Essential Genes Encoding Conserved Metabolic Pathway Function in Autotrophic Solventogenic Clostridial Species

Description

FIELD OF THE INVENTION

This invention relates to the cloning and expression of novel genetic sequences of microorganisms used in the biological conversion of CO, H₂, and mixtures comprising CO and/or H₂ to biofuel products, and functional characterization thereof. Further, this invention relates to a method of prescreening autotrophic homoacetogenic microorganisms for the ability to produce high ethanol titers from syngas components.

BACKGROUND

Synthesis gas (syngas) is a mixture of carbon monoxide (CO) gas, carbon dioxide (CO₂) gas, and hydrogen (H₂) gas, and other volatile gases such as CH₄, N₂, NH₃, H₂S and other trace gases. Syngas is produced by gasification of various organic materials including biomass, organic waste, coal, petroleum, plastics, or other carbon containing materials, or reformed natural gas.

Acetogenic Clostridial microorganisms grown in an atmosphere containing syngas are capable of absorbing the syngas components CO, CO₂, and H₂ and producing aliphatic C₂-C₆ alcohols and aliphatic C₂-C₆ organic acids. These syngas components activate Wood-Ljungdahl metabolic pathway 100, as shown in FIG. 1, which leads to the formation of acetyl coenzyme A 102, a key intermediate in the pathway. Under autotrophic fermentation conditions, the enzymes activating Wood-Ljundahl pathway 200, as shown in FIG. 2, are carbon monoxide dehydrogenase (CODH) 104 and hydrogenase (H₂ase) 106. These enzymes capture the electrons from the CO and H₂ in the syngas and transfer them to ferredoxin 108, an iron-sulfur (FeS) electron carrier protein. Ferredoxin 108 is the main electron carrier in metabolic pathway 100 in acetogenic Clostridia, primarily because the redox potential during syngas fermentation is very low (usually between −400 and −500 mV). Upon electron transfer, ferredoxin 108 changes its electronic state from Fe³⁺ to Fe²⁺. Ferredoxin-bound electrons are then transferred to cofactors NAD⁺ 110 and NADP 112 through the activity of ferredoxin oxidoreductases 114 (FORs). The reduced nucleotide cofactors (NAD⁺ and NADP⁺) are used for the generation of intermediate compounds in Wood-Ljungdahl pathway 100 leading to acetyl-CoA 102 formation.

The FOR-mediated ferredoxin reduction reaction additionally feeds an Rnf complex that maintains a proton-motive force (PMF) to generate ATP through interconnectivity of the electron-motive force (EMF) with the PMF since under autotrophic growth conditions homoacetogenic cells are generating net ATP through a PMF via an FIFO type ATP synthase consisting of seven genes (see Table 1, Gene ID Nos. 50-57). The net ATP generated through operation of the Rnf complex is then consumed for cell growth or maintenance.

Acetyl-CoA 102 formation through Wood-Ljungdahl pathway 100 is shown in greater detail in FIG. 2. Either CO₂ 202 or CO 208 provide substrates for the pathway. The carbon from CO₂ 202 is reduced to a methyl group through successive reductions first to formate, by formate dehydrogenase (FDH) enzyme 204, and then is further reduced to methyl tetrahydrofolate intermediate 206. The carbon from CO₂ 208 is reduced to carbonyl group 210 by carbon monoxide dehydrogenase (CODH) 104 through a second branch of the pathway. The two carbon moieties are then condensed to acetyl-CoA 102 through the action of acetyl-CoA synthase (ACS) 212, which is part of a carbon monoxide dehydrogenase (CODH/ACS) complex. Acetyl-CoA 102 is the central metabolite in the production of C₂-C₆ alcohols and acids in acetogenic Clostridia.

Ethanol production from Acetyl CoA 102 is achieved via one of two possible paths. Aldehyde dehydrogenase facilitates the production of acetaldehyde, which is then reduced to ethanol by the action of primary alcohol dehydrogenases. In the alternative, in some homoacetogenic microorganisms, a bifunctional NADPH-dependent ADH/acetyl CoA reductase (“AR”) thioesterase facilitates the production of ethanol directly from acetyl CoA.

Wood-Ljungdahl pathway 100 is neutral with respect to ATP production when acetate 214 is produced (FIG. 2). When ethanol 216 is produced, one ATP is consumed in a step involving the reduction of methylene tetrahydrafolate to methyl tetrahydrofolate 206 by a reductase, and the process is therefore net negative by one ATP. The pathway is balanced when acetyl-PO₄ 218 is converted to acetate 214.

Acetogenic Clostridia organisms generate cellular energy by ion gradient-driven phosphorylation. When grown in a CO atmosphere, a transmembrane electrical and chemical potential is generated and used to synthesize ATP from ADP. Enzymes mediating the process include hydrogenase, NADH dehydrogenases, carbon monoxide dehydrogenase, and methylene tetrahydrofolate reductase. Membrane carriers that have been shown to be likely involved in the ATP generation steps include quinone, menaquinone, and cytochromes.

The acetogenic Clostridia produce a mixture of C₂-C₆ alcohols and acids, such as ethanol, n-butanol, hexanol, acetic acid, and butyric acid, that are of commercial interest through Wood-Ljungdahl pathway 100. For example, acetate and ethanol are produced by C. ragsdalei in variable proportions depending in part on fermentation conditions. However, the cost of producing the desired product, an alcohol such as ethanol, for example, can be lowered significantly if the production is maximized by reducing or eliminating production of the corresponding acid, in this example acetate. It is therefore desirable to metabolically engineer acetogenic Clostridia for improved production of selected C₂-C₆ alcohols or acids through Wood-Ljungdahl pathway 100 by modulating enzymatic activities of key enzymes in the pathway.

Acetogenesis as described above is a general metabolic trait that is not phylogenetically conserved. Therefore, production of liquid fuels via biocatalyst is the result of a unique collection of genes and functional protein activities that are expressed when grown in the presence of syngas under desirable growth conditions. Not all organisms that have the Wood-Ljungdahl pathway make ethanol, since some lack alcohol dehydrogenases or other genes to convert acetate to ethanol. Thus, the ability to convert syngas components to high ethanol titers is embodied in the unique collection of conserved genes described below.

SUMMARY OF THE INVENTION

The present invention is directed to an isolated and purified sequence encoding a series of polypeptides encoding polynucleotides which express the minimum set of required genes to maintain ethanologenic function, and which more particularly express the minimum set of required genes to maintain Acetyl-CoA to ethanol function and the minimum set of required genes to maintain Rnf complex function.

The present invention is additionally directed to a method of producing ethanol comprising: isolating and purifying anaerobic, ethanologenic microorganisms carrying the polynucleotides described above; fermenting syngas with said microorganisms in a fermentation bioreactor.

Further, the present invention is directed to a method of confirming high titer autotrophic solventogenesis function of a potentially commercially viable microorganism, said method comprising: sequencing the genome of said potentially commercially viable microorganism; comparing a resulting sequence of the genome of the microorganism to SEQ ID NO. 1.

Finally, the present invention is directed to a method of prescreening natural isolates with suspected autotrophic solventogenesis function for high ethanol titer potential, said method comprising: isolating and enriching a sample containing said natural isolates; subjecting said sample to a polymerase chain reaction using at least one set of degenerate primers capable of hybridizing to one or more of the genes of SEQ ID NO. 1; separating the amplified product of the polymerase chain reaction based on size; and determining the presence of said genes based on the results of said separation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating the electron flow pathway during syngas fermentation in acetogenic Clostridia including some of the key enzymes involved in the process;

FIG. 2 is a diagram illustrating the Wood-Ljungdahl (C₁) pathway for acetyl-CoA production and the enzymatic conversion of acetyl-CoA to acetate and ethanol;

FIG. 3 is a diagram illustrating the metabolic pathway utilized by acetogenic clostridia. The pathway is understood to consist of all the electron transfer reactions involved in the extraction of electrons from CO an H2 to carriers which feed into the Wood-Ljungdahl pathway to form acetyl-CoA and eventually on to ethanol biosynthesis. Also integral to the whole process is the maintenance of a proton-motive force (PMF) to generate ATP and the interconnectivity of the electron-motive force (EMF)with the PMF since under autotrophic growth conditions cells are generating net ATP through a PMF;

DETAILED DESCRIPTION

The present invention is directed to novel genetic sequences coding for acetogenic Clostridial microorganisms that produce ethanol and acids from syngas comprising CO, CO2, H2, or mixtures thereof, and functional characterizations thereof.

Specifically, the present invention is directed to a minimum set of metabolic pathway genes of biocatalysts involved in conversion of syngas to ethanol under autotrophic growth conditions. Further, the present invention is directed to a process for prescreening autotrophic homoacetogenic microorganisms for the ability to produce high ethanol titers from syngas components.

Several species of acetogenic Clostridia that produce C₂-C₆ alcohols and acids via the Wood-Ljungdahl pathway have been characterized: C. ragsdahlei, C. ljungdahlii, C. carboxydivorans, and C. autoethanogenum. The genomes of four of these micro-organisms were sequenced in order to locate and characterize the portions of the genome that code for the functions of interest, and that are conserved within the group of known organisms that produce high titers of ethanol when grown autotrophically on syngas.

The genes that code for the minimum set of metabolic pathway enzymes (including (1) electron transfer genes; (2) Wood-Ljungdahl pathway genes; (3) ethanol and acetate biosynthetic genes; and (4) energy [ATP] generation genes) are presented in Table 1. The first column identifies the broadly-categorized pathway associated with each gene. The gene identification numbers indicated in the second column correspond to the numbers representing the enzymes involved in the metabolic reactions in the pathway shown in FIG. 1. Any inclusion in an operon is noted in the fifth column.

The results of the sequence analysis and the creation of the minimum set of functional genes for the four primary functions inherent in metabolic function of homoacetogenic Clostridia (electron transfer, Wood-Ljungdahl pathway, ethanol and acetate biosynthesis, and ATP generation) indicate that certain Clostridial strains (i.e. those with a low G+C) can be categorized according to their ability to make ethanol from different substrates and that organisms characterized by high ethanol titers must contain at a minimum the genes contained in the minimum set to maintain function and autotrophic ethanol production from syngas.

The comparison and creation of the minimum set additionally indicates that the key differences between high ethanol producing strains and strains producing no ethanol or low levels of ethanol (i.e. “smears”) lies in the electron transfer reactions and the large collection of alcohol dehydrogenases.

Two operons coding for CODH function were identified as members of the minimum set (see Table 1), indicating that both are essential for proper metabolic pathway function in any acetogenic Clostridia. One operon (Gene ID Nos. 2-4) codes for a monofunctional CODH which transfers electrons from a reduced CO to ferredoxin carriers. The two carbon moieties are then condensed to acetyl-CoA 102 through the action of acetyl-CoA synthase (ACS) 212, which is part of a carbon monoxide dehydrogenase (CODH/ACS) complex, and makes up the second CODH operon, which codes for Wood-Ljungdahl function (Gene 1D Nos. 5-18) that is conserved across known species of acetogenic Clostridia. The CODH/ACS operon is also responsible for reducing the carbon from CO2 208 to a carbonyl group 210.

Additionally, there are three further genes that do not reside in an operon that have been identified as members of the minimum set for Wood-Ljungdahl function: A fourth CODH gene and two formate dehydrogenase genes complete the minimum set.

Ten genes coding for ethanol and acetate production enzymes (Gene ID Nos. 20-29) have been identified as the minimum set for proper production by acetogenic Clostridia. These ten genes code for acetate kinase, phosphotransacetylase, tungsten-containing aldehyde FOR, and alcohol and aldehyde dehydrogenases, all of which are required for ethanol and aldehyde production from the primary metabolite in autotrophic acetogenic microorganisms, acetyl CoA. Aldehyde dehydrogenase facilitates the production of acetaldehyde, which is then reduced to ethanol by the action of primary alcohol dehydrogenases.

Two operons, one consisting of six genes (Gene ID Nos. 44-49) and coding for electron transfer function, and the other consisting of eight genes and coding for ATP generation were found to be conserved across all known autotrophic acetogenic Clostridia. Electron transfer function in acetogenic microorganisms is ultimately controlled by an Rnf complex which mediates EMF/PMF function. The Rnf complex maintains a PMF to generate ATP through interconnectivity of the EMF with the PMF since under autotrophic growth conditions homoacetogenic cells are generating net ATP through a PMF via an FIFO type ATP synthase consisting of seven genes (see Table 1, Gene ID Nos. 50-57). The net ATP generated through operation of the Rnf complex is then consumed for cell growth or maintenance.

Key genes to promote production of ethanol in solventogenic Clostridia include:

SEQ ID NO 1(Gene ID Nos. 1-64, Table 1), the minimum set of genes required to maintain function of the metabolic pathway of acetogenic Clostridia, including the experimentally determined promoter regions for all monocistronic genes and the promoter regions for the first gene in all operons.
SEQ ID NO 2 (Gene ID Nos. 20-29, Table 1), the minimum set of genes required to maintain function of the Acetyl-CoA to ethanol step of the Clostridial metabolic process, including the experimentally determined promoter regions for all monocistronic genes and the promoter regions for the first gene in all operons;
SEQ ID NO 3 (Gene ID Nos. 44-64, Table 1), the minimum set of genes required to maintain function of the Rnf complex and corresponding PMF to ATP step of the Clostridial metabolic process, including the experimentally determined promoter regions for all monocistronic genes and the promoter regions for the first gene in all operons;

In order to create the minimum sets found in SEQ ID NOS. 1-3, the genomes of C. ragsdahlii, C. ljungdahlii, C. autoethanogenum, and C. carboxydivorans were fully sequenced. A sequence-level analysis and comparison was performed with a cutoff score of P=1×10⁻²⁰, and function was established for the genes present. At such a level of identity, one of skill in the art recognizes that there is virtually no probability that the alignment is the result of chance. Therefore, the minimum sets found in SEQ ID NOS. 1-3 represent fully conserved sets.

At the present time, screening potential microorganisms for high ethanol titer production capability is an extended and daunting task. Function maybe established, but often through costly and time-consuming bench assays. Using several embodiments of the present invention, however, a prospective microorganism may be prescreened for function, and such function may be confirmed.

To practice such embodiments, a sample is first collected that may contain anaerobic solventogenic microorganisms. The sample is amplified, and then undergoes an isolation and enrichment process that may comprise any number of steps according to techniques well-known in the art. Enrichment and isolation may include, but are not limited to, confirmation of autotrophic function, screening for syngas utilization, confirmation of the presence of an Acetyl-CoA reductase gene, or confirmation of the presence of a CODH/ACS operon. After isolating and enriching any microorganisms of interest, the microorganisms are plated for further phenotypic metagenomic analysis.

The narrowed microorganisms may then undergo a polymerase chain reaction with a sample of at least one degenerate primer that will bind to one of the essential genes for solventogenesis function. A kit with primers which will bind to all sixty-four essential metabolic genes may also be utilized. A separation is then performed based on size; in a preferred embodiment the product of the PCR will be electrophoreted. The results are then read to determine the presence or absence of each essential gene of interest. If one of the essential genes listed in Table 1 is absent, the metabolic pathway may not produce high titers of product.

In a further embodiment, to confirm de novo high ethanologenic function, the genome of the potentially ethanologenic microorganism is sequenced. A comparison is performed between the genes contained in the prospective microorganism's genome and the gene sequences of the minimum set with a cutoff score of P=1×10⁻²⁰. If the genome of the prospective microorganism comprises at least the minimum set, then ethanologenic function via the Wood-Ljungdahl pathway is preserved, and the microorganism is likely to produce high ethanol titers when fermented with syngas.