chromatography (Ikeda, A., 2003, Amino Acid Production Processes, in R. Fauric and J. Thommel (eds.) Microbial production of L-amin acids, 1-35).
Lactate, formate, 2-oxogluterate and other products can be recovered from the fermentation broth by any known method. However, by way of example, in the case of lactate, conventional fermentation process produces calcium lactate precipitate, which can be collect and re-acidified. Alternatively, membrane techniques, such as electrodialysis can be sued to separate lactate. Low concentrations of lactate can be separated from a fermentation broth by applying a suitable potential across a selective ion permeable membrane. Other suitable techniques include nanofiltration, wherein monovalent ions can selectively pass through a membrane under pressure.
It would be appreciated that in some situations, the method may be performed to produce and recover products other than ethanol (for example, one or more products comprising valine, leucine, succinate, pyruvate, lactate and formate). Accordingly, the invention should be understood to include methods for the production of one or more of these products.
EXAMPLES
The invention will now be described in more detail with reference to the following non-limiting examples.
Example 1
Deletion of C. autoethanogenum budA Gene by Homologous Recombination
Genetic modifications were carried out using a plasmid containing the 5′ and 3′ homology arms of budA gene of C. autoethanogenum DSM23693 (FIG. 1a, FIG. 1b, and FIG. 2). This plasmid was methylated in vivo using a novel methyltransferase and then transformed into C. autoethanogenum DSM23693 (DSMZ, Germany). The budA gene knockout has been shown by PCR and by the inhibition of 2,3-butanediol production in C. autoethanogenum DSM23693 ΔbudA strains.
Conjstruction of Expression Plasmid:
Standard Recombinant DNA and molecular cloning techniques were used in this invention and are described by Sambrook et al, 1989 and Ausubel et al, 1987. DNA sequences of 5′ upstream flanking homology arm (Seq. ID 3) and 3′ downstream flanking homology arm (Seq. ID 4) of Clostridum autoethanogenum DSM23693 budA gene were obtained from NCBI.
Genomic DNA from Clostridum autoethanogenum DSM23693 was isolated using Purelink Genomic DNA mini kit from Invitrogen, according to the manufacturer's instruction.
The 5′ (Seq. ID. 3) and 3′ (Seq. ID. 4) flanking homology arms were amplified by PCP with oligonucleotides in Table 1 using Clostridum autoethanogenum DSM23693 genomic DNA as template, iProof High Fidelity DNA Polymerase (Bio-Rad Labratories) and the following program: initial denaturation at 98° C. for 30 seconds, followed by 25 cycles of denaturation (98° C. for 10 seconds), annealing (60° C. for 15 seconds) and elongation (7° C. for 30 seconds), before a final extension step (72° C. for 7 minutes).







TABLE 1







Oligonucleotides for cloning













SEQ_ID


Target
Oligonucleotide Name
DNA Sequence (5′ to 3′)
NO.













5′ Homology arm
Og09f
attcatcctgcaggTTTCTTCACAGGAAAATATACTTCAG
5


5′ Homology arm
Og10r
gactgcggccgcATTACATTCACCTCTATGTCATTATAAC
6


 


3′ Homology arm
Og11f
atttgctagcACTAGACAGTGCTAATAACAATGTCTAG
7


3′ Homology arm
Og12r
atatggcgcgccTCATAAACCTGGATAACATAAGC
8


 


Plasmid
M13f
GTAAAACGACGGCCAG
10


Plasmid
M13r
CAGGAAACAGCTATGACC
11









The amplified 964 bp 5′ flanking homology arm (5′HA) of budA gene was cut with Sbfl and Notl resitriction enzymes and cloned into the E. coli-Clostridium shuttle vector pMTL 85141 (Seq. ID 9; FJ797651. 1; Nigel Minton, University of Nottingham; Heap et al., 2009) using Sbfl and Notl restriction sites and strain E. coli XLI-Blue MRF′ Kan (Stratagene). The created plasmid pMTL85141-budA-5′HA and the 977 bp PCR product of the 3′ homology arm of budA gene were both cut with Nhel and Ascl. A ligation of these digested DNA fragments was transformed into E. coli XL1-Blue MRF′ Kan (Stratagene) resulting in the plasmid pMTL85141-budA-ko. The insert in the resulting plasmid pMTL85141-budA-ko (SEQ_ID No. 12) was completely sequenced using oligonucleotides given in Table 1 and sequencing results confirmed that both 5′ and 3′ homology arms were free of mutations.
Methylation of DNA:
A hybrid methyltransferase gene fused to an inducible lac promoter (SEQ ID No. 31) was designed, by alignment of methyltransferase genes from C. autothanegenum, C. ljungdahlii, and C. ragsdalei, as described in U.S. patent application Ser. No. 13/049,263. Expression of the methyltransferase results in a protein having the sequence of SEQ ID No. 32). The hybrid methyltransferase gene was chemically synthesized and cloned into vector pGS20 (ATG:biosynthetics GmbH, Merzhausen, Germany—SEQ ID No. 33) using EcoRI. The resulting methylation plasmid pGS20-methyltransferase was double transformed with the plasmid pMTL85141-budA-ko into the restriction negative E. coli XL1-Blue MRF′ Kan (Stratagene). In vivo methylation was induced by addition of 1 mM IPTG, and methylated plasmids were isolated using the Zymo mini prep Kit (Zymo). The resulting methylated plasmid composition was used for transformation of C. autoethanogenum DSM23693.
Transformation:
During the complete transformation experiment, C. autoethanogenum DSM23693 was grown in YTF media (Tab. 2) in the presence of reducing agents and with 30 psi steel mill waste gas (collected from New Zealand Steel site in Glenbrook, NZ; composition: 44% CO, 32% N2, 22% CO2, 2% H2) at 37° C. using standard anaerobic techniques described by Hungate (1969) and Wolfe (1971).







TABLE 2





YTF media


















Media component
per L of Stock















Yeast Extract
10
g



Tryptone
16
g



Sodium chloride
0.2
g



Fructose
10
g



Distilled water
To 1
L














Reducing agent stock
per 100 mL of stock















NaOH
0.9
g



Cystein•HCl
4
g



Na2S
4
g



Distilled water
To 100
mL










To make competent cells, a 50 ml culture of C. autoethanogenum DSM23693 was subcultured to fresh YTF media for 5 consecutive days. These cells were used to inoculate 50 ml YTF media containing 40 mM DL-threortine at an OD600 nm of 0.05. When the culture reached an OD600 nm of 0.5, the cells were incubated on ice for 30 minutes and then transferred into an anaerobic chamber and harvested at 4,700×g and 4° C. The culture was twice washed with ice-cold electroporation buffer (270 mM sucrose, 1 mM MgCl2, 7 mM sodium phosphate, pH 7.4) and finally suspended in a volume of 600 μl fresh electroporation buffer. This mixture was transferred into a pre-cooled electroporation cuvette with a 0.4 cm electrode gap containing 2 μg of the methylated plasmid mix and 1 μl Type 1 restriction inhibitor (Epicentre Biotechnologies) and immediately pulsed using the Gene pulser Xcell electroporation system (Bio-Rad) with the following settings: 2.5 kV, 600 Ω, and 25 μF. Time constants of 3.7-4.0 ms were achieved. The culture was transferred into 5 ml fresh YTF media. Regeneration of the cells was monitored at a wavelength of 600 nm using a Spectronic Helios Epsilon Spectrophotometer (Thermo) equipped with a tube holder. After an initial drop in biomass, the cells started growing again. Once the biomass doubled from that point about 200 μl of culture was spread on YTF-agar plates and PETC agar plates containing 5 g/l fructose (Table 3) (both containing 1.2% BactoTM Agar (BD) and 15 μg/ml Thiamphenicol). After 3-4 days of incubation with 30 psi steel mill gas at 37° C., 500 colonies per plate were clearly visible.







TABLE 3





PETC media (ATCC media 1754; atcc.org/Attachments/2940.pdf)
















Media component
Concentration per 1.0 L of media












NH4Cl
1
g


KCl
0.1
g


MgSO4•7H2O
0.2
g


NaCl
0.8
g


KH2PO4
0.1
g


CaCl2
0.02
g


Trace metal solution
10
ml


Wolfe's vitamin solution
10
ml


Yeast Extract
1
g


Resazurin (2 g/L stock)
0.5
ml


MES
2
g


Reducing agent
0.006-0.008%
(v/v)








Distilled water
Up to 1 L, pH 5.5 (adjusted with HCl)












per L of Stock















Wolfe's vitamin solution





Biotin
2
mg



Folic acid
2
mg



Pyridoxine hydrochloride
10
mg



Thiamine•HCl
5
mg



Riboflavin
5
mg



Nicotinic acid
5
mg



Calcium D-(+)-pantothenate
5
mg



Vitamin B12
0.1
mg



p-Aminobenzoic acid
5
mg



Thioctic acid
5
mg



Distilled water
To 1
L



Trace metal solution



Nitrilotriacetic Acid
2
g



MnSO4•H2O
1
g



Fe (SO4)2(NH4)2•6H2O
0.8
g



CoCl2•6H2O
0.2
g



ZnSO4•7H2O
0.2
mg



CuCl2•2H2O
0.02
g



NaMoO4•2H2O
0.02
g



Na2SeO3
0.02
g



NiCl2•6H2O
0.02
g



Na2WO4•2H2O
0.02
g



Distilled water
To 1
L














Reducing agent stock
per 100 mL of stock















NaOH
0.9
g



Cystein•HCl
4
g



Na2S
4
g



Distilled water
To 100
mL










The colonies were streaked on fresh PETC agar plates also containing 5 g/L fructose and 15 μg/ml Thiamphenicol. After 2 days of incubation with 30 psi steel mill gas at 37° C. single colonies from these plates were restreaked on fresh non-selective PETC agar plates containing 5 g/l fructose only. The resreaking on PETC agar plates with 5 g/l fructose was repeated once again and plates incubated with 30 psi steel mill gas at 37° C. After 3 days 6 single colonies growing on non-slective media were inoculated in 2 ml PETC liquid media containing 5 g/l fructose. When growth occurred, the culture was sequentially upscaled to 5 ml, 25 ml and then to 50 ml PETC media containing 5 g/l fructose and 30 psi steel mill gas as carbon source.
Coryformation of the Successful Transformation:
C. autoethanogenum: To verify the identity of the six clones and the DNA transfer, genomic DNA was isolated from all 6 colonies/clones in PETC liquid media using PurelinkTM Genomic DNA mini kit (Invitrogen) according to manufacturer's instruction. These genomic DNA along with that of C. autoethanogenum DSM23693 wildtype were used as template in PCR. The PCR was performed with iproof High Fidelity DNA Polymerase (Bio-Rad Laboratories), primers as listed in Table 4 and the following program: initial denaturation at 98° C. for 2 minutes, followed by 25 cycles of denaturation (98° C. for 10 seconds), annealing (61° C. for 15 seconds) and elongation (72° C/ for 90 seconds), before a final extension step (72° C. for 7 minutes). The genomic DNA from wildtype C. autoethanogenum DSM23693 was used as template in control PCR.







TABLE 4







Oligonucleotides for PCR confirmation of plasmid and species











Oligonucleotide

Seq ID


Target region
Name
DNA Sequence (5′ to 3′)
No.













16s rRNA gene
FD1
CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG
27


16s rRNA gene
rP2
CCCGGGATCCAAGCTTACGGCTACCTTGTTACGACTT
28


 


Homology arm
og09f
attcatcctgcaggTTTCTTCACAGGAAAATATACTTCAG
5


Homology arm
Og12r
atatggcgccTCATAAACCTGGATAACATAAGC
8


 


budA gene
Og44f
TTGCTGTAGTCACTGAACTGGAAAA
29


budA gene
Og45r
AATCAGGACACCTAAATCCAACCAC
30









To confirm the identity of the 6 clones, PCR was performed against the 16s rRNA gene using, primers fD1 (Seq. ID. 27) and rP2 (Seq. ID 28) and using PCR conditions as described above. The PCR products were purified using Zymo Clean and ConcentratorTM kit and sequenced using primer rP2 (Seq. ID 28). Sequences of all 6 clones (Seq. ID. 13-19) showed at least 90% identity against the 16S rRNA gene of C. autoethanogenum (Seq. ID 15; Y18178, GI:7271109).
PCR of 6 analyzed clones with primers specific to the budA target region using primers Og09f (Seq. ID. 5) and Og12r (Seq. ID. 8) resulted in amplification of 2.2 kb DNA fragment from 5 out of 6 clones. PCR product of 2.7 kb was amplified with wildtype C. autoethanogenum DSM23693 genomic DNA. The identity of the 2.2 kb PCR products from potential budA knockout clones was confirmed by sequencing (Seq ID 20-26) with primers listed in Table 5 and no sequence of budA gene was detected in these fragments. The lacZ DNA fragment had replaced the budA gene. The absence of budA gene in these 6 clones was confirmed again by PCR with primers, Og44f (Seq. ID. 29) and Og45r (Seq. ID. 30) specific to the 275 bp internal region of C. autoethanogenum DSM23693 budA gene which was amplified only from wild type C. autoethanogenum DSM23693.
Absence of 2,3 Butanediol Production and Increase in Ethanol Yield:
To demonstrate the lack of acetoin and subsequently 2,3-butanediol production, serum bottle experiments were carried out with clone 1 in triplicates with steel mill waste gas (composition, 44% CO, 12% N2, 22% CO2, and 2% H2; collected from a steel site in Glenbrook, New Zealand) and PETC media as described above. Unmodified wild type strain of C. autoethanogenum DSM23693 was grown under the same conditions as control.
Analysis of metabolites was performed by HPLC using an Agilent 1100 Series HPLC system equipped with a RID operated at 35° C. (Refractive Index Detector) and an Alltech IOA-2000 Organic acid column (150×6.5 mm, particle size 5 μm) kept at 32° C. Slightly acidified water was used (0.005 M H2SO4) as mobile phase with a flow rate of 0.25 ml/min. To remove proteins and other cell residues, 400 μl samples were mixed with 100 μl of a 2%) (w/v) 5-Sulfosalicylic acid and centrifuged at 14,000×g for 3 min to separate precipitated residues. 10 μl of the supernatant were then injected into the HPLC for analyses.
The results of serum bottle experiments with ΔbudA C. autoethanogenum DSM23693 clone 1 and unmodified wild type C. autoethanogenum DSM23693 are shown in Table 5. Maximum biomass of strain ΔbudA C. autoethanogenum DSM23693 was with an OD600 nm of 0.32 relatively lower than the unmodified wild-type, which grew to an OD600 nm of 0.58. Compared to the wild type, no 2,3-butanediol was detected in the culture of the ΔbudA C. autoethanogenum DSM23693 clone 1, and the ethanol yield was significantly higher in ΔbudA C. autoethanogenum DSM23693 clone 1 than in the unmodified C. autoethanogenum DSM23693 (Table 5).







TABLE 5







Metabolites produced by ΔbudA C. autoethanogenum


DSM23693 clone 1 and unmodified wildtype C. autoethanogenum


DSM23693 relative to the biomass












Metabolite


ΔbudA



(g/l)
Media
Wildtype
Clone 1







Ethanol
1.395
2.500




Acetic acid
2.296
0.180



2,3-butanediol
0.085
0.000



Lactic acid
0.020
0.197



Formic acid
0.002
1.647



Succinic acid
0.002
0.344










Production of Other Metabolites—Lactate, Formate, Succinate, 2-oxogluterate Valine, Leucine, Isoleucine:

At the same time, interestingly, while the unmodified C. autoethanogenum DSM23693 produced only 0.02 g/l lactic acid as other by-product, ΔbudA C. autoethanogenum DSM23693 produced a significantly higher amount of lactic acid 0.07 g/l (0.197 g/l normalized to biomass) as well as 0.53 g/l (1.647 normalized to biomass) formic acid and 0.13 g/l (0.344 g/l normalized to biomass) succinic acid (Table 5). This increase is likely from the pyruvate, early precursor of 2,3-butanediol FIGS. 1a and 1b, accumulation because of knockout of budA gene which has blocked the production of 2,3-butanediol.
The production of succinate and lactate by ΔbudA C. autoethanogenum DSM23693 was also confirmed by Gas Chromatography-Mass spectrometry (GC-MS). For this, about 2.5 ml culture of ΔbudA C. autoethanogenum DSM23693 clone 1 grown with steel mill waste gas (composition, 44% CO, 32% N2, 22% CO2, and 2% H2; collected from a steel site in Glenbrook, New Zealand) at an optical density of 0.32 was centrifuged and supernatant was filtered through 0.2 uM filter (Smart K F, Aggio R B, Van Houtte J R, Villas-Bôas S G, Analytical platform for metabotome analysis of microbial cells using methyl chlorothrmate derivatization followed by gas chromatography-mass spectrometry, Nat Protoc. 2010 September; 5(10):1709-29. 2010). About 0.65 ml culture of wild type C. autoethanogenum DSM23693 and 2.5 ml of media blank were processed similarly. The samples were freeze-dried and analyzed by GC-MS in triplicates at the University of Auckland. As seen in Table 6 the peak intensity of the succinate and lactate signal was stronger in ΔbudA C. autoethanogenum DSM23693 clone 1 compared to unmodified C. autoethanogenum DSM23693 and the control media blank. The GC-MS results for succinate and lactate are consistent with HPLC results.
GC-MS results (Table 6) not only confirmed production of lactate and succinate with ΔbudA C. autoethanogenum DSM23693 clone 1, but also show production of 2-oxogluterate, the other incomplete TCA cycle endproduct besides succinate, and branched-chain amino acids valine, leucine, isoleucine, which are produced from pyruvate and acetolactate, the precursors of 2,3-butanediol which are likely to be present at elevated levels in the ΔbudA C. autoethanogenum strain. TCA cycle intermediates such as malate, fumerate, citrate, cis-Aconitate, iso-Citrate haven't been tested, but are likely to be elevated, as end-products succinate and 2-oxogluterate have been found to be produced (FIG. 1b).







TABLE 6





Metabolite analysis of ΔbudA C. autoethanogenum DSM23693 clone 1 (ΔbudA) and unmodified wild


type C. autoethanogenum DSM23693 (Wild type) by GC-MS, Media was included in the analyses as a control.


The values given in table correspond to the normalized peak intensity obtained for each replicate (R).



















Metabolite
Media
Average

















Lactate
0.547053273
0.474988
0.431645
0.48



Succinate
1.036264929
0.960478
1.243932
1.08



2-Oxogluterate
ND
ND
ND
0.00



Valine
5.970408365
5.446962
5.937764
5.79



Leucine
3.418425725
3.154261
3.237803
3.27



Isoleucine
ND
ND
0.607184
0.20















Metabolite
Wild type
Average

















Lactate
0.801302932
0.691344
0.853559
0.78



Succinate
0.547053273
0.474988
0.431645
0.48



2-Oxogluterate
ND
0.003092
0.0028
0.00



Valine
0.018545724
0.011764
0.014182
0.01



Leucine
0.0307755
0.024291
0.023099
0.03



Isoleucine
0.008136206
0.005305
0.00643
0.01














Metabolite
ΔbudA Clone 1 (Sample 1)
ΔbudA Clone 1 (Sample 2)
Average

















Lactate
5.017350825
5.672474
5.237064
5.987887
5.138095
4.39521
5.24


Succinate
2.535447097
2.984226
2.516218
5.017351
5.672474
5.237064
3.99


2-Oxogluterate
0.522265764
0.462277
ND
1.22281
0.021205
ND
0.37


Valine
11.13216958
9.419048
7.824351
10.08887
10.66202
9.192138
9.72


Leucine
10.92981831
5.478571
4.497006
4.70419
11.36585
4.441235
6.90


Isoleucine
6.087638048
9.397619
0.895459
10.59162
2.912456
9.976735
6.64





ND = not detected.






Production of acetoin and 2,3-butanediol is usually associated with deacidification of strong pyruvic acid (Xiao, Z., and P. Xu. 2007. Acetoin metabolism in bacteria. Crit. Rev. Biochem. Microbiol, 33:127-140), which can pose a serious threat to the cell by destroying the internal pH and proton gradient needed for energy conservation. Both acetoin and 2,3-butanediol are pH neutral compounds. Production of 2,3-butanediol also serves as electron sink to offload surplus reducing equivalents produced during the fermentation process.
While not wishing to be bound by any particular theory, the inventors believe that by knocking-out production of acetoin and 2,3-butanediol, the cell needs to find other ways to deacidify pyruvic acid (pKa=2.50) and offload reducing equivalents and thus is shifting it's metabolism to production of other (novel) products such as branched-chain amino acids valine, leucine or isoleucine, succinate (pKa1=4.20, pKa2=5.60), lactic acid (pKa=3.86), and formic acid (pka=3.77). Production of succinic acid also gives the chance to offload 4 reducing equivalents, while 2 reducing equivalents can be offloaded by production of lactic acid.
Example 2
Succinate Pathway
The pathway for production of succinate is described in FIG. 1b. Respective genes were identified in Clostridium autoethanogenum and enzyme activity was demonstrated.
In a first step, pyruvate is converted to malate, either directly catalyzed by a malic enzyme or via oxaloacetate catalyzed by a malate dehydrogenase. Oxaloacetate (OAA) can be produced from pyruvate by action of a Pyruvate carboxylase, or via Phosphoenolpyruvate (PEP) in a two step conversion catalyzed by Pyruvate phosphate dikinase (PPDK) and PEP carboxykinase (PCK). Malate is subsequently converted to succinate in a two-step process catalysed by Fumarate hydratase and fumarate reductase. Respective genes were identified in C. autoethanogenum and homologous genes are present in other carboxydotrophic acetogens as C. ljungdahlii and C. ragsdalei (Table 7).







TABLE 7







Genes and Enzymes identified to be


involved in Succinate production











C. auto-
C.
C.



ethanogenum
ljungdahlii
ragsdalei














Malic enzyme 1
Seq. ID 38-39
CP001666.1
Seq. ID 60-61




CLJU_c04160;




ADK13498.1


Malic enzyme 2
Seq. ID 40-41
CP001666.1
—




CLJU_c38460;




ADK16871.1


Malate
Seq. ID 42-43
CP001666.1
Seq. ID 62-63


dehydrogenase

CLJU_c05920;




ADK13674.1


Pyruvate phosphate
Seq. ID 44-45
CP001666.1
Seq. ID 64-65


dikinase (PPDK)

CLJU_c08140;




ADK13882.1


Pyruvate carboxylase
Seq. ID 46-47
CP001666.1
Seq. ID 66-67


(PYC)

CLJU_c37390;




ADK16765.1


PEP carboxykinase
Seq. ID 48-49
CP001666.1
Seq. ID 68-69


(PCK)

CLJU_c06210;




ADK13703.1


Fumarate hydratase
Seq. ID 50-51
CP001666.1
Seq. ID 70-71


subunit A

CLJU_c40600;




ADK17084.1


Fumarate hydratase
Seq. ID 52-53
CP001666.1
Seq. ID 72-73


subunit B

CLJU_c40590;




ADK17083.1


Fumarate reductase 1,
Seq. ID 54-55
CP001666.1
Seq. ID 74-75


flavoprotein

CLJU_c22800;




ADK15338.1


Fumarate reductase 2,
Seq. ID 56-57
CP001666.1
—


flavoprotein

CLJU_c30250;




ADK16073.1


Fumarate reductase 3,
Seq. ID 58-59
CP001666.1
Seq. ID 76-77


flavoprotein

CLJU_c08670;




ADK13935.1









Assay of Enzyme Activities:

Cells (Clostridium autoethanogenum) were harvested in the exponential phase of anaerobic growth. Cultures (A600 ̃0.45), and pelleted at 8000×q, 4° C. for 10 min. The supernatant was discarded, and the pellet was washed twice in wash buffer (0.1 M Tris-HCl, 10 mM dithiothreitol (DTT), pH 6.5, 4° C.). Finally, the pellet was resuspended in wash buffer containing protease inhibitor and mixed with 1.44 g of zirconia beads (Ambion RiboPure Bacteria Kit). Tubes were chilled on ice for 5 mins prior to disruption in a Vortex Mixer with a vortex adapter (Vortex Genie 2, Scientific Industries, Inc.) through 5 cycles of 1 min beating at 3200 rpm followed by 1 min on ice between cycles. After lysis, the sample was centrifuged (13,000×g, 4° C. for 10 min), and the supernatant was atiquoted and stored at −80° C. until analysis.
All assays were based on the oxidation of NADH to NAD (ε=6.2 mM−1 cm−1) under aerobic conditions in a cuvette with a path length of 1 cm. Enzyme activities were obtained from three replicates of at least two independent cell extractions. Protein content of the extracts was determined using a commercial kit (Pierce® Microplate BCA Protein Assay Kit-Reducing Agent Compatible, Thermo Scientific). One unit of enzyme activity was defined as the amount of enzyme that could convert a nanomole of substrate into product per minute per mg of total protein.
The activity of malate dehydrogenase was measured spectrophotornetrically by following the oxidation of reduced pyridine nucleotides with oxaloacetate (OAA) (Sridhar J. et al, 2000, Elucidation of enzymes in fermentation pathways used by Clostridium thermosuccinogenes growing on inulin. Appl. Environ. Microbiol. 66, 246-51). The reaction mixture contained the following: 0.1M Tris-Cl pH 6.5, 10 mM DTT, 0.15 mM NADH, 5 mM fumarate, 0.3 mM NADH and cell-free extract. The reaction was initiated by the addition of OAA and was monitored at room temperature. The specific activity of this enzyme in cell-free extracts of Clostridium autoethanogenum was measured as 160±17 nmol min−1 mg protein−1. This activity was comparable with the malate dehydrogenase found in Clostridium thermosuccinogenes measured at 3° C. (Sridhar J. et al, 2000, Elucidation of enzymes in fermentation pathways used by Clostridium thermosuccinogenes growing on inulin. Appl. Environ. Microbiol. 66, 246-51).
The activity of fumarate reductase was measured based on the conversion of fumarate to succinate (Sridhar J. et al, 2000. Elucidation of enzymes in fermentation pathways used by Clostridium thermosuccinogenes growing on inulin. Appl. Environ. Microbiol. 66, 246-51). The reaction mixture contained the following: 0.1M Tris-Cl pH 6.5, 10 mM DTT, 0.15 mM NADH, 5 mM fumarate and cell-free extract. The reaction was initiated by the addition of fumarate and was monitored at room temperature. The specific activity of this enzyme in cell-free extracts of Clostridium autoethanogenum was measured as 17.3±1.3 nmol min−1 mg protein−1.
The assays confirmed that Clostridium autoethanogenum posseses malate dehydrogenase activity, fumarate reductase/succinate dehydrogenase
As described herein, the invention provides microorganisms and methods which allow for increased production of ethanol by microbial fermentation of substrates comprising carbon monoxide. It also provides for the production of succinate. There have been no previous reports of the production of succinate by acetogens, let alone carboxydotrophic acetogens. The potential to produce succinate by microbial fermentation may have a number of advantages over the current petrochemical production methods. The microorganisms also produce formate and branched chain amino acids which have not (previously been described as products of fermentation by acetogenic microorganisms,
Succinate is used as a bulk platform chemical for the production of a number of industrial chemicals including 1,4-butanediol, tetrahydrofuran, gamma-butyrolactone, ethylene diamine disuccinate, diethyl succinate, and adipic acid. Formate is used in preservation of animal food and in leather tanning processes, as well as a bleaching solution in the pulp and paper industry. Branched chain amino acids have a number of uses in industrial biotechnology.
The microorganisms of the invention also produce one or more other products. The use of these products has been described elsewhere herein.
Example 3
Group II Intron Based Insertional Inactivation of Genes Involved in 2,3-BDO Biosynthesis in C. autoethanogenum DSM23693
Design and Construction of ClosTron Constructs Targeting budA and 2,3bdh Gene:
The acetolactate decarboxylase (budA) and 2,3-butanediol dehydrogenase (2,3-bdh) genes involved in 2,3-Butanediol. production in C. autoethanogenum DSM23693 were inactivated using ClosTron group II intron mediated gene disruption tool (Heap et al., 2010). The Perutka algorithm hosted at ClosTron.com was used to identify the group II intron target site between bases 450/451 and 468/469 on the sense strand of budA and 2,3-bdh genes, respectively. The same algorithm was used to design the intron targeting regions (Seq. ID. 82 and 83) which was commercially synthesized by DNA2.0 and delivered in pMTL007C-E5 vector. The final vectors, pMTL007C-E5-budA-450!451s and pMTL007C-E5-2,3bdh-468!469s, contain a Retro-tranposition-Activated ermB Marker (RAM) which confers resistance to antibiotic Clarithromycin upon insertion into the target site.
The pMTL007C-E5-budA-450!451s and pMTL007C-E5-2,3bdh-468!469s plasmids were introduced into C. autoethanogenum DSM23693 by conjugating with donor E. coli strain CA434 as donor. Briefly, the donor strain was grown overnight in LB media supplemented with 25 μg/ml chloramphenicol and 100 μg/ml spectinomycin. Cells from 1.5 ml culture were harvested and washed in phosphate buffered saline. The donor cells pellet was resuspended in 200 μl culture of exponentially growing recipient C. autoethanogenum DSM23693. The mixture was spotted on PETC agar media supplemented with fructose and incubated at 37° C. in pressurized gas jar. After 24 hours the cells were scrapped and resuspended in 500 μl PETC broth and spread on PETC agar media supplemented with 15 μg/ml thiamphenicol (Sigma) and 10 μg/ml trimethoprim (Sigma). C. autoethanogenum transconjugants were selected using 15 μg/ml thiamphenicol and E. coli CA434 strain was counter selected using 10 μg/ml trimethoprim. Colonies were observed after 3 days of incubation at 37° C. in pressurized gas jars.
Streaks of single colonies were made sequentially first on PETC-MES media containing 15 μg/ml thiamphenicol and 10 μg/ml trimethoprim followed by on agar plates with PETC media containing 5 μg/ml Clarithromycin. 4 colonies per plasmid were randomly screened for group II intron insertion by PCR using primers Og44f (Seq. ID. 29) and Og45r (Seq. ID. 30), flanking the group II intron insertion site in budA gene, and primers Og42f (Seq. ID. 84) and Og43r (Seq. ID. 85), flanking the group II intron insertion site in 2,3-bdh gene. The Maxime PCR PreMix Kit was used for PCR. 16s rDNA was also PCR amplified using primers fD1 (Seq. ID. 27) and rP2 (Seq. ID.28) and Maxime PCR PreMix Kit.
Confirmation of budA and 2,3bdh Gene Disruption Using ClosTron Group II Insertional Inactivation Tool:
Amplification of PCR products of 273 and 375 bp with primers Og44f/Og45r and Og42f/Of43r indicates the unmodified wild type budA and 2.3-bdh genes, respectively. Amplification of PCR products of  ̃2 kb using the same set of primers indicates insertion of ClosTron group II intron in the target genes. In case of clones targeting budA gene, clones 1 and 3 had bands of expected size. Clone 4 appears to be a mix with both the wild type and disrupted gene (FIG. 6). All 4 clones which were targeted for 2,3-bdh gene appear positive for gene disruption as seen by the amplification of 2 kb PCR product (FIG. 6). These results confirm the disruption of budA and 2,3-bdh genes in C. autoethanogenum DSM23693.
The 16s rDNA PCR product of Δ2,3bdh ClosTron clones 2 (Seq ID. 86, and 87) and 4 (Seq ID. 88 and 89) and ΔbudA ClosTron clones 1 (Seq ID. 90 and 91) and 3 (Seq ID. 92 and 93) were sequence confirmed to be of C. autoethanogenum DSM23693.
Thus the inventors have demonstrated targeted gene disruption in acetogenic C. autoethanogenum DSM23693 using two different approaches—(i) gene knockout by homologous recombination and (ii) by gene disruption using group II intron based insertional inactivation tool.
Study of ΔbudA and Δ2,3bdhClosTron Mutants for 2,3BDO Production:
The metabolites from ΔbudA and Δ2,3bdh mutants growing in serum bottles were analysed by HPLC (as explained earlier). The ΔbudA Clostron mutant like the ΔbudA knockout mutant did not produce 2,3-BDO (Table 8). The disruption of budA gene by two different methods in C. autoethanogenum confirms the role of budA gene in 2,3-BDO biosynthesis.







TABLE 8







Metabolites production by ΔbudA and Δ2,3 bdh


ClosTron C. autoethanogenum DSM23693 mutants












ΔbudA

Δ2,3 bdh














Metabolites
Clone 1
Clone 3
Clone 2
Clone 4

















Ethanol
0.09
0.08
0.37
0.23



Acetic Acid
2.56
2.63
3.78
3.34



2-3-Butanediol
0.0
0.0
0.01
0.01



Lactic Acid
0.0
0.0
0.0
0.0










The Δ2,3bdh ClosTron mutant still produced 2,3-BDO (Table 8) indicating the participation of a second gene in converting acetoin to 2,3-BDO.
Yan et al have shown that a secondary alcohol dehydrogenase from C. beijerinckii and three other organisms can also convert acetoin to 2,3-BDO (Yan. Lee & Liao, 2009). A similar secondary alcohol dehydrogenase (SecAdh) gene is found in C. autothenogenum DSM23693 (Seq ID 34 and 35), C. ljungdahlii (Seq ID 36) and C. ragsdalei (Seq ID 37).
In the absence of 2,3-bdh gene in C. autoethanogenum DSM23693, the SecAdh would most likely convert acetoin to 2,-3BDO.
Role of a Second Dehydrogenase in Converting Acetoin to 2,3-BDO:
To test the role of a second gene in converting acetoin to 2,3-BDO, wild type C. autoethanogenum DSM23693 and Δ2,3bdh ClosTron mutant were fed with 10 g/L acetoin in fermentation experiments.
Fermentation with Wild Type and Δ2,3bdh ClosTron Mutant:
Fermentations were carried out in 1.5 L bioreactors at 37° C. and CO-containing steel mill gas as sole energy and carbon source as described below. A defined medium containing per litre: MgCl, CaCl2 (0.5 mM), KCl (2 mM), H3PO4 (5 mM), Fe (100 μM), Ni, Zn (5 μM), Mn, B, W, Mo, Se(2 μM) was used for culture growth. The media was transferred into the bioreactor and autoclaved at 121° C. for 45 minutes. After autoclaving, the medium was supplemented. with Thiamine, Pantothenate (0.05 mg), Biotin (0.02 mg) and reduced with 3 mM Cysteine-HCl. To achieve anacrobicity the reactor vessel was sparged with nitrogen through a 0.2 μm filter. Prior to inoculation, the gas was switched to CO-containing steel mill gas, feeding continuously to the reactor. The feed gas composition was 2% H2 42% CO 20% CO2 36% N2. The pH of the culture was maintained between 5 and 5.2. The gas flow was initially set at 80 ml/min, increasing to 200 ml/min during mid-exponential phase, while the agitation was increased from 200 rpm to 350. Na2S was dosed into the bioreactor at 0.25 ml/hr. Once the OD600 reached 0.5, the bioreactor was switched to a continuous mode at a rate of 1.0 ml/min (Dilution rate 0.96 d−1). When the growth was stable, the reactor was spiked with 10 g/L racemic mix of acetoin. Media samples were taken to measure the biomass and metabolites by HPLC.
The metabolites were analysed by HPLC regularly until the disappearance of acetoin. The wild type C. autoethanogenum DSM23693 converted all acetoin to meso-BDO and 2,3-BDO in less than 1 h (FIG. 7). The rate of conversion of acetoin to meso-BDO and 2,3-BDO was relatively slow in Δ2,3bdh ClosTron mutant. The Δ2,3bdh ClosTron mutant reduced 10 g/L acetoin in more than 2 h. These results indicate the role of a second dehydrogenase in complementing for the disruption of 2,3bdh gene, albeit at slower rate.
Example 4
Modified C. autoethanogenum DSM23693 Strain Producing Only Acetoin
Industrial separation of acetoin from ethanol is technically more feasible compared to its downstream product 2,3-BDO. It is thus desirable to have a C. autoethanogenum strain producing acetoin and not its reduced form, 2,3-BDO. As Δ2,3bdh ClosTron mutant still produces 2,3-BDO, it is desirable to have a C. autoethanogenum DSM23693 strain in which both the 2,3bdh and SecAdh genes are disrupted. This can be achieved by two ways (a) homologous recombination and (b) marker less gene disruption using ClosTron tool as explained in Example 1 and Example 3.
(a) Δ2,3bdh ΔSecAdh Double Knockout C. autoethanogenum DSM23693 Strain by Homologous Recombination:
The  ̃1 kb 5′ (Seq. ID. 94) and 3′ (Seq. ID. 95) homology arms of 2,3bdh genes are PCR amplified using C. autoethanogenum DSM23693 genomic DNA. Primers Og13f (Seq. ID. 96)/Og14r (Seq. ID. 97) and Og15f (Seq. ID. 98)/Og16r (Seq. ID. 99) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151. plasmids between the Sbfl/Notl. and Nhel/Ascl sites to get pMTL85151-2,3bdh-KO. This plasmid is introduced into C. autoethanogenum DSM23693 either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transformants are screened for 2,3bdh knockout using the primers Og33f (Seq. ID.100) and Og34r (Seq. ID.101) that flank the homology arms of 2,3bdh for PCR and sequencing of this PCR product.
The plasmid for SecAdh gene knockout is similarly constructed. The  ̃1 kb 5′ (Seq. ID. 102) and 3′ (Seq. ID. 103) homology arms of SecAdh genes are PCR amplified using C. autoethanogenum DSM23693 genomic DNA. Primers Sec5f (Seq. ID. 104)/Sec5r (Seq. ID. 105) and Sec3f (Seq. ID. 106)/Sec3r (Seq. ID. 107) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-SecAdh-KO. Following selection on thiamphenicol plates the transformants are screened for SecAdh knockout using the primers SecOf (Seq. ID.108) and SecOr (Seq. ID.109) that flank the homology arms of SecAdh gene for PCR.
Once having achieved the knockout of either 2,3bdh or the SecAdh genes in C. autoethanogenum DSM23693, the second gene in these single mutants is targeted using either pMTL85151-2,3bdh-KO or pMTL85151-SecAdh-KO plasmids. The plasmid is introduced into the single gene knockout mutant either by electroporation or by conjugation as already described in Example 1 and 3. The transforrnants are screened for the knockout of the second gene using the primers flanking the homology arms of the corresponding genes.
(b) Δ2,3bdh ΔSecAdh Double Gene Disruption Using ClosTron:
The RAM ermB cassette in the ClosTron group II intron construct is flanked by Flippase Recombination sites (Frt). By introducing flippase recombinase into Δ2,3bdh ClosTron mutant either by conjugation or by electroporation, the RAM ermB marker of  ̃1.3 kb is removed from the genome of the mutant and thus the ermB marker is recycled. A  ̃0.8 kb fragment of group II intron will be left on the genome. This is confirmed by (i) testing its susceptibility to clarithromycin and (ii) by PCR with the primers flanking the group II intron insertion site with the primers Og42f (Seq. ID. 84) and Og43r (Seq. ID. 85) and sequencing of the PCR product. Once obtaining the Δ2,3bdh ClosTron mutant without RAM ermB marker (Δ2,3bdh-ermB ClosTron), the SecAdh gene in the mutant is targeted in a similar way using ClosTron group II intron insertional inactivation tool. The intron insertion site between bases 399 and 400 on the sense strand is identified in the SecAdh gene using Perutka algorithm hosted at ClosTron.com and the intron targeting cassette has been designed (Seq. ID. 110). The intron targeting cassette is commercially synthesized by DNA2.0 and delivered in pMTL007C-E2 vector as pMTL007C-E5-SecAdh-399!400s which is introduced into Δ2,3bdh-ermB ClosTron mutant by either conjugation or electroporation. The transformants are sequentially selected on thiamphenicol and clarithromycin agar plates and screened by PCR with primers SecCTf (Seq. ID. 111) and SecCTr (Seq. ID. 112) as explained earlier in Example 3.
The Δ2,3bdh ΔSecAdh double gene disrupted C. autoethanogenum DSM23693 mutant is created by using either homologous recombination technique or by ClosTron group II intron insertional inactivation tool as explained in the above paragraphs.
The disruption of 2,3bdh and SecAdh genes and the production of acetoin, other metabolites and 2,3-BDO is confirmed by performing enzyme activity assays for the conversion of acetoin to 2,3-BDO and also by analysing the products produced by the mutant by HPLC, as previously described.
Example 5
Modified C. autoethanogenum DSM23693 Strain Producing Reduced or No 2,3-BDO
As shown in FIGS. 1a and 1b, acetolactate is one of the intermediates in 2,3-BDO biosynthesis and is also the precursor for the synthesis of branched chain amino acids. The enzyme acetolactate synthase catalyses the reaction leading to acetolactate from 2 molecules of pyruvate as substrates. The enzyme acetolactate synthase is broadly classified into two groups; (i) anabolic acetolactate synthase is associated with the genes involved in the synthesis of branched amino acids like valine, isoleucine and leucine and (ii) catabolic acetolactate synthase is associated with 2,3-BDO synthesis (alsS; amino acid—AE190719.1 and nucleic acid—HQ876013.1).
The genome of C. autoethanogenum DSM23693 has 3 putative anabolic acetolactate synthase genes, ilvC, ilvl and ilvB. Exemplary amino acid sequence from C. autoethanogenum (AEI90719.1AE190730.1, AEI90731.1, AEI90713.1, AEI90714,1), C. ljungdahlii (ADK15104.1, ADK15104.1 ADK15105.1ADK15400.1, ADK15400.1), and C. ragsdalei (AEI90734.1, AEI90734.1, AEI90735.1, AEI90727.1, AEI90727.1) and respective nucleic acid sequences from C. autoethanogenum (HQ876013.1, HQ876023.1, HQ876021.1), C. Ijungdahiii (CP001666.1—CLJU_c38920, CLJU_c32420, CLJU_c20420-30), and C. ragsdalei (HQ876014.1, HQ876024.1, HQ876022.1) are obtained from GenBank.
The disruption of all 4 acetolactate synthase genes or any combination of these 4 genes should lead to a decrease in acetoin and 2,3-BDO production. In order to ensure the growth of these mutants the media is supplemented with the three branched chain amino acids valine, leucine and isoleucine.
As described in Examples 1, 3 and 4 single mutants of C. autoethanogenum DSM23693 alsS, ilvC, ilvl and ilvB mutants can be created by either homologous recombination or using ClosTron group II intron mutagenesis tool.
Design of alsS, ilvC, ilvi and ilvB Knockout Cassettes:
The knockout constructs for alsS, ilvC, livl and ilvB genes are designed as explained above. The  ̃1 kb 5′ (Seq. ID. 113) and 3′ (Seq. ID. 114) homology arms of alsS gene are PCR amplified using C. autoethanogenum DSM23693 genomic DNA. Primers alsS5f (Seq. ID. 115)/alsS5r (Seq. ID. 116) and alsS3f (Seq. ID. 117)/alsS3r (Seq. ID. 118) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-alsS-KO. This plasmid is introduced into C. autoethanogenum DSM23693 either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transformants are screened for alsS knockout using the primers alsSOf (Seq. ID.119) and alsSOr (Seq. ID.120) that flank the homology arms of alsS for PCR and sequencing of this PCR product.
For knockout of ilvC gene, the  ̃1 kb 5′ (Seq. ID. 121) and 3′ (Seq. ID. 122) homology arms of ilvC gene are PCR amplified using C. autoethanogenum DSM23693 genomic DNA. Primers ilvC5f (Seq. ID. 123)/ilvC5r (Seq. ID. 124) and ilvC3f (Seq. ID. 125)/ilvC3r (Seq. ID. 126) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-ilvC-KO. This plasmid is introduced into C. autoethanogenum DSM23693 either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transformants are screened for ilvC knockout using the primers ilvCOf (Seq. ID. 127) and ilvCOr (Seq. ID. 128) that flank the homology arms of ilvC gene for PCR and sequencing of this PCR product.
For knockout of ilvl gene, the ̃1 kb 5′ (Seq. ID. 129) and 3′ (Seq. ID. 130) homology arms of ilvl gene are PCR amplified using C. autoethanogenum DSM23693 genomic DNA. Primers ilvi5f (Seq. ID. 131)/ilvl5r (Seq. ID, 132) and ilvl3f (Seq. ID. 133)/ilvI3r (Seq. ID. 134) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-ilvl-KO. This plasmid is introduced into C. autoethanogenum DSM23693 either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transthrmants can be screened for ilvl knockout using the primers ilvlOf (Seq. ID.135) and ilvIOr (Seq. ID. 136) that flank the homology arms of ilvI gene for PCR and sequencing of this PCR product.
For knockout of ilvB gene, the  ̃1 kb 5′ (Seq. ID. 137) and 3′ (Seq. ID. 138) homology arms of ilvB gene are PCR amplified using C. autoethanogenum DSM23693 genomic DNA. Primers ilvB5f (Seq. ID. 139)/ilvB5r (Seq. ID. 140) and ilvB3f (Seq. ID. 141)/ilvB3r (Seq. ID. 142) are used to amplify the 5′ and 3′ homology aims, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-ilvB-KO. This plasmid are introduced into C. autoethanogenum DSM23693 either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transformants are screened for ilvB knockout using the primers ilvBOf (Seq. ID.143) and ilvBOr (Seq. ID.144) that flank the homology arms of ilvB gene for PCR and sequencing of this PCR product.
Once the single gene knockout mutants are obtained the other 3 acetolactate synthase genes are sequentially targeted to create a mutant having all 4 acetolactate synthase genes deleted. The growth of these mutants may be auxotrophic to branched chain amino acids. The production or lack of production of acetoin, 2,3-BDO and other metabolites in these mutants can be analysed by HPLC, as described for the previous examples. The enzyme activity assays with pyruvate as substrate and thiamine diphosphate and flavin adenine dinucleotide as cofactors can be performed to confirm for the loss of acetolactate synthase activity in these mutants (rittinann, Vyazmensky, Hubner, Barak & Chipman, 2005; Vinogradov et al, 2006).
Design of ClosTron Group II Intron Targeting Cassettes for alsS, ilvC, ilvl and Genes:
C. autoethanogenum DSM23693 alsS, ilvC, ilvl and ilvB genes can also be disrupted or inactivated using ClosTron group II intron mediated gene disruption tool (Heap et al., 2010). The Perutka algorithm hosted at ClosTron.com is used to identify the group II intron target site between bases 303/304, 228/229, 975/976 and 157/158 on the sense strand of alsS, ilvC, ilvl and on the antisense strand of ilvB genes, respectively. Other sites identified by the algorithm can also be targeted. The same algorithm has been used to design the intron targeting regions (alsS—Seq. ID.145; ilvC—Seq. ID.146; ilvl—Seq. ID.147 and ilvB—Seq. ID.148) which can be commercially synthesized by DNA2.0 and delivered in pMTL007C-E2 vector. The final vectors, pMTL007C-E2-alsS-303!304s, pMTL007C-E2-ilvC-228!229s, pMTL007C-E2-livl-975!976s and pMTL007C-E2-ilvB-157!158a, contain a Retro-tranposition-Activated ermB Marker (RAM) which confers resistance to antibiotic Clarithromycin upon insertion into the target site. These plasmids are introduced into C. autoethanogenum DSM23693 by either conjugation or electroporation. The transformants are sequentially selected on thiamphenicol and clarithromycin agar plates and screened by PCR with primers alsSCTf (Seq. ID. 149) and alsSCTr (Seq. ID. 150), ilvCCTf (Seq. ID. 151) and ilvCCTr (Seq. ID. 152), ilvICTf (Seq. ID. 153) and ilvICTr (Seq. ID. 154) and ilvBCTf (Seq. ID. 155) and ilvBCTr (Seq. ID. 156) for inactivation of alsS, ilvC, ilvl and ilvB genes, respectively.
Once ClosTron mutants with single gene disrupted are obtained, the RAM ermB cassette is removed from the genome of these mutants using pMTL plasmids carrying a flippase gene which is introduced into the mutant by either electroporation or by conjugation. The resulting transformants are screened for the toss of ermB cassette by testing its susceptibility to clarithromycin and (ii) by PCR with the primers flanking the group II intron insertion site with alsSCTf (Seq. ID. 149) and alsSCTr (Seq. ID. 150), ilvCCTf (Seq. ID. 151) and ilvCCTr (Seq. ID. 152), iivICTf (Seq. ID. 153) and ilvBICTr (Seq. ID. 154) and ilvBCTf (Seq. ID. 155) and ilvBCTr (Seq. ID. 156) in alsS, ilviC, and ilvB2 genes, respectively, and by further sequencing of these PCR product.
After confirming the loss of ermB cassette, the ClosTron mutants like the knockout mutants are sequentially targeted for the inactivation of other acetolactate synthase genes. In one embodiment, these ClosTron mutants are grown in the presence of branched chain amino acids. The production or lack of production of acetoin, 2,3-BDO and other metabolites in these mutants can be analysed by HPLC as described in previous examples. The enzyme activity assays with pyruvate as substrate and thiamine diphosphate and flavin adenine dinucleotide as cofactors can be performed to confirm for the loss of acetolactate synthase activity in these mutants (Tittman et al, 2005; Vinogradov et al, 2006).
Example 6
Disruption of 2,3-BDO Pathway Genes in C. ljungdhalii and C. ragsdalei 
The pathway for 2,3-BDO production is conserved across acetogens including C. autoethanogenum, C. ljungdahlii and C. ragsdalei. The alsS, ilvC, ivvI, ilvB, budA, 2,3bdh and SecAdh genes in the three acetogens share high degree of sequence homology. Hence these genes can be genetically modified to increase or decrease the 2,3-BDO production in the three acetogens. Method to genetically modify C. ljungdahlii by electroporation have been described (Kapke et al., 2010) (PCT/NZ2011/000203). Electroporation and conjugation methods that have been described above for C. autoethanogenum can be applied to C. ragsdalei by any skilled person.
The amino acid and nucleic acid sequences for C. ljungdahlii and C. ragsdalei alsS, ilvC, ilvB1, ilvB2, budA, and 2,3bdh genes can be obtained from GenBank. The C. ljungdahlii (Seq. ID. 36) and C. ragsdalei (Seq. ID. 37) SecAdh nucleotide sequences are provided.
The knockout and ClosTron plasmids that were used to disrupt alsS, ilvC, ilvB1 ilvB2, budA, 2,3bdh and SecAdh genes by homologous recombination and ClosTron group II intron based insertional inactivation in C. autoethanogenum can also be used to disrupt the same genes C. ljungdahlii and C. ragsdalei. For example pMTL85141-budA-ko, pMTL007C-E5-budA-450!451 s and pMTL007C-E5-2,3bdh-468!469s can be introduced into C. ljungdahlii (explained below in Example 6a) and C. ragsdalei (explained below in Example 6b) by either electroporation or conjugation as described above for C. autoethanogenum in Examples 1 and 3. Similar mutant screening and characterization methods can be applied in C. ljungdahlii and C. ragsdalei. 
Example 6a
Disruption of budA and 2,3bdh Genes in C. ljungdahlii by Homologous Recombination and Group II Intron Based Insertional Inactivation Tool for No and Reduced 2,3-BDO Production
Plasmids pMTL85141-budA-ko is introduced into C. ljungdahlii by electroporation (Koepke et al 2010). The transformants are selected on PETC-agar plates containing 15 μg/ml thiamphenicol and screened for budA knockout using primers Og44f (Seq. ID. 29) and Og45r (Seq. ID. 30)
For budA and 2,3bdh gene disruptions in C. ljungdahlii using ClosTron group II intron based insertional inactivation tool, plasmids pMTL007C-E5-budA-450!451s and pMTL007C-E5-2,3bdh-468!469s are introduced into C. ljungdahlii by conjugation. Streaks of single colonies following conjugation are made sequentially first on PETC agar media containing 15 μg/ml thiamphenicol and 10 μg/ml trimethoprim followed by on agar plates with PETC media containing 5 μg/ml Clarithromycin. Colonies per plasmid are randomly screened for group II intron insertion by PCR using primers Og44f (Seq. ID. 29) and Og45r (Seq. ID. 30), flanking the group II intron insertion site in budA gene, and primers Og42f (Seq. ID. 84) and Og43r (Seq. ID. 85), flanking the group II intron insertion site in 2,3-bdh gene.
The budA and 2,3bdh knockout and ClosTron C. ljungdahlii mutants generated above are analyzed for 2,3-BDO and acetoin production by HPLC and fermentation in bioreactors as explained in Examples 1 and 3.
Example 6b
Disruption of budA and 2,3bdh Genes in C. ragsdalei by Homologous Recombination and Group II Intron Based Insertional Inactivation Tool for No and Reduced 2,3-BDO Production
Plasmids pMTL85141-budA-ko is introduced into C. ragsdalei by electroporation as described above for C. autoethaogenum or C. ljungdahlii, either by electroporation or conjugation. The transformants are selected on PETC-agar plates containing 15 μg/ml thiamphenicol and screened for budA knockout using primers Og44f (Seq. ID. 29) and Og45r (Seq. ID. 30)
For budA and 2,3bdh gene disruptions in C. ragsdalei using ClosTron group II intron based insertional inactivation tool, plasmids pNITL007C-E5-budA-450!45 is and pMIL007C-E5-2,3bdh-468!469s are introduced into C. ragsdalei by conjugation. Streaks of single colonies following conjugation are made sequentially first on PETC agar media containing 15 μg/ml thiamphenicol and 10 μg/ml trimethoprim followed by on agar plates with PETC media containing 5 μg/ml Clarithromycin. Colonies per plasmid are randomly screened for group II intron insertion by PCR using primers Og44f (Seq. ID. 29) and Og45r (Seq. ID. 30), flanking the group II intron insertion site in budA gene, and primers Og42f (Seq. ID. 84) and Og43r (Seq, ID. 85), flanking the group II intron insertion site in 2,3-bdh gene.
The budA and 2,3bdh knockout and ClosTron C. ragsdalei mutants generated above are anlyzed for 2,3-BDO and acetoin production by HPLC and fermentation in bioreactors as explained in Examples 1 and 3.
Example 7
Modified C. ljungdahlii Producing Only Acetoin
As explained earlier, separation of acetoin from ethanol is technically more feasible compared to 2,3-BDO. It is thus desirable to have a C. ljungdahlii strain producing acetoin and not 2,3-BDO. This will be achieved by deleting or disrupting both 2,3bdh and SecAdh genes in two ways as explained in Example 6a.: (a) homologous recombination and (b) marker less gene disruption using ClosTron tool.
(a) Δ2,3bdh ΔSecAdh Double Knockout C. ljungdahlii Strain by Homologous Recombination:
The  ̃1 kb 5′ (Seq. ID. 94) and 3′ (Seq. ID. 95) homology arms of 2,3bdh genes are PCR amplified using C. ljungdahlii genomic DNA. Primers Og13f (Seq, ID. 96)/Og14r (Seq. ID. 97) and Og15f (Seq. ID. 98)/Og16r (Seq. ID. 99) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-2,3bdh-KO. This plastaid is introduced into C. ljungdahlii either by conjugation or by electroporation as described in the above Example 6a. Following selection on thiamphenicol plates the transformants are screened for 2,3bdh knockout using the primers Og33f (Seq. ID.100) and Og34r (Seq. ID.101) that flank the homology arms of 2,3bdh for PCR and sequencing of this PCR product.
The plasmid for SecAdh gene knockout is similarly constructed. The  ̃1 kb 5′ (Seq. ID. 102) and 3′ (Seq. ID. 103) homology arms of SecAdh genes are PCR amplified using C. ljungdahlii genomic DNA. Primers Sec5f (Seq. ID. 104)/Sec5r (Seq. ID. 105) and Sec3f (Seq. ID. 106)/Sec3r (Seq. ID. 107) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-SecAdh-KO. Following selection on thiamphenicol plates the transformants are screened for SecAdh knockout using the primers SecOf (Seq. ID.108) and SecOr (Seq. ID.109) that flank the homology arms of SecAdh gene for PCR.
Once having achieved the knockout of either 2,3bdh or the SecAdh genes in C. ljungdahlii, the second gene in these single mutants is targeted using either pMTL85151-2,3bdh-KO or ‘pMTL85151-SecAdh-KO plasmids. The plasmid is introduced into the single gene knockout mutant either by electroporation or by conjugation as already described in Example 6a. The transformants are screened for the knockout of the second gene using the primers flanking the homology arms of the corresponding genes.
(b) Δ2,3bdh ΔSecAdh Double Gene Disruption Using ClosTron in C. ljungdahlii: 
The RAM ermB cassette in the ClosTron group II intron construct is flanked by Flippase Recombination sites (Frt). By introducing flippase recombinase into Δ2,3bdh ClosTron mutant either by conjugation or by electroporation, the RAM ermB marker of  ̃1.3 kb is removed from the genome of the mutant and thus the ermB marker can be recycled. A  ̃0.8 kb fragment of group II intron will be left on the genome. This is confirmed by (i) testing its susceptibility to clarithromycin and (ii) by PCR with the primers flanking the group II intron insertion site with the primers Og42f (Seq. ID. 84) and Og43r (Seq. ID. 85) and sequencing of the PCR product. Once obtaining the Δ2,3bdh ClosTron mutant without RAM ermB marker (Δ2,3bdh-ermB ClosTron), the SecAdh gene in the mutant is targeted in a similar way using ClosTron group II intron insertional inactivation tool. The intron insertion site between bases 399 and 400 on the sense strand is identified in the SecAdh gene using Perutka algorithm hosted at ClosTron.com and the intron targeting cassette is designed (Seq. ID. 110). The intron targeting cassette is commercially synthesized by DNA2.0 and delivered in pMTL007C-E2 vector as pMTL007C-E5-SecAdh-399!400s which is introduced into Δ2,3bdh-ermB ClosTron mutant by either conjugation or electroporation. The transformants are sequentially selected on thiamphenicol and clarithromycin agar plates and screened by PCR with primers SecCTf (Seq. ID. 111) and SecCTr (Seq. ID. 112) as explained earlier in Example 6a.
The Δ2,3bdh ΔSecAdh double gene disruption C. ljungdahlii mutant is created by using either homologous recombination technique or by ClosTron group II intron insertional inactivation tool as explained in the above paragraphs.
The disruption of 2,3bdh and SecAdh genes and the production of metabolites and 2,3-BDO is confirmed by performing enzyme activity assays for the conversion of acetoin to 2,3-BDO and also by analysing the products produced by the mutant by HPLC, as previously described.
Example 8
Modified C. ljungdahlii Strain Producing Reduced or No 2,3-BDO
As shown in FIGS. 1a and 1b, acetolactate is one of the intermediates in 2,3-BDO biosynthesis and is also the precursor for the synthesis of branched chain amino acids. The enzyme acetolactate synthase catalyses the reaction leading to acetolactate from 2 molecules of pyruvate as substrates. The enzyme acetolactate synthase is broadly classified into two groups; (i) anabolic acetolactate synthase is associated with the genes involved in the synthesis of branched amino acids like valine, isoleucine and leucine and (ii) catabolic acetolactate synthase is associated with 2,3-BDO synthesis.
The genome of C. ljungdahlii has 3 putative anabolic acetolactate synthase genes, ilvC, ilvl and ilvB and 1 catabolic acetolactate synthase, alsS. Exemplary amino acid sequence from C. ijungdahlii (ADK15104.1, ADK15104.1, ADK15105.1, ADK15400.1, ADK15400.1) and respective nucleic acid sequences from C. ljungdahlii (CP001666.1, (CLJU_c38920, CLJU_c32420, CLJU_c20420-30) are obtained from GenBank.
The disruption of all 4 acetolactate synthase genes or any combination of these 4 genes should lead to a decrease in acetoin and 2,3-BDO production. In order to ensure the growth of these mutants the media is supplemented with the three branched chain amino acids valine, leucine and isoleucine.
As described in Examples 6a, and 7 single mutants of C. ljungdahlii alsS, ilvC, ilvI and ilvB mutants can be created by either homologous recombination or using ClosTron group II intron mutagenesis tool.
Design of alsS, IlvC, ilvl and ilvB Knockout Cassettes:
The knockout constructs for alsS, ilvC, ilvl and ilvB genes are designed as explained above.
The  ̃1 kb 5′ (Seq. ID. 113) and 3′ (Seq. ID. 114) homology arms of alsS gene are PCR amplified using C. ljungdahlii genomic DNA. Primers alsS5f (Seq. ID. 115)/alsSr (Seq. ID. 116) and alsS3f (Seq. ID. 117) / alsS3r (Seq. ID. 118) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-alsS-KO. This plasmid is introduced into C. ljungdahlii either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transformants are screened for alsS knockout using the primers alsSOf (Seq. ID.119) and alsSOr (Seq. ID.120) that flank the homology arms of alsS for PCR and sequencing of this PCR product.
For knockout of ilvC gene, the  ̃1 kb 5′ (Seq. ID. 121) and 3′ (Seq. ID. 122) homology arms of ilvC gene are PCR amplified using C. ljungdahlii genomic DNA. Primers ilvC5f (Seq. ID. 123)/ilvC5r (Seq. ID. 124) and ilvC3f (Seq. ID. 125)/ilvC3r (Seq. ID. 126) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-ilvC-KO. This plasmid is introduced into C. ijungdahlii either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transformants are screened for ilvC knockout using the primers iivCOf (Seq. ID. 127) and ilvCOr (Seq. ID. 128) that flank the homology arms of ilvC gene for PCR and sequencing of this PCR product.
For knockout of ilvl gene, the  ̃1 kb 5′ (Seq. ID. 129) and 3′ (Seq. ID. 130) homology arms of ilvl gene are PCR amplified using C. ljungdahlii genomic DNA. Primers ilvl5f (Seq. ID. 131)/ilvI5r (Seq. ID. 132) and ilvI3f (Seq. ID. 133)/ilvI3r (Seq. ID. 134) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-ilvl-KO. This plasmid is introduced into C. ljungdahlii either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transforrnants are screened for ilvI knockout using the primers ilvIOf (Seq. ID.135) and ilvIOr (Seq. ID. 136) that flank the homology arms of ilvi gene for PCR and sequencing of this PCR product.
For knockout of ilvB gene, the  ̃1 kb 5′ (Seq. ID. 137) and 3′ (Seq. ID. 138) homology arms of ilvB gene are PCR amplified using C. ljungdahlii genomic DNA. Primers ilvB5f (Seq. ID. 139)/ilvB5r (Seq. ID. 140) and ilvB3f (Seq. ID. 141)/ilvB3r (Seq. ID. 142) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-ilvB-KO. This plasmid is introduced into C. ljungdahlii either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transformants are screened for ilvB knockout using the primers ilvBOf (Seq. ID.143) and ilvBOr (Seq. ID.144) that flank the homology arms of ilvB gene for PCR and sequencing of this PCR product.
Once the single gene knockout mutants are obtained the other 3 acetolactate synthase genes are sequentially targeted to create a mutant having all 4 acetolactate synthase genes deleted. The growth of these mutants may be auxotrophic to branched chain amino acids. The production or lack of production of acetoin, 2,3-BDO and other metabolites in these mutants can be analysed by HPLC, as described for the previous examples. The enzyme activity assays with pyruvate as substrate and thiamine diphosphate and flavin adenine dinucleotide as cofactors can be performed to confirm for the loss of acetolactate synthase activity in these mutants (Tittmann, Vyazmensky, Hubner, Barak, & Chipman, 2005; Vinogradov et al., 2006).
Design of ClosTron Group II Intron Targeting Cassettes for alsS, ilvC, ilvl and ilvB Genes:
C. ljungdahlii alsS, ilvC, ilvI and ilvB genes can also be disrupted or inactivated using ClosTron group II intron mediated gene disruption tool (Heap et al., 2010). The Perutka algorithm hosted at ClosTron.com is used to identify the group II intron target site between bases 303/304, 228/229, 975/976 and 157/158 on the sense strand of alsS, ilvC, ilvI and antisense strand of ilvB genes, respectively. Other sites identified by the algorithm can also be targeted. The same algorithm is used to design the intron targeting regions (alsS—Seq. ID.145; ilvC—Seq. ID.1.46; ilvI—Seq. ID.147 and ilvB—Seq. ID.148) which is commercially synthesized by DNA2.0 and delivered in pMTL007C-E2 vector. The final vectors, pMTL007C-E2-alsS-303!304s, pMTL007C-E2-ilvC-228!229s, pMTL007C-E2-ilvI-975!976s and pMTL007C-E2-ilvB-157!158a, contain a Retro-tranposition-Activated ermB Marker (RAM) which confers resistance to antibiotic Clarithromycin upon insertion into the target site. These plasmids are introduced into C. ljungdahlii by either conjugation or electroporation. The transformants are sequentially selected on thiamphenicol and clarithromycin agar plates and screened by PCR with primers alsSCTf (Seq. ID. 149) and alsSCTr (Seq. ID. 150), ilvCCTf (Seq. ID. 151) and ilvCCTr (Seq. ID. 152), ilvICTf (Seq. ID. 153) and ilvICTr (Seq. ID. 154) and ilvBCTf (Seq. ID. 155) and ilvBCTr (Seq. ID. 156) for inactivation of alsS, ilvC, ilvI and ilvB genes, respectively.
Once ClosTron mutants with single gene disrupted are obtained, the RAM ermB cassette is removed from the genome of these mutants using pMTL plasmids carrying a flippase gene which is introduced into the mutant by either electroporation or by conjugation. The resulting transformants are screened for the loss of ermB cassette by testing its susceptibility to clarithromycin and (ii) by PCR with the primers flanking the group II intron insertion site with alsSCTf (Seq. ID. 149) and alsSCTr (Seq. ID. 150), ilvCCTf (Seq. ID. 151) and ilvCCTr (Seq. ID. 152), ilvICTf (Seq. ID. 153) and ilvICTr (Seq. ID. 154) and ilvBCTf (Seq. ID. 155) and ilvBCTr (Seq. ID. 156) in alsS, ilvC, ilvB1 and ilvB2 genes, respectively, and by further sequencing of these PCR product.
After confirming the loss of ermB cassette, the ClosTron mutants like the knockout mutants are sequentially targeted for the inactivation of other acetolactate synthase genes. In one embodiment, these ClosTron mutants are grown in the presence of branched chain amino acids. The production or lack of production of acetoin, 2,3-BDO and other metabolites in these mutants is analysed by HPLC as described in previous examples and studied by performing enzyme activity assays with pyruvate as substrate and thiamine diphosphate and flavin adenine dinucleotide as cofactors can be performed to confirm for the loss of acetolactate synthase activity in these mutants (Tittmann et al., 2005; Vinogradov et al., 2006).
Example 9
Modified C. ragsdalei Producing Only Acetoin
As explained earlier, separation of acetoin from ethanol is technically more feasible compared to 2,3-BDO. It is thus desirable to have a C. ragsdalei strain producing acetoin and not 2,3-BDO. This will be achieved by deleting or disrupting both 2,3bdh and SecAdh genes in two ways as explained in Example 6b,: (a) homologous recombination and (b) marker less gene disruption using ClosTron tool.
(a) Δ2,3bdh ΔSecAdh Double Knockout C. ragsdalei Strain by Homologous Recombination:
The  ̃1 kb 5′ (Seq. ID. 94) and 3′ (Seq. ID. 95) homology arms of 2,3bdh genes are PCR amplified using C. ragsdalei genomic DNA. Primers Og13f (Seq. ID. 96)/Og14r (Seq. ID. 97) and Og15f (Seq. ID. 98)/Og16r (Seq. ID. 99) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-2,3bdh-KO. This plasmid is introduced into C. ragsdalei either by conjugation or by electroporation as described in the above Example 6b. Following selection on thiamphenicol plates the transformants are screened for 2,3bdh knockout using the primers Og33f (Seq. ID.100) and Og34r (Seq. ID.101) that flank the homology arms of 2,3bdh for PCR and sequencing of this PCR product.
The plasmid for SecAdh gene knockout is similarly constructed. The  ̃1 kb 5′ (Seq. ID. 102) and 3′ (Seq. ID. 103) homology arms of SecAdh genes are PCR amplified using C. ragsdalei genomic DNA. Primers Sec5f (Seq. ID. 104)/Sec5r (Seq. ID. 105) and Sec3f (Seq. ID. 106) Sec3r (Seq. ID. 107) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-SecAdh-KO. Following selection on thiamphenicol plates the transformants are screened for SecAdh knockout using the primers SecOf (Seq. ID.108) and SecOr (Seq. ID.109) that flank the homology arms of SecAdh gene for PCR.
Once having achieved the knockout of either 2,3bdh or the SecAdh genes in C. ragsdalei, the second gene in these single mutants is targeted using either pMTL85151-2,3bdh-KO or pMTL85151-SecAdh-KO plasmids. The plasmid is introduced into the single gene knockout mutant either by electroporation or by conjugation as already described in Example 6b. The transformants are screened for the knockout of the second gene using the primers flanking the homology arms of the corresponding genes.
(b) Δ2,3bdh ΔSecAdh Double Gene Disruption Using ClosTron in C. ragsdalei: 
The RAM ermB cassette in the ClosTron group II intron construct is flanked by Flippase Recombination sites (Frt). By introducing flippase recombinase into Δ2,3bdh ClosTron mutant either by conjugation or by electroporation, the RAM ermB marker of  ̃1.3 kb is removed from the genome of the mutant and thus the ermB marker can be recycled. A  ̃0.8 kb fragment of group II intron will be left on the genome. This is confirmed by (i) testing its susceptibility to clarithromycin and (ii) by PCR with the primers flanking the group II intron insertion site with the primers Og42f (Seq. ID. 84) and Og43r (Seq. ID. 85) and sequencing of the PCR product. Once obtaining the Δ2,3bdh ClosTron mutant without RAM ermB marker (Δ2,3bdh-ermB ClosTron), the SecAdh gene in the mutant is targeted in a similar way using ClosTron group II intron insertional inactivation tool. The intron insertion site between bases 399 and 400 on the sense strand is identified in the SecAdh gene using Perutka algorithm hosted at ClosTron.com and the intron targeting cassette has been designed (Seq. ID. 110). The intron targeting cassette is commercially synthesized by DNA2.0 and delivered in pMTL007C-E2 vector as pMTL007C-E5-SecAdh-399!400s which is introduced into Δ2,3bdh-ermB ClosTron mutant by either conjugation or electroporation. The transformants can be sequentially selected on thiamphenicol and clarithromycin agar plates and screened by PCR with primers SecCTf (Seq. ID. 111) and SecCTr (Seq. ID. 112) as explained earlier in Example 6b.
The Δ2,3bdh ΔSecAdh double gene disruption C. ragsdalei mutant is created by using either homologous recombination technique or by ClosTron group II intron insertional inactivation tool as explained in the above paragraphs.
The disruption of 2,3bdh and SecAdh genes is confirmed by performing enzyme activity assays for the conversion of acetoin to 2,3-BDO and also by analysing the metabolites and 2,3-BDO produced by the mutant by HPLC, as previously described.
Example 10
Modified C. ragsdalei Strain Producing Reduced or No 3-BDO
As shown in FIG. 1 acetolactate is one of the intermediates in 2,3-BDO biosynthesis and is also the precursor for the synthesis of branched chain amino acids. The enzyme acetolactate synthase catalyses the reaction leading to acetolactate from 2 molecules of pyruvate as substrates. The enzyme acetolactate synthase is broadly classified into two groups; (i) anabolic acetolactate synthase is associated with the genes involved in the synthesis of branched amino acids like valine, isoleucine and leucine and (ii) catabolic acetolactate synthase is associated with 2,3-BDO synthesis).
The genome of C. ragsdalei has 3 putative anabolic acetolactate synthase genes, ilvC, ilvI and ilvB and 1 catabolic acetolactate synthase, alsS. Exemplary amino acid sequence from C. ragsdalei (AEI90734.1, AEI90734.1, AEI90735.1, AE190727.1, AE190727.1) and respective nucleic acid sequences HQ876014.1, HQ876024.1, HQ876022.1) are obtained from GenBank.
The disruption of all 4 acetolactate synthase genes or any combination of these 4 genes should lead to a decrease in acetoin and 2,3-BDO production. In order to ensure the growth of these mutants the media is supplemented with the three branched chain amino acids valine, leucine and isoleucine.
As described in Examples 6b, and 9 single mutants of C. ragsdalei alsS, ilvC, ilvl and ilvB mutants can be created by either homologous recombination or using ClosTron group II intron mutagenesis tool.
Design of alsS, ilvC, ilvI and ilvB Knockout Cassettes:
The knockout constructs for alsS, ilvC, ilvI and ilvB Genes are Designed as Explained Above.
The  ̃1 kb 5′ (Seq. ID. 113) and 3′ (Seq. ID. 114) homology arms of alsS gene are PCR amplified using C. ragsdalei genomic DNA. Primers alsS5f (Seq. ID. 115)/alsSr (Seq. ID. 116) and alsS3f (Seq. ID. 117)/alsS3r (Seq. ID. 118) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-alsS-KO. This plasmic is introduced into C. ragsdalei either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transformants are screened for alsS knockout using the primers alsSOf (Seq. ID.119) and alsSOr (Seq. ID.120) that flank the homology arms of alsS for PCR and sequencing of this PCR product.
For knockout of ilvC gene, the  ̃1 kb 5′ (Seq. ID. 121) and 3′ (Seq. ID. 122) homology arms of ilvC gene are PCR amplified using C. ragsdalei genomic DNA. Primers ilvCSf (Seq. ID. 123)/ilvC5r (Seq. ID. 124) and ilvC3f (Seq. ID. 125)/ilvC3r (Seq. ID. 126) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-ilvC-KO. This plasmid is introduced into C. ragsdalei either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transformants are screened for ilvC knockout using the primers ilvCOf (Seq. ID. 127) and ilvCOr (Seq. ID. 128) that flank the homology arms of ilvC gene for PCR and sequencing of this PCR product.
For knockout of ilvI gene, the  ̃1 kb 5′ (Seq. ID. 129) and 3′ (Seq. ID. 130) homology arms of ilvI gene are PCR amplified using C. ragsdalei genomic DNA. Primers ilvI5f (Seq. ID. 131)/ilvI5r (Seq. ID. 132) and ilvI3f (Seq. ID. 133)/ilvI3r (Seq. ID. 134) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-ilvI-KO. This plasmid is introduced into C. ragsdalei either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transformants are screened for ilvI knockout using the (primers ilvIOf (Seq. ID. 135) and ilvIOr (Seq. ID. 136) that flank the homology arms of ilvI gene for PCR and sequencing of this PCR product.
For knockout of ilvB gene, the  ̃1 kb 5′ (Seq. ID. 137) and 3′ (Seq. ID. 138) homology arms of ilvB gene are PCR amplified using C. ragsdalei genomic DNA. Primers ilvB5f (Seq. ID. 139)/ilvB5r (Seq. ID. 140) and ilvB3f (Seq. ID. 141)/ilvB3r (Seq. ID. 142) are used to amplify the 5′ and 3′ homology arms, respectively. The two PCR products are cloned into pMTL85151 plasmids between the Sbfl/Notl and Nhel/Ascl sites to get pMTL85151-ilvB-KO. This plasmid is introduced into C. ragsdalei either by conjugation or by electroporation as described in the above examples. Following selection on thiamphenicol plates the transformants are screened for ilvB knockout using the primers ilvBOf (Seq. ID.143) and ilvIBOr (Seq. ID.144) that flank the homology arms of ilvB gene for PCR and sequencing of this PCR product.
Once the single gene knockout mutants are obtained the other 3 acetolactate synthase genes are sequentially targeted to create a mutant having all 4 acetolactate synthase genes deleted. The growth of these mutants may be auxotrophic to branched chain amino acids. The production or lack of production of acetoin, 2,3-BDO and other metabolites in these mutants can be analysed by HPLC, as described for the previous examples. The enzyme activity assays with pyruvate as substrate and thiamine diphosphate and flavin adenine dinucleotide as cofactors can be performed to confirm for the loss of acetolactate synthase activity in these mutants (Tittmann, Vyazmensky, Hübner, Barak, & Chipman, 2005; Vinogradov et al., 2006).
Design of ClosTron Group II Intron Targeting Cassettes for alsS, ilvC, ilvl and ilvB Genes:
C. ragsdalei alsS, ilvC, ilvl and ilvB genes can also be disrupted or inactivated using ClosTron group II intron mediated gene disruption tool (Heap et al., 2010). The Perutka algorithm hosted at ClosTron.com is used to identify the group II intron target site between bases 303/304, 228/229, 975/976 and 157/158 on the sense strand of alsS, ilvC, ilvl and antisense strand of ilvB genes, respectively. Other sites identified by the algorithm can also be targeted. The same algorithm is used to design the intron targeting regions (alsS—Seq. ID.145; ilvC—Seq. ID.146; ilvI—Seq. ID.147 and ilvB—Seq. ID.148) which is commercially synthesized by DNA2.0 and delivered in pMTL007C-E2 vector. The final vectors, pMTL007C-E2-alsS-303!304s, pMTL007C-E2-ilvC-228!229s, pMTL007C-E2-ilvl-975!976s and pMTL007C-E2-ilvB-157!158a, contain a Retro-tranposition-Activated ermB Marker (RAM) which confers resistance to antibiotic Clarithromycin upon insertion into the target site. These plasmids are introduced into C. ragsdalei by either conjugation or electroporation. The transformants are sequentially selected on thiamphenicol and clarithromycin agar plates and screened by PCR with primers alsSCTf (Seq. ID. 149) and alsSCTr (Seq. ID. 150), ilvCCTf (Seq. ID. 151) and ilvCCTr (Seq. ID. 152), ilvICTf (Seq. ID. 153) and ilvICTr (Seq. ID. 154) and ilvBCIf (Seq. ID. 155) and ilvBCTr (Seq. ID. 156) for inactivation of alsS, ilvC, ilvI and ilvB genes, respectively.
Once ClosTron mutants with single gene disrupted are obtained, the RAM ermB cassette is removed from the genome of these mutants using pMTL plasmids carrying a flippase gene which is introduced into the mutant by either electroporation or by conjugation. The resulting transformants are screened for the loss of ermB cassette by testing its susceptibility to clarithromycin and (ii) by PCR with the primers flanking the group II intron insertion site with alsSCTf (Seq. ID. 149) and alsSCTr (Seq. ID. 150), ilvCCTf (Seq. ID. 151) and ilvCCTr (Seq. ID. 152), ilvICTf (Seq. ID. 153) and ilvICTr (Seq. ID. 154) and ilvBCTf (Seq. ID. 155) and ilvBCTr (Seq. ID. 156) in alsS, ilvC, ilvB1 and ilvB2 genes, respectively, and by further sequencing of these PCR product.
After confirming the loss of ermB cassette, the ClosTron mutants like the knockout mutants are sequentially targeted for the inactivation of other acetolactate synthase genes. In one embodiment, these ClosTron mutants are grown in the presence of branched chain amino acids. The production or lack of production of acetoin, 2,3-BDO and other metabolites in these mutants is analysed by HPLC as described in previous examples and studied by performing enzyme activity assays with pyruvate as substrate and thiamine diphosphate and flavin adenine dinucleotide as cofactors can be performed to confirm for the loss of acetolactate synthase activity in these mutants (Tittmann et al., 2005; Vinogradov et al., 2006).
The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent or substituted for known equivalents without departing from the scope of the invention. It should be appreciated that such modifications and equivalents are herein incorporated as if individually set forth. Titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read as limiting the scope of the present invention.
The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference. However, the reference to any applications, patents and publications in this specification is not, and should not be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.
Throughout this specification and any claims which follow, unless the context requires otherwise, the words “comprise”, “comprising” and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of “including, but not limited to”.
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## Claims

1. A method of producing a product comprising culturing a recombinant carboxydotrophic acetogenic microorganism in the presence of a gaseous substrate to produce at least one product, wherein the microorganism comprises at least one genetic modification that disrupts the expression and/or activity of an enzyme in the 2,3-butanediol biosynthesis pathway selected from the group consisting of (i) an enzyme capable of converting pyruvate to acetolactate, (ii) an enzyme capable of converting acetolactate to acetoin, and (iii) an enzyme capable of converting acetoin to 2,3-butanediol.

2. The method of claim 1, wherein the product is selected from the group consisting of ethanol, formate, lactate, pyruvate, succinate, valine, leucine, isoleucine, acetolactate, malate, fumarate, 2-oxogluterate, and citrate.

3. The method of claim 2, wherein the product is ethanol.

4. The method of claim 1, wherein the microorganism comprises at least one genetic modification that disrupts the expression and/or activity of at least two enzymes in the 2,3-butanediol biosynthesis pathway selected from the group consisting of (i) an enzyme capable of converting pyruvate to acetolactate, (ii) an enzyme capable of converting acetolactate to acetoin, and (iii) an enzyme capable of converting acetoin to 2,3-butanediol.

5. The method of claim 1, wherein the enzyme capable of converting pyruvate to acetolactate is acetolactate synthase.

6. The method of claim 5, wherein the acetolactate synthase is AlsS.

7. The method of claim 1, wherein the enzyme capable of converting acetolactate to acetoin is acetolactate decarboxylase.

8. The method of claim 7, wherein the acetolactate decarboxylase is BudA.

9. The method of claim 1, wherein the enzyme capable of converting acetoin to 2,3-butanediol is 2,3-butanediol dehydrogenase, acetoin reductase, or primary:secondary alcohol dehydrogenase.

10. The method of claim 9, wherein the 2,3-butanediol dehydrogenase is 2,3-Bdh.

11. The method of claim 1, wherein the microorganism produces an increased amount of the product compared to a parental microorganism and/or a reduced amount of 2,3-butanediol or a precursor thereof compared to a parental microorganism.

12. The method of claim 11, wherein the parental microorganism is Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, or Clostridium coskatii.

13. The method of claim 11, wherein the parental microorganism is Clostridium Autoethanogenum DSM23693.

14. The method of claim 1, wherein the gaseous substrate is an industrial waste gas or an industrial off gas.

15. The method of claim 1, wherein the gaseous substrate is syngas.

16. The method of claim 1, wherein the gaseous substrate comprises CO.

17. The method of claim 16, wherein the gaseous substrate comprises at least 20% CO by volume.