US20150210987A1

US20150210987A1 - Recombinant microorganisms and methods of use thereof

Info

Publication number: US20150210987A1
Application number: US14/609,420
Authority: US
Inventors: Shilpa Nagaraju; Bakir Al-Sinawi; Sashini De Tissera; Michael Koepke
Original assignee: Lanzatech New Zealand Ltd
Current assignee: Lanzatech NZ Inc
Priority date: 2014-01-30
Filing date: 2015-01-29
Publication date: 2015-07-30
Also published as: BR112016017092B1; US11549103B2; EA201691383A1; MY178451A; JP2017504342A; KR20160113129A; AU2015210892A1; EP3099783A4; WO2015116874A1; EA036071B1; US20200248152A1; CA2936252C; JP6783658B2; KR20220056885A; CN106133132A; EP3099783A1; KR102533454B1; CA2936252A1; ZA201604870B; AU2015210892B2

Abstract

The invention provides a carboxydotrophic acetogenic bacterium comprising a disrupting mutation in a lactate biosynthesis pathway enzyme and a method of producing a product by culturing the bacterium in the presence of a substrate comprising carbon monoxide. Preferably, the lactate biosynthesis pathway enzyme is lactate dehydrogenase (LDH) or another enzyme that converts pyruvate to lactate, wherein the disrupting mutation reduces or eliminates the expression or activity of the enzyme such that the bacterium produces a reduced amount of lactate or no lactate.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application 61/933,815 filed Jan. 30, 2014 and U.S. Provisional Patent Application 61/944,541 filed Feb. 25, 2014, the entirety of which are incorporated herein by reference.

SEQUENCE LISTING

This application includes a nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 23,322 byte ASCII (text) file named “LT102US1_ST25.txt” created on Jan. 29, 2015, the entirety of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

An acetogen is a microorganism that generates or is capable of generating acetate as a product of anaerobic respiration. Typically, acetogens are obligately anaerobic bacteria that use the Wood-Ljungdahl pathway as their main mechanism for energy conservation and for synthesis of acetyl-CoA and acetyl-CoA-derived products, such as acetate and ethanol (Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008).
Many acetogens naturally produce at least two or more products. However, this is not necessarily desirable on a commercial scale, since the production of multiple products is detrimental to the efficiency and yield of each individual product. In particular, byproducts may divert carbon away from the biosynthetic pathways of a target product, introduce toxicity concerns, impede the recovery and separation of a target product, complicate the control of fermentation conditions favoring a target product, and serve as a substrate for contaminating microorganisms.
For instance, acetogens such as Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei (Köpke, Appl Environ Microbiol, 77: 5467-5475, 2011), and Butyribacterium methylotrophicum (Heiskanen, Enzyme Microb Technol, 41 362-367, 2007) may produce lactate as a byproduct. This production of lactate reduces the efficiency and yield of target products, such as ethanol, butanol, or 2,3-butanediol. Additionally, lactate may be toxic to acetogens such as Clostridium autoethanogenum even at low concentrations (Köpke, Appl Environ Microbiol, 77: 5467-5475, 2011) and may serve as a substrate for other bacteria, increasingly the likelihood of bacterial contamination when lactate is produced. Furthermore, separating lactate from other products, such as ethanol, may require cumbersome processing steps.
Accordingly, there is a strong need for microorganisms and methods that reduce or eliminate the production of byproducts, such as lactate.

SUMMARY OF THE INVENTION

The invention provides a carboxydotrophic acetogenic bacterium comprising a disrupting mutation in a lactate biosynthesis pathway enzyme. In one embodiment, the disrupting mutation reduces or eliminates the expression or activity of the lactate biosynthesis pathway enzyme.
The disrupting mutation affects the ability of the bacterium to produce lactate. In one embodiment, the bacterium of the invention produces a reduced amount of lactate compared to a parental bacterium. In one embodiment, the bacterium of the invention produces substantially no lactate.
The bacterium of the invention may produce products, such as one or more of ethanol, 2,3-butanediol, formate, pyruvate, succinate, valine, leucine, isoleucine, malate, fumarate, 2-oxogluterate, citrate, and citramalate. In one embodiment, the bacterium of the invention produces an increased amount of one or more of ethanol, 2,3-butanediol, formate, pyruvate, succinate, valine, leucine, isoleucine, malate, fumarate, 2-oxogluterate, citrate, and citramalate compared to a parental bacterium.
In one embodiment, the lactate biosynthesis pathway enzyme is an enzyme that natively converts pyruvate to lactate. In a preferred embodiment, the lactate biosynthesis pathway enzyme is lactate dehydrogenase (LDH).
The bacterium of the invention may be derived from a parental bacterium, such as Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei. In a preferred embodiment, the parental bacterium is Clostridium autoethanogenum deposited under DSMZ accession number DSM23693.
The invention further provides a method of producing a product comprising culturing the bacterium of the invention in the presence of a substrate comprising CO whereby the bacterium produces a product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a LDH knockout strategy and the primers used for screening.

FIG. 2 is a set of gel images. The first gel image shows screening for single crossover integration of knockout plasmid using primers Og24r/Og35f for 5′ crossover and Og21f/Og36r for 3′ crossover in wild type (w) and transconjugant clone 6 (6). The second gel image shows screening for double crossover using outer flanking primers Og35f/Og36r and Og21f/Og24r.

FIG. 3 is a gel image showing colony PCR for Gene ID: 126803 Target 129S using primers LdhAF/R. A PCR product of 100 bp indicated a wild-type genotype, while a product size of approximately 1.9 kb confirmed the insertion of the group II intron in the target site.

FIG. 4 is a gel image showing plasmid loss with primers CatPR/RepHF. The plasmid loss was checked by amplification of the resistance marker (catP) and the gram positive origin of replication (pCB102).

FIG. 5A is a graph showing HPLC analysis of C. autoethanogenum after 6 days of growth in serum bottles with 30 psi steel mill off-gas (44% CO, 22% CO₂, 2% H₂, 32% N₂) as substrate. FIG. 5B is a graph showing HPLC analysis of C. autoethanogenum with inactivated lactate dehydrogenase after 6 days of growth in serum bottles with 30 psi steel mill off-gas (44% CO, 22% CO₂, 2% H₂, 32% N₂) as substrate.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that disruption of the lactate biosynthesis pathway in an acetogenic bacterium results in increased or more efficient production of products, such as ethanol, 2,3-butanediol, formate, succinate, 2-oxogluterate, valine, leucine, and isoleucine, compared to a parental microorganism, and may also result in increased or more efficient production of pyruvate, malate, fumarate, and citrate, which are precursors of succinate, 2-oxogluterate, valine, leucine, and isoleucine. The production of valine, leucine, formate, and pyruvate also obviates the need to supplement culture media with these compounds, which may result in further cost savings. Furthermore, reduction or elimination of lactate production by a bacterium reduces or eliminates the toxic effects of lactate on the bacterium.
The invention provides a carboxydotrophic acetogenic bacterium comprising a disrupting mutation in a lactate biosynthesis pathway enzyme.
“Mutation” refers to a modification in a nucleic acid or protein in the bacterium of the invention compared to the wild-type or parental microorganism from which the bacterium of the invention is derived. The term “genetic modification” encompasses the term “mutation.” In one embodiment, the mutation may be a deletion, insertion, or substitution of one or more nucleotides in a gene encoding an enzyme. In another embodiment, the mutation may be a deletion, insertion, or substitution of one or more amino acids in an enzyme.
Typically, the mutation is a “disrupting mutation” that reduces or eliminates (i.e., “disrupts”) the expression or activity of a lactate biosynthesis pathway enzyme. The disrupting mutation may partially inactivate, fully inactivate, or delete a lactate biosynthesis pathway enzyme or a gene encoding the enzyme. The disrupting mutation may be a knockout (KO) mutation. The disrupting mutation may be any mutation that reduces, prevents, or blocks the biosynthesis of lactate. The disrupting mutation may include, for example, a mutation in a gene encoding a lactate biosynthesis pathway enzyme, a mutation in a genetic regulatory element involved in the expression of a gene encoding a lactate biosynthesis pathway enzyme, the introduction of a nucleic acid which produces a protein that reduces or inhibits the activity of a lactate biosynthesis pathway enzyme, or the introduction of a nucleic acid (e.g., antisense RNA, siRNA, CRISPR) or protein which inhibits the expression of a lactate biosynthesis pathway enzyme.
The disrupting mutation results in a bacterium of the invention that produces no lactate or substantially no lactate or a reduced amount of lactate compared to the parental bacterium from which the bacterium is derived. For example, the bacterium of the invention may produce no lactate or at least about 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% less lactate than the parental bacterium. For example, the bacterium of the invention may produce less than about 0.001, 0.01, 0.10, 0.30, 0.50, or 1.0 g/L lactate. In contrast, depending on fermentation conditions, unmodified C. autoethanogenum LZ1561 may produce up to about 2 g/L lactate. Other unmodified bacterial strains may produce even more lactate.
The disrupting mutation may be introduced using any method known in the art. Exemplary methods include heterologous gene expression, gene or promoter insertion or deletion, altered gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, and codon optimization. Such methods are described, for example, in Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Pleiss, Curr Opin Biotechnol, 22: 611-617, 2011; and Park, Protein Engineering and Design, CRC Press, 2010. The disrupting mutation may be introduced using nucleic acids, such as single-stranded or double-stranded DNA, RNA, cDNA, or combinations thereof, as is appropriate. The nucleic acids may be referred to as constructs or vectors, and may include one or more regulatory elements, origins of replication, multicloning sites, and/or selectable markers. In one embodiment, the nucleic acid may be adapted to disrupt a gene encoding a lactate biosynthesis pathway enzyme in a parental bacterium. In one embodiment, the nucleic acid may be adapted to allow expression of one or more genes encoded by the nucleic acid. Constructs or vectors may include plasmids (e.g., pMTL, pIMP, pJIR), viruses (including bacteriophages), cosmids, and artificial chromosomes. The constructs may remain extra-chromosomal upon transformation of a parental bacterium or may be adapted for integration into the genome of the bacterium. Accordingly, constructs may include nucleic acid sequences adapted to assist integration (e.g., a region which allows for homologous recombination and targeted integration into the host genome) or expression and replication of an extrachromosomal construct (e.g., origin of replication, promoter, and other regulatory sequences).
The nucleic acids may be introduced using homologous recombination. Such nucleic acids may include arms homologous to a region within or flanking the gene to be disrupted (“homology arms”). These homology arms allow homologous recombination and the introduction, deletion, or substitution of one or more nucleotides within the gene to be disrupted. While it is preferred that the homology arms have 100% complementarity to the target region in the genome, 100% complementarity is not required so long that the sequence is sufficiently complementary to allow for targeted recombination with the target region in the genome. Typically, the homology arms will have a level of homology which would allow for hybridization to a target region under stringent conditions (Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Knowledge of the target nucleic acid sequences in a parental bacterium (i.e., the sequence of a target gene or target region in a parental bacterium) is generally sufficient to design appropriate homology arms. For example, to disrupt LDH, the flanking homology arms described herein may be used (e.g., SEQ ID NOs: 1-2). In C. ljungdahlii, homology arms may be designed based on GenBank CP001666.1. For other strains, homology arms may be designed based on other publically available nucleic acid sequence information.
The “lactate biosynthesis pathway” is a pathway of reactions resulting in the production of lactate. In one embodiment, the lactate biosynthesis pathway comprises one or more enzymes that convert pyruvate to lactate. In one embodiment, the lactate biosynthesis pathway comprises a lactate dehydrogenase enzyme. Depending on the bacterium, a number of different enzymes may be involved in the lactate biosynthesis pathway. When a bacterium comprises two or more enzymes in the lactate biosynthesis pathway, e.g., two or more enzymes capable of converting pyruvate to lactate, disrupting more than one such enzyme may have the effect of increasing the production of a product above the level that may be achieved by disrupting a single enzyme. In one embodiment, the bacterium comprises disrupting mutations in two, three, four, five, or more enzymes capable of converting pyruvate to lactate. While disrupting expression and/or activity of all such enzymes may provide some advantage in terms of product production, it is not generally necessary to disrupt expression and/or activity of all such enzymes to gain the benefits of the invention, namely increased production of one or more main or target products.
In one embodiment, the lactate biosynthesis pathway enzyme natively (i.e., endogenously or naturally) converts pyruvate to lactate, such that the enzyme has lactate dehydrogenase activity. The enzyme may have additional catalytic functions so long as it also converts pyruvate to lactate. For example, the enzyme may be any dehydrogenase having lactate dehydrogenase activity. The introduction of a disrupting mutation to the enzyme that converts pyruvate to lactate reduces or eliminates (i.e., “disrupts”) the expression or activity of that enzyme.
In a preferred embodiment, the lactate biosynthesis pathway enzyme is lactate dehydrogenase (LDH). The introduction of a disrupting mutation to LDH reduces or eliminates (i.e., “disrupts”) the expression or activity of LDH.
The bacterium of the invention may comprise one or more other genetic modifications in addition to a disrupting mutation in a lactate biosynthesis pathway enzyme, including genetic modifications of one or more genes or proteins not associated with the lactate biosynthesis pathway.
In one particular embodiment, the bacterium of the invention may express an inhibitor of a lactate biosynthesis pathway enzyme in addition to or instead of comprising a disrupting mutation in a lactate biosynthesis pathway enzyme.
“Enzyme activity” refers broadly to enzymatic activity, including, but not limited, to the activity of an enzyme, the amount of an enzyme, or the availability of an enzyme to catalyze a reaction. Accordingly, “decreasing” or “reducing” enzyme activity includes decreasing or reducing the activity of an enzyme, the amount of an enzyme, or the availability of an enzyme to catalyze a reaction. An enzyme is “capable of converting” a first compound or substrate into a second compound or product, if it can catalyze a reaction in which at least a portion of the first compound is converted into the second compound.
The term “variants” includes nucleic acids and proteins whose sequence varies from the sequence of a reference nucleic acid and protein, such as a sequence of a reference nucleic acid and protein disclosed in the prior art or exemplified herein. The invention may be practiced using variant nucleic acids or proteins that perform substantially the same function as the reference nucleic acid or protein. For example, a variant protein may perform substantially the same function or catalyze substantially the same reaction as a reference protein. A variant gene may encode the same or substantially the same protein as a reference gene. A variant promoter may have substantially the same ability to promote the expression of one or more genes as a reference promoter.
Variant nucleic acids or proteins with substantially the same level of activity as a reference nucleic acid or protein may be referred to herein as “functionally equivalent variants.” By way of example, functionally equivalent variants of a nucleic acid may include allelic variants, fragments of a gene, mutated genes, polymorphisms, and the like. Homologous genes from other microorganisms are also examples of functionally equivalent variants. These include homologous genes in species such as Clostridium acetobutylicum, Clostridium beijerinckii, or Clostridium ljungdahlii, the details of which are publicly available on websites such as Genbank or NCBI. Functionally equivalent variants also includes nucleic acids whose sequence varies as a result of codon optimization for a particular organism. A functionally equivalent variant of a nucleic acid will preferably have at least approximately 70%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 98%, or greater nucleic acid sequence identity (percent homology) with the referenced nucleic acid. A functionally equivalent variant of a protein will preferably have at least approximately 70%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 98%, or greater amino acid identity (percent homology) with the referenced protein. The functional equivalence of a variant nucleic acid or protein may be evaluated using any method known in the art.
However, variant nucleic acids or proteins may also have a reduced level of activity compared to a reference nucleic acid or protein. For example, a variant nucleic acid may have a reduced level of expression or a variant enzyme may have a reduced ability to catalyze a particular reaction compared to a reference nucleic acid or enzyme, respectively. Enzyme assays and kits for assessing the activity of enzymes in the lactate biosynthesis pathway are known in the art (Wang, J Bacteriol, 195: 4373-4386, 2013; Sigma-Aldrich (MAK066), Thermo (88953); Worthington Biochemical Corporation (LS002755)).
Nucleic acids may be delivered to a bacterium of the invention using any method known in the art. For example, nucleic acids may be delivered as naked nucleic acids or may be formulated with one or more agents (e.g., liposomes). Restriction inhibitors may be used in certain embodiments (Murray, Microbiol Molec Biol Rev, 64: 412-434, 2000). By way of example, transformation (including transduction or transfection) may be achieved by electroporation, ultrasonication, polyethylene glycol-mediated transformation, chemical or natural competence, protoplast transformation, prophage induction, or conjugation (see, e.g., Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). The use of electroporation has been reported for several carboxydotrophic acetogens, including Clostridium ljungdahlii (Koepke, PNAS, 107:13087-13092, 2010; WO/2012/053905), Clostridium autoethanogenum (WO/2012/053905), Clostridium aceticum (Schiel-Bengelsdorf, Synthetic Biol, 15: 2191-2198, 2012), and Acetobacterium woodii (Strätz, Appl Environ Microbiol, 60: 1033-1037, 1994). The use of electroporation has also been reported in Clostridia, including Clostridium acetobutylicum (Mermelstein, Biotechnol, 10: 190-195, 1992), and Clostridium cellulolyticum (Jennert, Microbiol, 146: 3071-3080, 2000). Prophage induction has been demonstrated for carboxydotrophic acetogens, including Clostridium scatologenes (Parthasarathy, Development of a Genetic Modification System in Clostridium scatologenes ATCC 25775 for Generation of Mutants, Masters Project, Western Kentucky University, 2010), and conjugation been described for many Clostridia, including Clostridium difficile (Herbert, FEMS Microbiol Lett, 229: 103-110, 2003) and Clostridium acetobuylicum (Williams, J Gen Microbiol, 136: 819-826, 1990). In certain embodiments having active restriction enzyme systems, it may be necessary to methylate a nucleic acid before introduction of the nucleic acid into the bacterium of the invention (WO 2012/105853).
The term “recombinant” indicates that a nucleic acid, protein, or microorganism is the product of genetic modification, mutation, or recombination. Generally, the term “recombinant” refers to a nucleic acid, protein, or microorganism that contains or is encoded by genetic material derived from multiple sources, such as two or more different strains or species of microorganisms. As used herein, the term “recombinant” may also be used to describe a microorganism that comprises a mutated nucleic acid or protein, including a mutated form of an endogenous nucleic acid or protein.
A “parental bacterium” is a bacterium used to generate a bacterium of the invention. The parental bacterium may be a naturally-occurring bacterium (i.e., a wild-type bacterium) or a bacterium that has been previously modified (i.e., a mutant or recombinant bacterium). The bacterium of the invention may be modified to express a lower amount of an enzyme compared to the parental bacterium, or the bacterium of the invention may be modified to not express an enzyme that is expressed by the parental bacterium. In one embodiment, the parental bacterium is Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment, the parental bacterium is Clostridium autoethanogenum deposited under DSMZ accession DSM23693 (i.e., Clostridium autoethanogenum LZ1561).
The term “derived from” indicates that a nucleic acid, protein, or microorganism is modified or adapted from a different (e.g., a parental or wild-type) nucleic acid, protein, or microorganism, so as to produce a new nucleic acid, protein, or microorganism. Such modifications or adaptations typically include insertion, deletion, mutation, or substitution of nucleic acids or genes. Generally, the bacterium of the invention is derived from a parental bacterium. In one embodiment, the bacterium of the invention is derived from Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment, the bacterium of the invention is derived from Clostridium autoethanogenum LZ1561, which is deposited under DSMZ accession DSM23693.
In one embodiment, the parental bacterium is selected from the group of carboxydotrophic acetogenic bacteria comprising the species Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium coskatii, 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, and Thermoanaerobacter kiuvi. These carboxydotrophic acetogenic bacteria are defined by their ability to grow chemoautotrophically on gaseous one-carbon sources such as carbon monoxide (CO) and carbon dioxide (CO₂), use carbon monoxide (CO) and/or hydrogen (H₂) as energy sources under anaerobic conditions, and produce 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-tetrahydrofolate reductase, and carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS), which combination is characteristic and unique to this type of bacteria (Drake, The Prokaryotes, 354-420, Springer, New York, N.Y., 2006). In contrast to chemoheterotrophic growth of sugar-fermenting bacteria that convert a substrate into biomass, secondary metabolites, and pyruvate, from which products are formed directly or via acetyl-CoA, acetogens channel a substrate directly into acetyl-CoA, from which products, biomass, and secondary metabolites are formed.
In a preferred embodiment, the bacterium of the invention is derived from a parental microorganism comprising a lactate dehydrogenase, wherein the bacterium of the invention comprises a disrupting mutation in the lactate dehydrogenase. For example, the parental microorganism may be C. autoethanogenum comprising a nucleic acid sequence comprising GenBank AEI90736.1 or an amino acid sequence comprising GenBank CP006763.1, KEGG CAETHG_—1147, or GenBank HQ876025.1. The parental microorganism may be C. ljungdahlii comprising a nucleic acid sequence comprising GenBank YP_—003781368.1 or an amino acid sequence comprising GenBank CP001666.1 or KEGG CLJU_c32190. The parental microorganism may be C. ragsdalei comprising a nucleic acid sequence comprising GenBank AEI90737.1 or an amino acid sequence comprising GenBank HQ876026.1. Other parental bacteria may have other nucleic acid and amino acid sequences.
A “carboxydotroph” is a microorganism capable of tolerating a high concentration of carbon monoxide (CO). Typically, the bacterium of the invention is a carboxydotroph.
The bacterium of the invention may be derived from the cluster of carboxydotrophic Clostridia comprising the species Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, and related isolates, including, but not limited to, strains Clostridium autoethanogenum JAI-1T (DSM10061) (Abrini, Arch Microbiol, 161: 345-351, 1994), Clostridium autoethanogenum LBS1560 (DSM19630) (WO 2009/064200), Clostridium autoethanogenum LZ1561 (DSM23693), Clostridium ljungdahlii PETCT (DSM13528=ATCC 55383) (Tanner, Int J Syst Bacteriol, 43: 232-236, 1993), Clostridium ljungdahlii ERI-2 (ATCC 55380) (U.S. Pat. No. 5,593,886), Clostridium ljungdahlii C-01 (ATCC 55988) (U.S. Pat. No. 6,368,819), Clostridium ljungdahlii O-52 (ATCC 55989) (U.S. Pat. No. 6,368,819), Clostridium ragsdalei P11T (ATCC BAA-622) (WO 2008/028055), related isolates such as “Clostridium coskatii” (U.S. Publication 2011/0229947), or mutated strains such as Clostridium ljungdahlii OTA-1 (Tirado-Acevedo, Production of Bioethanol from Synthesis Gas Using Clostridium ljungdahlii, PhD thesis, North Carolina State University, 2010).
These strains form a subcluster within the Clostridial rRNA cluster I and their 16S rRNA gene is more than 99% identical with a similar low GC content of around 30%. However, DNA-DNA reassociation and DNA fingerprinting experiments showed that these strains belong to distinct species (WO 2008/028055). 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. Furthermore, the strains of this cluster lack cytochromes and conserve energy via an Rnf complex. All species of this cluster 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 are strictly anaerobic (Abrini, Arch Microbiol, 161: 345-351, 1994; Tanner, Int J Syst Bacteriol, 43: 232-236, 1993; and 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 similar metabolic profile with ethanol and acetic acid as main fermentation end products, and small amounts of 2,3-butanediol and lactic acid formed under certain conditions (Abrini, Arch Microbiol, 161: 345-351, 1994; Köpke, Curr Opin Biotechnol, 22: 320-325, 2011; Tanner, Int J Syst Bacteriol, 43: 232-236, 1993; and WO 2008/028055). Indole production was observed with all three species as well.
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). Moreover, some of the species were found to be auxotrophic to certain vitamins (e.g., thiamine, biotin) while others were not. 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, Curr Opin Biotechnol, 22: 320-325, 2011). Also, reduction of carboxylic acids into their corresponding alcohols has been shown in a range of these microorganisms (Perez, Biotechnol Bioeng, 110:1066-1077, 2012). These traits are therefore not specific to one microorganism, like Clostridium autoethanogenum or Clostridium ljungdahlii, but rather general traits for carboxydotrophic, ethanol-synthesizing Clostridia and it can be anticipated that mechanisms work similarly across these strains, although there may be differences in performance.
An “acetogen” is a microorganism that generates or is capable of generating acetate as a product of anaerobic respiration. Typically, acetogens are obligately anaerobic bacteria that use the Wood-Ljungdahl pathway as their main mechanism for energy conservation and for synthesis of acetyl-CoA and acetyl-CoA-derived products, such as acetate (Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008). In a preferred embodiment, the bacterium of the invention is an acetogen.
The invention further provides a method of producing a product comprising culturing the bacterium of the invention in the presence of a substrate comprising CO whereby the bacterium of the invention produces a product.
The term “substrate” refers to a carbon and/or energy source for the bacterium of the invention. Typically, the substrate is a gaseous substrate that comprises carbon monoxide (CO). The substrate may comprise a major proportion of CO, such as about 20% to 100%, 20% to 70%, 30% to 60%, or 40% to 55% CO by volume. In particular embodiments, the substrate comprises about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% CO by volume. The bacterium of the invention generally converts at least a portion of the CO in the substrate to a product.
While it is not necessary for the substrate to contain any hydrogen (H₂), the presence of H₂should not be detrimental to product formation and may result improved overall efficiency. For example, in particular embodiments, the substrate may comprise an approximate ratio of H₂:CO of 2:1, 1:1, or 1:2. In one embodiment, the substrate comprises less than about 30%, 20%, 15%, or 10% H₂by volume. In other embodiments, the substrate comprises low concentrations of H₂, for example, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% H₂. In further embodiments, the substrate contains substantially no H₂.
The substrate may also contain carbon dioxide (CO₂), for example, about 1% to 80% or 1% to 30% CO₂by volume. In one embodiment, the substrate comprises less than about 20% CO₂by volume. In further embodiments, the substrate comprises less than about 15%, 10%, or 5% CO₂by volume. In another embodiment, the substrate contains substantially no CO₂.
Although the substrate is typically gaseous, the substrate may also be provided in alternative forms. For example, the substrate may be dissolved in a liquid saturated with a CO-containing gas using a microbubble dispersion generator (Hensirisak, Appl Biochem Biotechnol, 101: 211-227, 2002). By way of further example, the substrate may be adsorbed onto a solid support.
The substrate may be a waste gas obtained as a by-product of an industrial process or from some other source, such as from automobile exhaust fumes or biomass gasification. In certain embodiments, the industrial process is selected from the group consisting of ferrous metal products manufacturing, such as a steel mill manufacturing, non-ferrous products manufacturing, petroleum refining processes, coal gasification, electric power production, carbon black production, ammonia production, 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, i.e., a gas comprising carbon monoxide and hydrogen. The CO produced from industrial processes is normally flared off to produce CO₂and therefore the invention has particular utility in reducing CO₂greenhouse gas emissions. The composition of the substrate may have a significant impact on the efficiency and/or cost of the reaction. For example, the presence of oxygen (O₂) may reduce the efficiency of an anaerobic fermentation process. Depending on the composition of the substrate, it may be desirable to treat, scrub, or filter the substrate to remove any undesired impurities, such as toxins, undesired components, or dust particles, and/or increase the concentration of desirable components.
The bacterium of the invention may be cultured to produce one or more products. Generally, the bacterium of the invention produces one or more products selected from the group consisting of ethanol, 2,3-butanediol, formate, pyruvate, succinate, valine, leucine, isoleucine, malate, fumarate, 2-oxogluterate, citrate, and citramalate. The bacterium of the invention may also produce other products, such as acetolactate or acetoin malate.
In a preferred embodiment, the bacterium of the invention produces an increased amount of one or more of ethanol, 2,3-butanediol, formate, pyruvate, succinate, valine, leucine, isoleucine, malate, fumarate, 2-oxogluterate, citrate, and citramalate compared to a parental bacterium. For example, the bacterium of the invention may produce about 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500% more of one or more products compared to the parental bacterium from which the bacterium of the invention is derived. This increase in product production may be due, at least in part, to the disrupting mutation in the lactate biosynthesis pathway enzyme, which diverts carbon and energy away from the production of lactate and towards the production of other products.
The term “main product” refers to the single product produced in the highest concentration and/or yield. In one embodiment, the main product is ethanol or 2,3-butanediol.
Additionally, it is possible to engineer the bacterium of the invention to favor the production of one or more products over one or more other products. For example, disrupting the conversion of pyruvate to lactate may favor the production of 2,3-butanediol, formate, malate, fumarate, citrate, succinate and 2-oxogluterate over the production of valine, leucine and isoleucine.
Herein, recitation of a product (e.g., citrate) includes both salt (e.g., citrate) and acid (e.g., citric acid) forms of the product. Oftentimes, a mixture of the salt and acid forms of the product will be present in a fermentation broth, in a ratio that varies depending on the pH of the broth. As further examples, the term “acetate” encompasses acetate and acetic acid, the term “formate” encompasses formate and formic acid, the term “malate” encompasses malate and malic acid, and the term “lactate” encompasses lactate and lactic acid.
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 forms) should be taken generally to encompass any one or more such isomers of the compound. For example, reference to “lactate” generally encompasses both the D and L isomers of lactate.
Typically, the culture is performed in a bioreactor. The term “bioreactor” includes a culture/fermentation device consisting of one or more vessels, towers, or piping arrangements, such as a 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. In some embodiments, the bioreactor may comprise a first growth reactor and a second culture/fermentation reactor. The substrate may be provided to one or both of these reactors. As used herein, the terms “culture” and “fermentation” are used interchangeably. These terms encompass both the growth phase and product biosynthesis phase of the culture/fermentation process.
The culture is generally maintained in an aqueous culture medium that contains nutrients, vitamins, and/or minerals sufficient to permit growth of the bacterium. 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. No. 5,173,429, U.S. Pat. No. 5,593,886, and WO 2002/008438.
The culture/fermentation should desirably be carried out under appropriate conditions for production of the target product. Reaction conditions to consider include pressure (or partial pressure of CO), 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 particular, the rate of introduction of the CO-containing substrate may be controlled to ensure that the concentration of CO in the liquid phase does not become limiting, since products may be consumed by the culture under CO-limited conditions.
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 catalyzing 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 produced compared with other by-products of the fermentation.
Operating a bioreactor at elevated pressures allows for an increased rate of CO mass transfer from the gas phase to the liquid phase. Accordingly, it is generally preferable to perform the culture/fermentation at pressures higher than atmospheric pressure. Also, since a given CO conversion rate is, in part, a function of the substrate retention time and retention time 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 culture/fermentation equipment. According to examples in U.S. Pat. No. 5,593,886, reactor volume can be reduced in linear proportion to increases in reactor operating pressure. In other words, a bioreactor operated at 10 atmospheres of pressure need only be one tenth the volume of a bioreactor operated at 1 atmosphere of pressure. Additionally, WO 2002/008438 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. In contrast, 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.
The method of the invention may further comprise recovering or purifying one or more products. For example, ethanol or a mixed alcohol stream containing ethanol and/or other products may be recovered from a fermentation broth by any method known in the art, including fractional distillation, evaporation, pervaporation, or extractive fermentation (e.g., liquid-liquid extraction). Byproducts, such as acetate or acids, may also be recovered from a fermentation broth using any method known in the art, including activated charcoal adsorption systems, electrodialysis, or continuous gas stripping. In one embodiment, a product may be recovered from a fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration), and recovering the product from the broth. The separated microbial cells may be returned to the bioreactor. Additionally, cell-free permeate may also be returned to the bioreactor after the product has been removed, optionally with supplementation of nutrients, such as B vitamins.
Succinate can be recovered from a fermentation broth using, for example, acidification, electrodialysis coupled with ion-exchange chromatography (Song, Enzyme Microb Technol, 39: 352-361, 2006), precipitation with Ca(OH) coupled with filtration and addition of sulfuric acid (Lee, Appl Microbiol Biotechnol, 79: 11-22, 2008), or reactive extraction with amine-based extractants such as tri-n-octylamine (Huhet, Proc Biochem 41: 1461-1465, 2006). For all methods, it is crucial to have the free acid form, not the salt. Most biotechnological production processes for succinic acid, however, operate at a neutral or slightly acidic pH of 6-7. Given the pKa of succinic acid (pKa=4.16 and 5.61), the majority of succinic acid is present as salt and not as free acid under these conditions. C. autoethanogenum and other carboxydotrophic acetogens, however, are known to tolerate and grow at a desirably low pH of 4-6.
Branched-chain amino acids, such as valine, leucine, and isoleucine, may be recovered from a fermentation broth using concentration (e.g., via reverse osmosis), crystallization or removal of the biomass (e.g., via ultrafiltration or centrifugation), or ion exchange chromatography (Ikeda, Microbial Production of L-Amino Acids, 1-35, 2003).
2,3-butanediol, formate, 2-oxogluterate, and other products may be recovered from a fermentation broth using any method known in the art. For example, low concentrations of 2,3-butanediol may be recovered using membrane techniques, such as electrodialysis, involving the application of a suitable potential across a selective ion permeable membrane. Other suitable techniques include nanofiltration, wherein monovalent ions selectively pass through a membrane under pressure.

EXAMPLES

The following examples further illustrate the invention but, of course, should not be construed to limit its scope in any way.

Example 1

This example describes general materials and methods.
C. autoethanogenum DSM10061 and DSM23693 (a derivate of DSM10061) and C. ljungdahlii DSM13528 were sourced from DSMZ (The German Collection of Microorganisms and Cell Cultures, Inhoffenstraβe 7 B, 38124 Braunschweig, Germany). C. ragsdalei ATCC BAA-622 was sourced from ATCC (American Type Culture Collection, Manassas, Va. 20108, USA). E. coli DH5α was sourced from Invitrogen (Carlsbad, Calif. 92008, USA).
E. coli was grown aerobic at 37° C. in LB (Luria-Bertani) medium. Solid media contained 1.5% agar.


		Amount per 1.0 L
	LB medium component	of LB medium

	Tryptone	10 g
	Yeast extract	5 g
	NaCl	10 g

Clostridium strains were grown at 37° C. in PETC medium at pH 5.6 using standard anaerobic techniques (Hungate, Methods Microbiol, 3B: 117-132, 1969; Wolfe, Adv Microbiol Physiol, 6: 107-146, 1971). Fructose (heterotrophic growth) or 30 psi CO-containing steel mill gas (collected from New Zealand Steel site in Glenbrook, NZ; composition: 44% CO, 32% N₂, 22% CO₂, 2% H₂) in the headspace (autotrophic growth) was used as substrate. For solid media, 1.2% bacto agar (BD, Franklin Lakes, N.J. 07417, USA) was added.


		Amount per 1.0 L of
	PETC medium component	PETC medium

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 (see below)	10	ml
Wolfe's vitamin solution (see below)	10	ml
Yeast extract (optional)	1	g
Resazurin (2 g/L stock)	0.5	ml
NaHCO₃	2	g

Reducing agent solution (see below)

0.006-0.008% (v/v)

	Fructose (for heterotrophic growth)	5	g


		Amount per 1.0 L of
	Trace metal solution component	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


		Amount per 1.0 L of
	Wolfe's vitamin solution component	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


	Reducing agent	Amount per 100 mL of
	solution component	reducing agent solution

NaOH	0.9	g
Cysteine-HCl	4	g
Na₂S	4	g

Fermentations with C. autoethanogenum DSM23693 were carried out in 1.5 L bioreactors at 37° C. using CO-containing steel mill gas as sole energy and carbon source. A defined medium was prepared, containing: 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). The medium 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/l), and biotin (0.02 mg/l) and reduced with 3 mM cysteine-HCl. To achieve anaerobic conditions, 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 gas flow was initially set at 80 ml/min and increased to 200 ml/min during mid-exponential phase, while the agitation was increased from 200 rpm to 350 rmp. Na₂S was dosed into the bioreactor at 0.25 ml/hr. Once the OD600 reached 0.5, the bioreactor was switched to continuous mode at a rate of 1.0 ml/min (dilution rate 0.96 d⁻¹). Samples were taken to measure the biomass and metabolites. Additionally, headspace analysis of the in- and out-flowing gas was performed on regular basis.
Gas composition of the headspace was measured on a Varian CP-4900 micro GC with two installed channels. Channel 1 was a 10 m Mol-sieve column running at 70° C., 200 kPa argon and a backflush time of 4.2 s, while channel 2 was a 10 m PPQ column running at 90° C., 150 kPa helium and no backflush. The injector temperature for both channels was 70° C. Runtimes were set to 120 s, but all peaks of interest would usually elute before 100 s.
HPLC analysis of metabolic end products was performed using an Agilent 1100 Series HPLC system equipped with a RID (Refractive Index Detector) operated at 35° C. and an Alltech IOA-2000 organic acid column (150×6.5 mm, particle size 5 μm) kept at 60° C. Slightly acidified water was used (0.005 M H₂SO₄) as mobile phase with a flow rate of 0.7 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.
GC analysis of metabolic end products was performed using an Agilent 6890N headspace GC equipped with a Supelco PDMS 100 1 cm fiber, an Alltech EC-1000 (30 m×0.25 mm×0.25 μm) column, and a flame ionization detector (FID). 5 ml samples were transferred into a Hungate tube, heated to 40° C. in a water bath and exposed to the fiber for exactly 5 min. The injector was kept at 250° C. and helium with a constant flow of 1 ml/min was used as carrier gas. The oven program was 40° C. for 5 min, followed by an increase of 10° C./min up to 200° C. The temperature was then further increased to 220° C. with a rate of 50° C./min followed by a 5 min hold at this temperature, before the temperature was decreased to 40° C. with a rate of 50° C./min and a final 1 min hold. The FID was kept at 250° C. with 40 ml/min hydrogen, 450 ml/min air and 15 ml/min nitrogen as make up gas.
During the complete transformation experiment, C. autoethanogenum DSM23693 was grown in YTF medium 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 (Hungate, Methods Microbiol, 3B: 117-132, 1969; Wolfe, Adv Microbiol Physiol, 6: 107-146, 1971).


	YTF medium component	Amount per 1.0 L of YTF medium

Yeast extract	10	g
Tryptone	16	g
Sodium chloride	0.2	g
Fructose	10	g
Distilled water	to 1.0	L


	Reducing agent	Amount per 100 mL of
	solution component	reducing agent solution

NaOH	0.9	g
Cysteine-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-threonine at an OD_{600 nm}of 0.05. When the culture reached an OD_{600 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, 600Ω, and 25 μF. Time constants of 3.7-4.0 ms were achieved. The culture was transferred into 5 ml fresh YTF medium. 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/1 fructose (both containing 1.2% bacto agar 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.
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 PURELINK™ Genomic DNA mini kit (Invitrogen) according to manufacturer's instruction. These genomic DNA along with that of wild-type C. autoethanogenum DSM23693 were used as a template in PCR. The PCR was performed with iproof High Fidelity DNA Polymerase (Bio-Rad Labratories), specific primers as described in examples below and the following program: initial denaturation at 98° C. for 2 min, followed by 25 cycles of denaturation (98° C. for 10 s), annealing (61° C. for 15 s) and elongation (72° C. for 90 s), before a final extension step (72° C. for 7 min). The genomic DNA from wild-type C. autoethanogenum DSM23693 was used as template in control PCR.
To confirm the identity of the clones, PCR was also performed against the 16s rRNA gene using primers fD1 (SEQ ID NO: 10) and rP2 (SEQ ID NO: 11) and using PCR conditions as described above. The PCR products were purified using Zymo CLEAN AND CONCENTRATOR™ kit and sequenced using primer rP2.

Example 2

This example demonstrates the genetic modification of C. autoethanogenum to eliminate lactate dehydrogenase activity.
Demonstration of inactivation of the identified (Köpke, Appl Environ Microbiol, 77: 5467-5475, 2011) lactate dehydrogenase (AEI90736.1) gene ldh (HQ876025.1) of C. autoethanogenum was demonstrated by using two methodologies: homologous recombination and ClosTron.
Homologous Recombination:
To create a C. autoethanogenum strain that can no longer produce lactate, a knock out construct was designed to disrupt ldh by double homologous recombination. Approximately 1 kb homology arms (SEQ ID NOs: 1-2) flanking the ldh gene were cloned into pMTL85151 plasmid (FIG. 1) and the resulting plasmid pMTL85151-ldh-ko (SEQ ID NO: 3). Standard recombinant DNA and molecular cloning techniques are known in the art (Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Ausubel, Current Protocols in Molecular Biology. Wiley, 1987). Genomic DNA from C. autoethanogenum DSM23693 was isolated using Purelink Genomic DNA mini kit from Invitrogen, according to the manufacturer's instruction.
Transformation to introduce DNA was carried out as described above or in WO 2012/053905.
Following selection, colonies was screened for single crossover integration (FIG. 2A) and then for double-crossover mutants (FIG. 2B). A 3′ crossover event was seen in clone 6 (FIG. 2A) and knockout of ldh gene was observed when screened with outer flanking primers (FIG. 2B). Oligonucleotides Og21f (SEQ ID NO: 4), Og24r (SEQ ID NO: 5), Og35f (SEQ ID NO: 6, and Og36r (SEQ ID NO: 7) were used for identification of the double-crossover lactate dehydrogenase deletion.
The same strategy and plasmid can also be used, for example, in C. ljungdahlii or C. ragsdalei. Transformation protocols have been described in the art (WO 2012/053905 Leang, Applied Environ Microbiol, 79: 1102-1109, 2013).
ClosTron:
ClosTron (Heap, J Microbiol Methods, 70: 452-464, 2007), an intron design tool hosted on the ClosTron website, was used to design a 344 bp targeting region 129s (SEQ ID NO: 8) and identify a target site (SEQ ID NO: 9). The targeting region was chemically synthesized in the vector pMTL007C-E2 containing a retro-transposition activated ermB marker (RAM) by DNA2.0 (Menlo Park) (SEQ ID NO: 12).
The vectors were introduced into C. autoethanogenum as described in WO 2012/053905. Single colonies grown on PETC MES with 15 μg/ml thiamphenicol were streaked on PETC MES with 5 μg/ml clarothromycin. Colonies from each target were randomly picked and screened for the insertion using flanking primers 155F (SEQ ID NO: 4), and 939R (SEQ ID NO: 5). Amplification was performed using the iNtron Maxime PCR premix. A PCR product of 100 bp indicated a wild-type genotype, while a product size of approximately 1.9 kb suggests the insertion of the group II intron in the target site (FIG. 3). The loss of the plasmid was checked by amplification of the resistance marker (catP) and the gram positive origin of replication (pCB102) (FIG. 4).


SEQ ID NO	Description

1	left homology arm for disruption of the
	lactate dehydrogenase gene
2	right homology arm for disruption of the
	lactate dehydrogenase gene
3	plasmid pMTL85151-ldh-ko
4	oligonucleotide Og21f
5	oligonucleotide Og24r
6	oligonucleotide Og35f
7	oligonucleotide Og35f
8	ClosTron targeting region
9	ClosTron target site
10	oligonucleotide fD1
11	oligonucleotide rP2
12	ClosTron plasmid pMTL007C-E2-ldh::129s

The same strategy and plasmid can also be used in C. ljungdahlii or C. ragsdalei. Transformation protocols have been described in the art (WO 2012/053905; Leang, Appl Environ Microbiol, 79: 1102-1109, 2013).

Example 3

This example describes growth experiments comparing the product profile of C. autoethanogenum strains with inactivated lactate dehydrogenase to unmodified C. autoethanogenum.
Cultures of C. autoethanogenum and a C. autoethanogenum strain with an inactivated lactate dehydrogenase were grown in PETC media with 10 g/L MES buffer in serum bottles. The inoculum was 10% of the media volume and the volume of the media was 10 ml. The cultures were gassed with steel mill off-gas (44% CO, 22% CO₂, 2% H₂, 32% N₂) 30 psi and incubated at 37° C. The pH of the media was 5.7.
During the growth period, samples were taken for the measurement of OD600 and for analysis by HPLC. The bottles were gassed every day with 30 psi mill gas. The experiment was performed in triplicate.
While the unmodified C. autoethanogenum strain produced 0.263±0.041 g/L lactate after 6 days of growth (FIG. 5A), the C. autoethanogenum strain with inactivated lactate dehydrogenase produced no lactate after 6 days of growth (FIG. 5B). Additionally, the C. autoethanogenum strain with inactivated lactate dehydrogenase produced increased amounts of acetate, ethanol, and 2,3-butanediol. The two strains otherwise had a similar growth profile and reached a similar OD600 nm of 2.69 and 2.365, respectively.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement that that prior art forms part of the common general knowledge in the field of endeavour in any country.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A carboxydotrophic acetogenic bacterium comprising a disrupting mutation in a lactate biosynthesis pathway enzyme.

2. The bacterium of claim 1, wherein the mutation reduces or eliminates the expression or activity of the enzyme.

3. The bacterium of claim 1, wherein the bacterium produces a reduced amount of lactate compared to a parental bacterium.

4. The bacterium of claim 1, wherein the bacterium produces substantially no lactate.

5. The bacterium of claim 1, wherein the bacterium produces one or more of ethanol, 2,3-butanediol, formate, pyruvate, succinate, valine, leucine, isoleucine, malate, fumarate, 2-oxogluterate, citrate, and citramalate.

6. The bacterium of claim 1, wherein the bacterium produces an increased amount of one or more of ethanol, 2,3-butanediol, formate, pyruvate, succinate, valine, leucine, isoleucine, malate, fumarate, 2-oxogluterate, citrate, and citramalate compared to a parental bacterium.

7. The bacterium of claim 1, wherein the enzyme natively converts pyruvate to lactate.

8. The bacterium of claim 1, wherein the enzyme is lactate dehydrogenase (LDH).

9. The bacterium of claim 1, wherein the bacterium is derived from a parental bacterium selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei.

10. The bacterium of claim 9, wherein the parental bacterium is Clostridium autoethanogenum deposited under DSMZ accession number DSM23693.

11. A method of producing a product comprising culturing the bacterium of claim 1 in the presence of a substrate comprising CO whereby the bacterium produces a product.

12. The method of claim 11, wherein the mutation reduces or eliminates the expression or activity of the enzyme.

13. The method of claim 11, wherein the bacterium produces a reduced amount of lactate compared to a parental bacterium.

14. The method of claim 11, wherein the bacterium produces substantially no lactate.

15. The method of claim 11, wherein the product comprises one or more of ethanol, 2,3-butanediol, formate, pyruvate, succinate, valine, leucine, isoleucine, malate, fumarate, 2-oxogluterate, citrate, and citramalate.

16. The method of claim 11, wherein the bacterium produces an increased amount of the product compared to a parental bacterium.

17. The method of claim 11, wherein the enzyme natively converts pyruvate to lactate.

18. The method of claim 11, wherein the enzyme is lactate dehydrogenase (LDH).

19. The method of claim 11, wherein the bacterium is derived from a parental bacterium selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei.

20. The method of claim 19, wherein the parental bacterium is Clostridium autoethanogenum deposited under DSMZ accession number DSM23693.