WO2009155382A1 - Microorganisms and methods for the biosynthesis of fumarate, malate, and acrylate - Google Patents
Microorganisms and methods for the biosynthesis of fumarate, malate, and acrylate Download PDFInfo
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- WO2009155382A1 WO2009155382A1 PCT/US2009/047715 US2009047715W WO2009155382A1 WO 2009155382 A1 WO2009155382 A1 WO 2009155382A1 US 2009047715 W US2009047715 W US 2009047715W WO 2009155382 A1 WO2009155382 A1 WO 2009155382A1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
Definitions
- the present disclosure relates generally to the design of engineered organisms and, more specifically to organisms having selected genotypes for the production of fumarate, malate, and acrylate.
- Fumaric acid is used in industrial processes as a raw material in a wide range of chemical syntheses. The presence of a double bond and two carboxyl groups in this compound facilitates its use in making polyesters and other polymers. Some of its industrial applications include manufacturing of synthetic resins and biodegradable polymers. It also finds widespread use as a food acidulant, a dietary supplement and as a beverage ingredient. Fumaric acid is currently derived from maleic anhydride, which is produced by the catalytic oxidation of benzene or butene feedstocks.
- fumaric acid is approximately 10% more expensive than maleic anhydride, the non-toxic nature of the former and the special properties, such as greater hardness, that it imparts to the polymer structure makes it a good option for polymer industry as compared to maleic anhydride.
- fumaric acid can be used medicinally for treating a skin condition called psoriasis, and (ii) it can be used as a supplement for cattle feed.
- Malic acid is used as an acidulant and taste enhancer in the beverage and food industry. Racemic malic acid is synthesized petrochemically from maleic anhydride whereas enantiometrically pure L-malic acid (used in pharmaceutical production) is produced from fumarate by hydration with fumarase.
- Acrylic acid is a large volume petrochemical product.
- acrylic acid is a commodity monomer intermediate used for the production of polymeric materials such polyacrylic acid, which is a major component of superabsorbant diapers.
- Acrylic acid also is used for the production of acrylate esters, which are utilized in water-soluble latex coatings, adhesives and inks.
- Acrylic acid and acrylate esters are manufactured by petrochemical processes such as oxidation of propylene, followed by esterification with alcohols such as methanol, butanol, and 2-ethylhexanol.
- the present invention provides a non-naturally occurring microbial organism that includes one or more gene disruptions occurring in genes encoding enzymes selected from the group of fumarate reductase (FRD), alcohol dehydrogenase (ADHEr) and lactate dehydrogenase (LD H_D) such that the one or more gene disruptions confers increased production of fumarate onto said non-naturally occurring microbial organism.
- FBD fumarate reductase
- ADHEr alcohol dehydrogenase
- LD H_D lactate dehydrogenase
- the present invention provides a method for producing fumaric acid that includes culturing a non-naturally occurring microbial organism having one or more gene disruptions occurring in genes encoding enzymes selected from the group of fumarate reductase (FRD), alcohol dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D) such that the one or more gene disruptions confers increased production of fumarate onto said non-naturally occurring microbial organism.
- FBD fumarate reductase
- ADHEr alcohol dehydrogenase
- LDH_D lactate dehydrogenase
- the present invention provides a non-naturally occurring microbial organism that includes one or more gene disruptions occurring in genes encoding enzymes selected from a group of fumarate reducatse (FRD), alcohol dehydrogenase (ADHEr), fumarase (FUM) and lactate dehydrogenase (LDH_D), when the gene disruption reduces an activity of the enzyme it confers increased production of malate onto said non-naturally occurring microbial organism.
- FFD fumarate reducatse
- ADHEr alcohol dehydrogenase
- FUM fumarase
- LDH_D lactate dehydrogenase
- the present invention provides a method for producing malic acid that includes culturing a non-naturally occurring microbial organism having one or more gene disruptions occurring in genes encoding enzymes selected from a group of fumarate reducatse (FRD), alcohol dehydrogenase (ADHEr), fumarase (FUM) and lactate dehydrogenase (LD H_D), when the gene disruption reduces an activity of the enzyme it confers increased production of malate onto said non-naturally occurring microbial organism.
- FFD fumarate reducatse
- ADHEr alcohol dehydrogenase
- FUM fumarase
- L H_D lactate dehydrogenase
- the present invention provides a non-naturally occurring eukaryotic organism, comprising one or more gene disruptions occurring in genes encoding enzymes imparting increased fumarate production in the organism when the gene disruption reduces an activity of the enzyme, whereby the one or more gene disruptions confers increased production of fumarate onto the organism.
- the present invention provides a method for producing fumaric acid that includes culturing a non-naturally occurring eukaryotic organism having one or more gene disruptions occurring in genes encoding an enzyme providing increased fumarate production in the organism when the gene disruption reduces an activity of the enzyme, whereby the one or more gene disruptions confers increased production of fumarate onto the organism.
- the present invention provides a non-naturally occurring eukaryotic organism that includes one or more gene disruptions occurring in genes encoding enzymes imparting increased malate production in the organism when the gene disruption reduces an activity of the enzyme, whereby the one or more gene disruptions confers enhanced production of malate onto the organism.
- the present invention provides a method for producing malic acid that includes culturing a non-naturally occurring eukaryotic organism having one or more gene disruptions occurring in genes encoding enzymes imparting increased malate production to the organism when the gene disruption reduces an activity of the enzyme, whereby the one or more gene disruptions confers increased production of malate onto the organism.
- the present invention provides a non-naturally occurring eukaryotic organism that includes one or more gene disruptions occurring in genes encoding enzymes imparting increased acrylate production in the organism when the gene disruption reduces an activity of the enzyme, whereby the one or more gene disruptions confers increased production of acrylate onto the organism.
- the present invention provides a method for producing acrylic acid that includes culturing a non-naturally occurring eukaryotic organism having one or more gene disruptions occurring in genes encoding enzymes imparting enhanced acrylate production in the organism when the gene disruption reduces an activity of the enzyme, whereby the one or more gene disruptions confers increased production of acrlyate onto the organism.
- the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having an olefin pathway having at least one exogenous nucleic acid encoding an olefin pathway enzyme expressed in a sufficient amount to produce an olefin, the olefin pathway including a decarboxylase.
- the present invention provides a method for producing an olefin that includes culturing a non-naturally occurring microbial organism having an olefin pathway that includes at least one exogenous nucleic acid encoding an olefin pathway enzyme expressed in a sufficient amount to produce an olefin under conditions and for a sufficient period of time to produce an olefin, the olefin pathway including a decarboxylase.
- the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having an acrylate pathway having at least one exogenous nucleic acid encoding an acrylate pathway enzyme expressed in a sufficient amount to produce acrylate, the acrylate pathway including a decarboxylase.
- the present invention provides a method for producing acrylate that includes culturing a non-naturally occurring microbial organism having an acrylate pathway, the pathway includes at least one exogenous nucleic acid encoding an acrylate pathway enzyme expressed in a sufficient amount to produce acrylate under conditions and for a sufficient period of time to produce acrylate, the acrylate pathway including a decarboxylase.
- the present invention provides a method for producing acrylate that includes a) culturing a first non-naturally occurring microbial organism that includes one or more gene disruptions occurring in one or more genes encoding one or more enzymes that enhance fumarate production toin the organism when the one or more genes disruptions reduces an activity of the one or more enzymes, whereby the one or more gene disruptions confers increased production of fumarate onto the non-naturally occurring organism, and b) adding a decarboxylase to the cultured first non-naturally occurring microbial organism, the decarboxylase catalyzing the decarboxylation of fumarate.
- Figure 1 shows the hypothetical production envelopes of an OptKnock-designed strain contrasted against a typical non-growth-coupled production strain. Note that the potential evolutionary trajectories of the OptKnock strain are fundamentally different in that they will lead to a high producing phenotype.
- Figure 2 shows increased fumarate production characteristics of one strain (black, dashed) compared with those of the wild-type E. coli network (black). At the maximum rate of growth, the wild-type network is not expected to form any fumarate.
- Figure 3 shows increased fumarate production characteristics of another strain (black, dotted) compared with those of the wild-type E. coli network (black).
- the grey point shows the minimum amount of product formation expected from this strain.
- Figure 4 shows the production curve for still another strain (grey, dashed) compared with the production curve for the wild-type E. coli network (black). Note that this strain design is equivalent to design B if an additional deletion in THD2 is introduced.
- Figure 5 shows the production curve for yet another strain (grey, dashed) compared with the production curve of the wild type E. coli network (black). The black point indicates the minimum amount of product formation expected from this strain.
- Figure 6 shows the production curves for the strains in Figure 2 (black, dashed), Figure 3 (black, dotted), Figure 4 (grey, dashed) and Figure 5 (grey) compared with each other and with the production characteristics of the wild-type E. coli network (black). Note the reduction in feasible solution space as additional deletions are imposed on the network.
- Figure 7 shows the malate production curve for one strain (light grey) compared with the production curve for the wild type E. coli network (black).
- Figure 8 shows the production curve for a modified malate-producing strain design based on the strain of Figure 7, replacing deletion of FRD with deletion of ASPT,(grey) compared with that of the wild-type E. coli network (black).
- Figure 9 shows increased fumarate production characteristics of one strain (black, dotted) compared with those of the wild-type S. cerevisiae network (black). At the maximum rate of growth, the wild-type network is not expected to form any fumarate.
- Figure 10 shows increased fumarate production characteristics of another strain (light gray) compared with those of the wild-type S. cerevisiae network (black). The gray point shows the minimum amount of product formation expected from this strain.
- Figure 11 shows the production curve for yet another strain (dark gray, dashed) compared with the production curve for the wild-type S. cerevisiae network (black).
- the dark gray point shows the minimum amount of fumarate production expected from this design.
- Figure 12 shows the production curve for still another strain (light gray, dashed) compared with the production curve of the wild type S. cerevisiae network (black).
- the light gray point indicates the minimum amount of product formation expected from this strain.
- Figure 13 shows the production curves for various strains in Figure 9, black, dotted; Figure 10, light gray; Figure 11 dark gray, dashed; and and Figure 12 light gray, dashed, compared with each other and with the production characteristics of the wild-type S. cerevisiae network (black). Note the reduction in feasible solution space as additional deletions are imposed on the network.
- Figure 14 shows the acrylate production curve for one strain (dark gray, dashed) compared with the production curve for the wild type S. cerevisiae network (black).
- Figure 15 shows the acrylate production curve for another strain (black, dotted) compared with the production curve for the wild type S. cerevisiae network (black).
- Figure 16 shows the acrylate production curve for yet another strain (dark gray) compared with the production curve for the wild type S. cerevisiae network (black).
- Figure 17 shows the acrylate production curve for still another strain (light gray) compared with the production curve for the wild type S. cerevisiae network (black).
- Figure 18 shows the acrylate production curve for yet still another strain (light gray, dashed) compared with the production curve for the wild type S. cerevisiae network (black).
- Figure 19 shows the prophetic transformations of a) 1,1- and 1,2-substituted carboxylic acids to terminal olefins catalyzed by a decarboxylase and b) the transformation of a pentadienoic acid to 1,3 -butadiene.
- Figure 20 shows a biosynthetic pathway for the direct production of acrylate through decarboxylation of fumarate.
- Figure 21a shows the prophetic transformation of fumarate to acrylate catalyzed by a decarboxylase.
- Figure 21b shows the decarboxylation of aconitate to itaconate catalyzed by aconitate decarboxylase.
- Figure 21c shows the decarboxylation of 4-oxalocrotonate to 2-oxopentenoate catalyzed by 4- oxalocrotonate decarboxylase.
- Figure 21d shows the decarboxylation of cinnamate derivatives to styrene derivatives catalyzed by a decarboxylase.
- This invention is directed, in part, to engineered organisms having biosynthetic pathways to fumarate, malate, and acrylate.
- the invention utilizes optimization-based approaches based on in silico stoichiometric models of Escherichia coli and Saccharomyces cerevisiae metabolism that identify metabolic designs for increased production of fumarate, malate, and acrylate in these organisms.
- a bilevel programming framework, OptKnock is applied within an iterative algorithm to predict multiple sets of gene disruptions, that collectively result in increased production of fumarate, malate, or acrylate.
- various combinations of gene deletions or functional disruptions of genes significantly improve the fumarate, malate, or acrylate production capabilities of E. coli and S. cerevisiae.
- the invention is also directed, in part, to a developing a route to acrylate from fumarate by reaction with a decarboxylase enzyme.
- the decarboxylase enzyme can be introduced as an exogenous nucleic acid into the same organism that has been engineered for increased fumarate production via gene disruptions, or alternatively through a secondary transformation involving extracellular addition of a decarboxylase to a culture containing over-produced fumarate.
- Another alternative is to provide a second organism having decarboxylase activity. In such a case, the fumarate-producing organism can be co-cultured or serially cultured with the second organism possessing the requisite decarboxylase.
- the engineering designs are equally applicable if an organism other than E. coli or S. cerevisiae is chosen as the production host, even if the organism naturally lacks the activity or exhibits low activity of a subset of the gene products marked for disruption. In those cases, disruptions must only be introduced to eliminate or lessen the enzymatic activities of the gene products that are naturally present in the chosen production host. Production of fumarate, malate, or acrylate for the in silico designs are confirmed by construction of strains having the designed metabolic genotype. These metabolically engineered cells or organisms can also be subjected to adaptive evolution to further augment product production.
- the invention is directed to an integrated computational and engineering platform for developing metabolically altered microorganism strains having enhanced fumarate, malate, or acrylate producing characteristics. Strains identified via the computational component of the platform are put into actual production by genetically engineering the predicted metabolic alterations which lead to the enhanced production of fumarate, malate, or acrylate. Production of the desired product is optionally coupled to optimal growth of the microorganism. Strains exhibiting increased production of these products can be further subjected to adaptive evolution to further augment product biosynthesis. The levels of product production following adaptive evolution also can be predicted by the computational component of the system where, in this specific embodiment, the elevated product levels are realized following evolution.
- Rhizopus Tsao et al., Adv. Biochem. Eng. Biotechnol., 65:243-280 (1999); Lee et al., Macromolecular Bioscience, 4:157-164 (1999); Rhodes et al, Appl. Microbiol. 1962, 10(l):9-15; and Rhodes et al., Appl. Microbiol. 7(2):74-80 (1959)).
- Fumarate production in these organisms utilizes pyruvate carboxylase to fix carbon dioxide, converting pyruvate into oxaloacetate (Kenealy et al., Appl. Environ. Microbiol.
- Rhizopus sporangiospores Another difficulty involved in fumarate production is the tendency of Rhizopus sporangiospores to grow into mycelial mats or mycelial lumps (Zhou et al., Appl. Biochem. Biotechnol. 84-86:779-89 (2000)), interfering with the function of bafflers and propellers inside a reactor.
- a rotary biofilm contactor has been utilized in a simultaneous fermentation-adsorption process to obtain yields of 85 g/L of fumarate from 100 g/L of glucose.
- R. arrhizus NRRl 1526 immoblized on a polyurethane sponge was used to facilitate continuous fermentation for fumarate production.
- This level of malic acid production was achieved by introducing three genetic modifications: (i) overexpression of the native pyruvate carboxylase, (ii) increasing the expression of malate dehydrogenase and retargeting it to cytosol, and (iii) functional expression of a heterologous malate transporter gene.
- Other yeasts in which malic acid has been produced successfully include Aspergillus flavus, Rhizopus arrhizus, and Zygosaccharomyces rouxii. (Zelle, R.M., et al., Appl Environ Microbiol, 74(9):2766- 77 (2008)). The highest malic acid titer has been reported in A.
- This invention is also directed, in part, to methods for producing olefins by decarboxylation an alpha, beta-unsaturated carboxylic acids as exemplified in Figure 19a.
- the unsaturated carboxylic acid substrate can be of any structural olefin geometry.
- the unsaturated carboxylic acid may be substituted at either the alpha or beta position.
- beta- substituted unsaturated carboxylic acid substrates can have either E or Z olefin geometry.
- the product will typically be a terminal olefin.
- carboxylic acid substrate can be further conjugated as shown in Figure 19b, wherein pentadienoic acid is decarboxylated to the commercially valuable commodity chemical 1,3-butadiene.
- 1,3 -butadiene is an important chemical in the manufacture of synthetic rubbers, for example.
- this invention is directed to methods of producing acrylic acid involving primary metabolic production of fumaric acid, followed by decarboxylation.
- Figure 20 shows a biosynthetic scheme for producing acrylic acid which involves treatment of fumaric acid with a decarboxylase enzyme in a pathway leading directly to acrylate, or alternatively through a secondary transformation involving extracellular addition of a decarboxylase to a culture containing over-produced fumarate.
- substrates derived from renewable feedstocks such as glucose, xylose, arabinose, sorbitol, sucrose, glycerol, or even synthesis gas (a mixture carbon monoxide, hydrogen and carbon dioxide), can serve as carbon and energy sources for a fermentation process.
- synthesis gas a mixture carbon monoxide, hydrogen and carbon dioxide
- non- naturally occurring when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species.
- Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species.
- Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon.
- exemplary metabolic polypeptides include enzymes or proteins within a cyclohexanone biosynthetic pathway.
- the term "gene disruption,” or grammatical equivalents thereof, is intended to mean a genetic alteration that renders the encoded gene product inactive.
- the genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene with results in a truncated gene product or by any of various mutation methods that inactivate the encoded gene product.
- gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the non-naturally occurring eukaryotic organisms of the invention.
- gene disruption is also intended to mean a genetic alteration that lowers the activity of a given gene product relative to its activity in a wild-type organism.
- This attenuation of activity can be due to, for example, a deletion in a portion of the gene which results in a truncated gene product or any of various mutation methods that render the encoded gene product less active than its natural form, replacement or mutation of the promoter sequence leading to lower or less efficient expression of the gene, culturing the organism under a condition where the gene is less highly expressed than under normal culture conditions, or introducing antisense RNA molecules that interact with complementary mRNA molecules of the gene and alter its expression.
- a metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides or, functional fragments thereof. Exemplary metabolic modifications are disclosed herein.
- isolated when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature.
- the term includes a microbial organism that is removed from some or all components as it is found in its natural environment.
- the term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments.
- Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.
- microbial As used herein, the terms "microbial,” “microbial organism” or “microorganism” is intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.
- parent decarboxylase refers to both wild-type and previously engineered decarboxylases that serve as a starting point for further optimization of the decarboxylation activity. Optimizations can include not only changes made to the nucleic acid sequence encoding the decarboxylase, but also post-translational modifications to the enzyme product.
- acrylate and "acrylic acid” are used interchangeably.
- a typical carboxylic acid will depend on the pH of its environment. For example, with a pK a of approximately 4, acrylic acid can be significantly in its ionized acrylate form when the pH is 6 or more. While the final isolated product of any given process can be acrylic acid, the direct product of fermentation will frequently be the corresponding acrylate salt, although this can vary depending on the pH conditions employed.
- “fumarate” and “fumaric acid,” “malate” and “malic acid,” and “carboxylate” and “carboxylic acid” are used interchangeably.
- substantially anaerobic when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media.
- the term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1 % oxygen.
- Exogenous as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism.
- the molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism.
- the source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term “heterologous” refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.
- the non-naturally occurring microbal organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration.
- stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.
- E. coli metabolic modifications are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway.
- a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway.
- desired genetic material such as genes for a desired metabolic pathway.
- the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species.
- Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.
- ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms.
- mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides.
- Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor.
- Genes can also be considered orthologs if they share three- dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable.
- Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity.
- Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities.
- Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.
- Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism.
- An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species.
- a specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase.
- a second example is the separation of mycoplasma 5 '-3' exonuclease and Drosophila DNA polymerase III activity.
- the DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.
- paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions.
- Paralogs can originate or derive from, for example, the same species or from a different species.
- microsomal epoxide hydrolase epoxide hydrolase I
- soluble epoxide hydrolase epoxide hydrolase II
- Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor.
- Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.
- a nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species.
- a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein.
- Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.
- Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity.
- Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined.
- a computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art.
- Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% can represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.
- Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm can be as set forth below.
- amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11 ; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on.
- Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept-16- 1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11 ; filter: off.
- Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.
- the invention provides a non- naturally occurring microbial organism, that includes one or more gene disruptions.
- the disruptions occur in genes encoding an enzyme that is obligatory to coupling fumarate production to growth of the microorganism when the gene disruption reduces the activity of the enzyme, such that the gene disruptions confer stable growth-coupled production of fumarate onto the non-naturally occurring microorganism.
- engineered organisms that include one or more gene disruptions can enhance non-growth coupled production fumarate by linking the production of fumarate to energy generation and/or redox balance.
- the disruptions occur in genes encoding an enzyme obligatory to coupling malate production to growth of the microorganism when the gene disruption reduces the activity of the enzyme, such that the gene disruptions confer stable growth-coupled production of malate onto the non-naturally occurring microorganism.
- Engineered organisms that include one or more gene disruptions can also enhance non-growth coupled production malate by linking the production of malate to energy generation and/or redox balance.
- the invention provides a non-naturally occurring microbial organism that includes one or more gene disruptions.
- the disruptions occur in genes encoding an enzyme obligatory to coupling acrylate production to growth of the microorganism when the gene disruption reduces the activity of the enzyme, such that the gene disruptions confer stable growth-coupled production of acrylate onto the non-naturally occurring microorganism.
- engineered organisms that include one or more gene disruptions can also enhance non-growth coupled production acrylate by linking the production of acrylate to energy generation and/or redox balance.
- the invention provides a non-naturally occurring prokaryotic organism, that includes one or more gene disruptions.
- the disruptions occur in genes encoding an enzyme obligatory to coupling fumarate production to growth of the microorganism when the gene disruption reduces the activity of the enzyme, such that the gene disruptions confer stable growth-coupled production of fumarate onto the non-naturally occurring microorganism.
- an engineered prokaryotic organism that includes one or more gene disruptions can also enhance non-growth coupled production fumarate by linking the production of fumarate to energy generation and/or redox balance.
- the invention provides a non-naturally occurring prokaryotic organism that includes one or more gene disruptions.
- the disruptions occur in genes encoding an enzyme obligatory to coupling malate production to growth of the microorganism when the gene disruption reduces the activity of the enzyme, such that the gene disruptions confer stable growth-coupled production of malate onto the non-naturally occurring microorganism.
- an engineered prokaryotic organism that includes one or more gene disruptions can also enhance non-growth coupled production malate by linking the production of malate to energy generation and/or redox balance.
- the invention provides a non-naturally occurring prokaryotic organism that includes one or more gene disruptions.
- the disruptions occur in genes encoding an enzyme obligatory to coupling acrylate production to growth of the organism when the gene disruption reduces the activity of the enzyme, such that the gene disruptions confer stable growth-coupled production of acrylate onto the non-naturally occurring organism.
- an engineered prokaryotic organism that includes one or more gene disruptions can also enhance non-growth coupled production acrylate by linking the production of acrylate to energy generation and/or redox balance.
- the invention provides a non-naturally occurring eurakoytic organism, that includes one or more gene disruptions.
- the disruptions occur in genes encoding an enzyme obligatory to coupling fumarate production to growth of the organism when the gene disruption reduces the activity of the enzyme, such that the gene disruptions confer stable growth-coupled production of fumarate onto the non-naturally occurring organism.
- an engineered eukaryotic organism that includes one or more gene disruptions can also enhance non-growth coupled production fumarate by linking the production of fumarate to energy generation and/or redox balance.
- the invention provides a non-naturally occurring eukaryotic organism that includes one or more gene disruptions.
- the disruptions occur in genes encoding an enzyme obligatory to coupling malate production to growth of the organism when the gene disruption reduces the activity of the enzyme, such that the gene disruptions confer stable growth-coupled production of malate onto the non-naturally occurring organism.
- an engineered eukaryotic organism that includes one or more gene disruptions can also enhance non-growth coupled production malate by linking the production of malate to energy generation and/or redox balance.
- the invention provides a non-naturally occurring eukaryotic organism that includes one or more gene disruptions.
- the disruptions occur in genes encoding an enzyme obligatory to coupling acrylate production to growth of the organism when the gene disruption reduces the activity of the enzyme, such that the gene disruptions confer stable growth-coupled production of acrylate onto the non-naturally occurring organism.
- an engineered eukaryotic organism that includes one or more gene disruptions can also enhance non-growth coupled production acrylate by linking the production of acrylate to energy generation and/or redox balance.
- the present invention provides methods of producing such non-naturally prokaryotic or eukaryotic organisms having stable growth-coupled production of fumarate, malate, or acrylate.
- the method includes: (a) identifying in silico a set of metabolic modifications requiring fumarate production during cell growth, and (b) genetically modifying a microorganism to contain the set of metabolic modifications requiring fumarate production.
- the engineered organisms described herein are useful not only for enhancing growth-coupled production, but they are also well-suited for enhancing non-growth coupled production because they link the production of fumarate, malate and/or acrylate to energy generation and/or redox balance.
- Exemplary non-growth coupled production methods include implementing an aerobic growth phase followed by an anaerobic production phase.
- Vemuri et al. /. Ind. Microbiol. Biotechnol., 6:325-332 (2002) describe a dual-phase process for the production of succinate in E. CoIi.
- a similar non-growth couple production process in a strain of Corynebacterium glutamicum has been described (Okino et al., Appl. Microbiol. Biotechnol.
- Another such method involves withholding an essential nutrient from a propagated cell culture, thereby limiting growth, but not precluding production as described in Durner et al., Appl. Environ. Microbiol. 8:3408-3414( 2000).
- Yet another strategy aimed at decoupling growth from production involves replacing the growth substrate with another compound that is more slowly metabolizable as described in Altamirano et al., Biotechnol. Bioeng. 76:351-360 (2001). Growth decoupled-product formation can also be brought about by specific genetic modifications as described in Blombach et al. Appl. Microbiol. Biotechnol. 79:471-479 (2008).
- OptKnock is a metabolic modeling and simulation program that suggests gene disruption strategies that result in genetically stable microorganisms which overproduce the target product.
- the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth.
- the concept of growth-coupled biochemical production can be visualized in the context of the biochemical production envelopes of a typical metabolic network calculated using an in silico model. These limits are obtained by fixing the uptake rate(s) of the limiting substrate(s) to their experimentally measured value(s) and calculating the maximum and minimum rates of biochemical production at each attainable level of growth. Although exceptions exist, typically the production of a desired biochemical is in direct competition with biomass formation for intracellular resources. Thus, enhanced rates of biochemical production will necessarily result in sub-maximal growth rates.
- the knockouts suggested by OptKnock are designed to restrict the allowable solution boundaries forcing a change in metabolic behavior from the wild-type strain as depicted in Figure 1.
- OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism.
- the OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data.
- OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions.
- OptKnock computational framework allows the construction of model formulations that enable an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems.
- the metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. Patent Application Serial No. 10/043,440, filed January 10, 2002, and in International Patent No. PCT/US02/00660, filed January 10, 2002.
- SimPheny ® Another computational method for identifying and designing metabolic alterations favoring growth-coupled production of a product is metabolic modeling and simulation system termed SimPheny ® .
- This computational method and system is described in, for example, U.S. Patent Application Serial No. 10/173,547, filed June 14, 2002, and in International Patent Application No. PCT/US03/18838, filed June 13, 2003.
- SimPheny ® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system.
- This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions.
- the space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.
- Flux balance analysis is based on flux balancing in a steady state condition and can be performed as described in, for example, Varma and Palsson, Biotech. Bioeng. 12:994-998 (1994). Flux balance approaches have been applied to reaction networks to simulate or predict systemic properties of, for example, adipocyte metabolism as described in Fell and Small, /. Biochem. 138:781-786 (1986), acetate secretion from E. coli under ATP maximization conditions as described in Majewski and Domach, Biotech. Bioeng. 35:732-738 (1990) or ethanol secretion by yeast as described in Vanrolleghem et al., Biotech.
- metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny ® and OptKnock.
- SimPheny ® and OptKnock the computational systems exemplified above as SimPheny ® and OptKnock.
- the methods and strains will be described herein with reference to the OptKnock computation framework for modeling and simulation.
- Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art.
- the ability of a cell or organism to obligatory couple growth to the production of a biochemical product can be illustrated in the context of the biochemical production limits of a typical metabolic network calculated using an in silico model. These limits are obtained by fixing the uptake rate(s) of the limiting substrate(s) to their experimentally measured value(s) and calculating the maximum and minimum rates of biochemical production at each attainable level of growth. As shown in Figure 1 , the production of a desired biochemical generally is in direct competition with biomass formation for intracellular resources. Under these circumstances, enhanced rates of biochemical production will necessarily result in sub-maximal growth rates.
- the knockouts suggested by the above metabolic modeling and simulation programs such as OptKnock are designed to restrict the allowable solution boundaries forcing a change in metabolic behavior from the wild-type strain as depicted in Figure 1. Although the actual solution boundaries for a given strain will expand or contract as the substrate uptake rate(s) increase or decrease, each experimental point will lie within its calculated solution boundary. Plots such as these enable accurate predictions of how close the designed strains are to their performance limits which also indicates how much room is available for improvement.
- the OptKnock mathematical framework is exemplified herein for pinpointing gene deletions leading to growth-coupled biochemical production as illustrated in Figure 1.
- the procedure builds upon constraint-based metabolic modeling which narrows the range of possible phenotypes that a cellular system can display through the successive imposition of governing physico-chemical constraints, Price et al., Nat Rev Microbiol, 2: 886-97 (2004).
- constraint-based models and simulations are well known in the art and generally invoke the optimization of a particular cellular objective, subject to network stoichiometry, to suggest a likely flux distribution.
- v substrate v substrate uptake mmoi/gDW-hr V i e ⁇ limiting substrat ⁇ (s) ⁇ v atp ⁇ v at p_ ma ⁇ mmol/gDW-hr v j ⁇ 0' V j e ⁇ irrev. reactions ⁇
- S ⁇ is the stoichiometric coefficient of metabolite i in reaction j
- v is the flux of reaction j
- v SU bstrate_ U ptake represents the assumed or measured uptake rate(s) of the limiting substrate(s)
- v a t P _ m a m is the non-growth associated ATP maintenance requirement.
- the vector v includes both internal and external fluxes.
- reaction flux V 1 is not active ' assume a value of 1 if reaction j is active and a value of 0 if it is inactive.
- V j e M ensures that reaction flux y, is set to zero only if variable y ⁇ is equal to zero.
- y j - is equal to one
- v is free to assume any value between a lower v ⁇ mm and an upper v,TM" bound.
- y/" m and V j max are identified by minimizing and maximizing, respectively, every reaction flux subject to the network constraints described above, Mahadevan et al., Metab Eng, 5: 264-76 (2003).
- this bilevel optimization problem is illustrated in Figure 2.
- this bilevel optimization problem is expressed as the following bilevel mixed-integer optimization problem:
- any solution of the above described bilevel OptKnock problem will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in fumarate as an obligatory product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes.
- the set of reactions that are to be disrupted in order to achieve increased fumarate, malate, or acrylate production are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set.
- one particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene.
- These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the product coupling are desired or when genetic reversion is less likely to occur.
- integer cuts an optimization method, termed integer cuts. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatory couples product biosynthesis to growth.
- Constraints of the above form preclude identification of larger reaction sets that include previously identified sets. For example, employing the integer cut optimization method above in a further iteration would preclude identifying a quadruple reaction set that specified reactions 1, 2, and 3 for disruption since these reactions had been previously identified. To ensure identification of all possible reaction sets leading to growth-coupled production of a product, a modification of the integer cut method was employed.
- the modified integer cut procedure begins with iteration 'zero' which calculates the maximum production of the desired biochemical at optimal growth for a wild-type network. This calculation corresponds to an OptKnock solution with K equaling 0.
- the two parameter sets, objstore lter and ystoreuer j are introduced to store the objective function (y chemical) and reaction on-off information (y ⁇ ), respectively, at each iteration, iter.
- the following constraints are then successively added to the OptKnock formulation at each iteration.
- ⁇ and M are a small and a large numbers, respectively.
- ⁇ can be set at about 0.01 and M can be set at about 1000.
- numbers smaller and/or larger then these numbers also can be used.
- M ensures that the constraint can be binding only for previously identified knockout strategies, while censures that adding knockouts to a previously identified strategy must lead to an increase of at least ⁇ in biochemical production at optimal growth.
- the approach moves onto double deletions whenever a single deletion strategy fails to improve upon the wild-type strain. Triple deletions are then considered when no double deletion strategy improves upon the wild-type strain, and so on.
- the end result is a ranked list, represented as desired biochemical production at optimal growth, of distinct deletion strategies that differ from each other by at least one knockout.
- This optimization procedure as well as the identification of a wide variety of reaction sets that, when disrupted, lead to increased production of a biochemical product are exemplified in detail further below. Given the teachings and guidance provided herein, those skilled in the art will understand that the methods and metabolic engineering designs exemplified herein are applicable to linking cell or microorganism growth to any biochemical product.
- metabolic alterations have been identified that obligatorily couple the production of fumarate, malate, or acrylate to organism growth.
- Prokaryotic or eukaryotic organism strains constructed with the identified metabolic alterations produce elevated levels of fumarate, malate, or acrylate during the exponential growth phase. These strains can be beneficially used for the commercial production of fumarate, malate, or acrylate in continuous fermentation process without being subjected to the negative selective pressures described previously.
- the metabolic alterations also enable non-growth coupled production of fumarate, malate, or acrylate.
- the invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more genes associated with the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction and that reference to any of these metabolic constitutes also references the gene or genes encoding the enzymes that catalyze the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes as well as the reactants and products of the reaction.
- the methods of the invention provide a set of metabolic modifications that are identified by an in silico method selected from OptKnock.
- the set of metabolic modifications can include functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.
- Exemplary reactions, reaction nomenclature, reactants, products, cofactors and genes encoding enzymes catalyzing a reaction involved in the growth-coupled production of fumarate and malate in E. CoIi are set forth in Tables 1, 2, 3, and 4.
- the invention provides non naturally occurring microorganisms having increased production of fumarate or malate. Fumarate or malate production can be obligatorily linked to the exponential growth phase of the microorganism by genetically altering the metabolic pathways of the cell.
- the genetic alterations make fumarate an obligatory product during the growth phase.
- fumarate or malate production is not obligatorily linked to growth.
- the production of fumarate or malate takes place during a non-growth phase, for example.
- Tables 1 and 2 Sets of metabolic alterations or transformations that result in elevated levels of fumarate or malate biosynthesis are exemplified in Tables 1 and 2, respectively. Each alteration within a set corresponds to the requisite metabolic reaction that can be functionally disrupted. Functional disruption of all reactions within each set results increased production of fumarate or malate by the engineered strain.
- the corresponding reactions to the referenced alterations in Tables 1 and 2, and the gene or genes that potentially encode them in E. coli are set forth in Table 3.
- the metabolic alterations that can be generated for increased fumarate production are shown in each row. These alterations include the functional disruption of from one to six or more reactions.
- 348 strains are exemplified in Table 1 that have non-naturally occurring metabolic genotypes. Each of these non- naturally occurring alterations result in an enhanced level of fumarate production during the exponential growth phase of the microorganism compared to a wild-type strain, under appropriate culture conditions.
- Appropriate conditions include, for example, those exemplified further below in the Example I such as particular carbon sources or reactant availabilities and/or adaptive evolution.
- FFD fumarate reductase
- ADHEr alcohol dehydrogenase
- LD H_D lactate dehydrogenase
- GLUDy glutamate dehydrogenase
- Another strain shown in Figure 3 has three common deletions with the strain shown in Figure 2 and involves elimination of malic enzyme (ME2) and transhydrogenase (THD2) activity additionally.
- Malic enzyme catalyzes the decarboxylation of malate to form pyruvate with the concomitant reduction of a molecule of NADP to form NADPH.
- Transhydrogenase catalyzes the oxidation of NADH causing the reduction of NADP into NADPH.
- NADPH- forming malic enzyme and the membrane-bound proton-translocating transhydrogenase catalyzed by PntAB prevents or reduces the formation of NADPH, thus preventing or reducing carbon from being funneled into amino acids instead of being converted into fumarate.
- the efficacy of the two latter knockouts for fumarate production can be assessed and implemented sequentially based on the necessity to do so.
- the strain of Figure 3 is expected to have a maximum growth-coupled yield of 1.87 moles of fumarate per mole of glucose consumed at an expected maximum growth rate of 0.08/hr. Note also that the strain is has a minimum theoretical product yield of 0.48 moles per mole of glucose (the grey point on the black, dotted curve).
- Figure 3 shows the growth-coupled fumarate production characteristics of the strain (black, dotted) compared with those of the wild-type E. coli network (black). The grey point shows the minimum amount of product formation expected from this strain.
- FIG. 4 Another strain, shown in Figure 4, disrupts the GLCpts mechanism of glucose transport and instead relies on hexokinase activity. This disruption along with disruption of FRD, ADHEr, LDH_D, and ME2 leads to an expected maximum growth rate for the strain at approximately 0.1 per hour. The product yield is expected to be 1.82 moles per mole of glucose consumed. The strain is expected to start producing fumarate once it reaches approximately 36% of its maximum theoretical biomass formation rate.
- Figure 4 shows the production curve for this strain (grey, dashed) compared with the production curve for the wild-type E. coli network (black). Note that this strain is equivalent to the strain of Figure 2 if an additional deletion in THD2 is introduced.
- Another strain shown in figure 5, has deletions in FRD, ADHEr, LDH_D, ME2, THD2 and HEXl.
- the deletion in HEXl forces glucose flux through the PTS system, converting an equivalent molar amount of phosphoenolpyruvate (PEP) into pyruvate.
- PEP phosphoenolpyruvate
- the network is forced to convert most of the pyruvate back into PEP through PEP synthase. This is an energy-intensive step and limits the biomass formation in the network.
- the carbon distribution provides PEP to be used by PPCK and subsequent channeling into the reductive TCA cycle. This leads to the very high fumarate yields in the network of up to 1.97 moles per mole of glucose consumed as shown in Figure 5.
- strain is expected to have a minimum product yield of at least 1.25 moles per mole of glucose consumed as shown by the black point in Figure 5.
- Figure 5 shows the production curve for the strain (grey, dashed) compared with the production curve of the wild type E. coli network (black). The black point indicates the minimum amount of product formation expected from this strain.
- the anaerobic designs for the formation of malate are described below and utilize disruptions that have already been described for fumarate production.
- the strain designs for malate production have additional knockouts that preclude fumarate formation in the network.
- One strain, shown in Figure 7, allows for increased formation of malate by building upon the disruptions in the strain of Figure 2.
- deletions in ADHEr, LDH_D, FRD, ME2 and THD2 allow for the enhanced formation of either fumarate or malate.
- An additional deletion in fumarase (FUM) prevents or reduces the conversion of malate into fumarate, leading to increase malate production of 1.86 moles per mole of glucose consumed as shown in Figure 7.
- Small modifications in this strain lead to another high- yielding strain shown in Figure 8.
- this strain has a disruption in aspartase (ASPT).
- ASPT The deletion of ASPT reinforces the effect of the fumarase deletion by preventing the network from converting oxaloacetate into aspartate which can subsequently be transformed into fumarate via aspartase. Without the deletion in ASPT, the strain can produce approximately 1.55 moles of succinate per mole of glucose consumed.
- Several other strains with increased malate yields in E. CoIi have been identified and are listed in Table 2.
- the invention also provides a non-naturally occurring microorganism having a set of metabolic modifications coupling fumarate or malate production to growth of the microorganism, the set of metabolic modifications includes disruption of one or more genes selected from the set of genes encoding proteins that include: (a) a fumarate reductase (FRD), an alcohol dehydrogenase (ADHEr), and a lactate dehydrogenase (LD H_D).
- FFD fumarate reductase
- ADHEr alcohol dehydrogenase
- LD H_D lactate dehydrogenase
- the minimum enzyme disruption set based on the aforementioned strains includes disruption of fumarate reductase, alcohol dehydrogenase and lactate dehydrogenase. This corresponds to the following minimal exemplary gene disruption set:
- frd (b4151 or b4152 or b4153 or M154), adhE (bl421), and UhA (bl380)
- the minimal set of activities that need to be deleted can be expanded to include the following: Fumarate reductase, alcohol dehydrogenase, lactate dehydrogenase, and glutamate dehydrogenase, or
- the minimal gene set can be expanded to yield:
- frd (b4151 or b4152 or b4153 or M 154;, adhE (bl421), ldhA (bl380), and gdhA (bl761), or
- frd (b4151 or b4152 or b4153 or M 154), adhE (bl421), ldhA (bl380), and maeB (b2463), or
- frd (b4151 or b4152 or b4153 or M154), adhE (bl421), ldhA (JDUW), pntAB (bl602, bl603), and maeB (b2463)
- Fumarate reductase is Fumarate reductase, alcohol dehydrogenase, lactate dehydrogenase, transhydrogenase, malic enzyme, and hexokinase, or
- Fumarate reductase is Fumarate reductase, alcohol dehydrogenase, lactate dehydrogenase, malic enzyme, and the PTS transport mechanism of glucose, or
- Fumarate reductase is Fumarate reductase, alcohol dehydrogenase, lactate dehydrogenase, transhydrogenase, malic enzyme, and pyruvate formate lyase
- frd (b4151 or b4152 or b4153 or M 154), adhE (bl421), ldhA (JoUW), pntAB (bl602, bl603), maeB (b2463) and glk (b2388) frd (b4151 or b4152 or b4153 or M 154), adhE (bl421), UhA (bl380), maeB (b2463), and pts (bllOl or b2415 or b2416 or b2417)
- frd (b4151 or b4152 or b4153 or M 154), adhE (bl421), UhA (bl380), / ⁇ A5 (bl602, bl603), maeB (b2463), and pflAB (b0902, b0903)
- FUM disruption in fumarase
- adhE (bl421), UhA (bl380), and fumABC (bl611, bl612, b4122) and asp A (b4139)
- the present invention provides a non-naturally occurring microbial organism that includes one or more gene disruptions occurring in genes encoding enzymes that increase homomalate production when the gene disruption reduces an activity of the enzyme, whereby the one or more gene disruptions confers increased production of homomalate onto said non-naturally occurring microorganism.
- glutamate dehydrogenase glutamate dehydrogenase
- TDD2 transhydrogenase
- ME2 malic enzyme
- Fumarate reductase alcohol dehydrogenase, lactate dehydrogenase, fumarase, and glutamate dehydrogenase
- Fumarate reductase alcohol dehydrogenase, lactate dehydrogenase, fumarase, and malic enzyme
- each of these strains may be supplemented with additional deletions if it is determined that the strain does not sufficiently increase the formation of the product.
- some other enzymes not known to possess significant activity may become active due to adaptive evolution or random mutagenesis and they will also have to be disrupted as well.
- succinate dehydrogenase which oxidizes succinate to fumarate and is known to be active only under aerobic conditions may assume significant activity even under anaerobic conditions and may have to be disrupted.
- the list of gene disruption sets provided here serves as a starting point for construction of high- yielding malate and fumarate producing strains.
- the non-naturally occurring eukaryotic organism can have one or more gene disruptions included in a metabolic modification listed in Tables 5 or 6.
- the one or more gene disruptions can be a deletion.
- the non-naturally occurring eukaryotic organism of the invention can be selected from a group of eukaryotic organism having a metabolic modification listed in Tables 5 or 6.
- Non-naturally occurring eurkaryotic organisms of the invention include yeast, fungus, or any of a variety of other microorganisms applicable to fermentation processes.
- Exemplary eukaryotic species include those selected from S ⁇ cch ⁇ romyces cerevisi ⁇ e, Schizos ⁇ cch ⁇ romyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Rhizopus arrhizus, Rhizopus oryzae, and Pichiapastoris.
- the eukaryotic organisms having increased fumarate, malate, or acrylate production are exemplified herein with reference to an S. cerevisiae genetic background.
- the complete genome sequence available for now more than 550 species including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes
- the identification of an alternate species homolog for one or more genes including for example, orthologs, paralogs and nonorthologous gene displacements, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations enabling increased production of the products described herein with reference to a particular organism such as S.
- cerevisiae can be readily applied to other microorganisms, especially other eukaryotic organisms. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.
- homologues can include othologs and/or nonorthologous gene displacements.
- functional disruption can be accomplished by, for example, deletion of a paralog that catalyzes a similar, yet non-identical metabolic reaction which replaces the referenced reaction. Because there are differences among metabolic networks between different organisms, those skilled in the art will understand that the actual genes disrupted between different organisms may differ.
- the invention provides non naturally occurring eukaryotic organisms having growth-coupled production of fumarate, malate, or acrylate.
- Product production can be optionally obligatorily linked to the exponential growth phase of the microorganism by genetically altering the metabolic pathways of the cell.
- the genetic alterations can make the formation of the desired product obligatory to growth.
- Sets of metabolic alterations or transformations that result in elevated levels of fumarate, malate, or acrylate biosynthesis are exemplified in Tables 5 and 6, respectively. Each alteration within a set corresponds to the requisite metabolic reaction that should be functionally disrupted. Functional disruption of all reactions within each set results in the obligatory production of fumarate, malate, or acrylate by the engineered strain during the growth phase.
- the corresponding reactions to the referenced alterations in Tables 5 and 6, and the gene or genes that potentially encode them in S. cerevisiae, are set forth in Table 7.
- the metabolic alterations that can be generated for increase fumarate or malate production are shown in each row. These alterations include the functional disruption of from one to six or more reactions.
- 278 strains are exemplified in Table 5 that have non- naturally occurring metabolic genotypes. Each of these non- naturally occurring alterations result in an enhanced level of fumarate or malate production in the eukaryotic organism compared to a wild-type strain, under appropriate culture conditions. Appropriate conditions include, for example, those exemplified further below in the Example II such as particular carbon sources or reactant availabilities and/or adaptive evolution.
- 495 strains are exemplified in Table 6 that have non-naturally occurring metabolic genotypes. Each of these non-naturally occurring alterations result in an enhanced level of acrylate production during the exponential growth phase of the eukaryotic organism compared to a wild- type strain, under appropriate culture conditions.
- Disruption can occur by a variety of means including, for example, deletion of an encoding gene or incorporation of a genetic alteration in one or more of the encoding gene sequences as described previously in reference to the disruptions for E. CoIi.
- the encoding genes targeted for disruption can be one, some, or all of the genes encoding enzymes involved in the catalytic activity. For example, where a single enzyme is involved in a targeted catalytic activity disruption can occur by a genetic alteration that reduces or destroys the catalytic activity of the encoded gene product.
- disruption can occur by a genetic alteration that reduces or destroys the function of one or all subunits of the encoded gene products. Destruction of activity can be accomplished by loss of the binding activity of one or more subunits in order to form an active complex, by destruction of the catalytic subunit of the multimeric complex or by both. Other functions of multimeric protein association and activity also can be targeted in order to disrupt a metabolic reaction of the invention. Such other functions are well known to those skilled in the art. Further, some or all of the functions of a single polypeptide or multimeric complex can be disrupted according to the invention in order to reduce or abolish the catalytic activity of one or more enzymes involved in a reaction or metabolic modification of the invention. Similarly, some or all of enzymes involved in a reaction or metabolic modification of the invention can be disrupted so long as the targeted reaction is destroyed.
- an enzymatic reaction can be disrupted by reducing or eliminating reactions encoded by a common gene and/or by one or more orthologs of that gene exhibiting similar or substantially the same activity. Reduction of both the common gene and all orthologs can lead to complete abolishment of any catalytic activity of a targeted reaction. However, disruption of either the common gene or one or more orthologs can lead to a reduction in the catalytic activity of the targeted reaction sufficient to promote coupling of growth to product biosynthesis.
- Exemplified herein are both the common genes encoding catalytic activities for a variety of metabolic modifications as well as their orthologs.
- PPCK reversibility of PPCK allows for the fixing of carbon dioxide such that a yield of 2 moles per mole of glucose for each of these products can be attained under microaerobic/anaerobic conditions. More importantly, it allows for production of ATP in the process.
- the ATP generation accompanying the reverse operability of PPCK supports the energy requirements for biomass formation as well as for product formation and export under anaerobic conditions. Note that the production of fumaric, malic and acrylic acids is otherwise energetically neutral in the S. cerevisiae metabolic network.
- a heterologous enzyme for example from Mannheimia succiniciproducens (Lee et al., Appl Environ Microbiol, 72:1939- 1948 (2006)), Anaerobio spirillum succiniciproducens (Laivenieks et al., Appl Environ Microbiol, 63:2273-2280 (1997)), or Actinobacillus succinogenes (Kim, P.
- the ATPase in S. cerevisiae uses one ATP for exporting out each proton which makes fumarate and malate production energetically neutral under anaerobic conditions. A limited supply of oxygen therefore provides for favorable energetics that can enable growth and product export.
- microaerobic (substantially anaerobic) designs can be used based on increased formation of the desired product. To examine this, production cones were constructed for each strain by first maximizing and, subsequently minimizing the product yields at different rates of biomass formation feasible in the network. If the rightmost boundary of all possible phenotypes of the mutant network is a single point, it implies that there is a unique optimum yield of the product at the maximum biomass formation rate possible in the network.
- the rightmost boundary of the feasible phenotypes is a vertical line, indicating that at the point of maximum biomass the network can make any amount of the product in the calculated range, including the lowest amount at the bottommost point of the vertical line.
- Such designs were given a low priority.
- the fumarate-production strategies identified by the OptKnock framework were ranked on the basis of their (i) theoretical yields, and (ii) growth-coupled fumarate formation characteristics. All the strains with high product yields involve four or more knockouts because fewer knockouts were found to provide markedly lower yields. Strains with high yields include, for example, those with about 70% or more yield.
- the engineered strains can include further metabolic modifications aimed at limiting the production of the fumarate precursor, malate that is at the same redox state as fumarate.
- the fumarase enzyme(s) can be manipulated by using techniques such as directed evolution so that the overall kinetics favors the conversion of malate into fumarate.
- Another option is to use a fumarase enzyme from any of the Rhizopus species that are known to produce high concentrations of fumarate without malate formation (e.g.fumR from R. oryzae, GenBank accession number: X78576).
- a fumarase enzyme from any of the Rhizopus species that are known to produce high concentrations of fumarate without malate formation (e.g.fumR from R. oryzae, GenBank accession number: X78576).
- an additional enzyme activity is introduced into S. cerevisiae to channel fumarate into a different growth-coupled end product, it will drive the metabolism towards fumarate formation and prevent malate formation.
- a case in point is the production of acrylic acid.
- G3PD glycerol-3- phosphate dehydrogenase
- PYRDC pyruvate decarboxylase
- FUMm mitochondrial fumarase
- FUMm soluble fumarate reductase
- FUMm soluble fumarate reductase
- FUMm soluble fumarate reductase
- FRDcm soluble fumarate reductase
- This strain is predicted to have a growth-coupled yield of 1.47 moles of fumarate per mole of glucose consumed and the maximum growth rate is anticipated to be 0.07/hr as shown in Figure 9.
- the sorbitol reductase activity encoded by YHR104W
- All the proposed disruptions can be implemented sequentially based on the necessity to do so.
- the strain shown in Figure 10 has four disruptions including malic enzyme (MEIm), pyruvate kinase (PYK), fumarase (FUMm), and soluble fumarate reductase (FRDcm), two of which are the same as those in Figure 9. Under microaerobic conditions, this set of disruptions is expected to yield fumarate up to 1.71 moles/mole of glucose consumed.
- the disruptions in pyruvate kinase and the malic enzyme are targeted at preventing pyruvate formation in the network such that the maximum amount of PEP can be routed into the reductive TCA cycle using the energy- generating PPCK.
- the disruptions in mitochondrial fumarase and in the soluble fumarate reductase prevent the carbon flux from being routed into the reductive mitochondrial TCA cycle and prevent further reduction of fumarate into succinate, respectively.
- the strain is expected to produce a minimum of 18% of its maximum theoretical yield see gray point in Figure 10.
- the strain is predicted to have a maximum growth rate of 0.045/hr.
- the strain in Figure 11 has an additional disruption in glucose-6-phosphate dehydrogenase as compared to the strain in Figure 9.
- the disruption of G6PDH alters the cofactor balance in the network favorably for fumarate production at the cost of biomass production by preventing the NADPH formation required for biomass synthesis.
- These disruptions lead to an expected maximum growth rate of approximately 0.041 per hour for the strain as shown in Figure 11.
- the maximum theoretical fumarate yield is 1.79 moles per mole of glucose consumed.
- the imposed disruptions reduce the feasible phenotypes significantly such that the strain is anticipated to produce a minimum of 84% of its maximum theoretical yield just to grow as shown by the dark gray point in Figure 11.
- Another strain shown in Figure 12 has an additional disruption in isocitrate dehydrogenase as compared to the strain in Figure 11, leading to a marginal increase in the expected maximum theoretical fumarate yield to 1.83 moles per mole of glucose consumed.
- the rationale is analogous to that explained for the disruption of G6PDH in design.
- the maximum biomass formation rate is anticipated to decrease from 0.04 per hour to 0.03 per hour.
- Figure 13 shows their production curves on the same plot and compares them with those of the wild-type S. cerevisiae network. All the other designs for fumarate production are listed in Table 5. All the designs proposed for fumarate production described above and in Table 5 can be used for malate production under microaerobic conditions by introducing an additional disruption in the cytosolic fumarase gene that will prevent the conversion of malate into fumarate.
- One strain for acrylate production shown in Figure 14, has a disruption in pyruvate decarboxylase (PYRDC). Under anaerobic conditions, a disruption in pyruvate decarboxylase reduces ethanol formation significantly. All the carbon flux is instead redirected towards acrylate production which also allows for the regeneration of the NADH generated in the network, leading to a tight coupling with biomass formation in the network.
- the maximum product yield is predicted to be 1.55 moles per mole of glucose consumed at the highest growth rate of 0.21 per hour.
- PYK pyruvate kinase
- ATPSm mitochondrial ATP synthase
- the disruption in PYK prevents PEP conversion into pyruvate.
- the disruption of ATP synthase prevents ATP formation in the mitochondrion, removing the incentive for the network to route carbon flux into the mitochondrion.
- the product of YRJ121 W is directly involved in the formation of Fl-ATP synthase (beta subunit), while YMR064W is a translational regulator required for expression of the mitochondrial ATPase subunit 9 in yeast (Saltzgaber-Muller et al., J Biol Chem. 258:11465-11470 (1983)).
- Another strain shown in Figure 16, has disruptions in malic enzyme (NAD-dependent) (MElm)and in pyruvate kinase (PYK). These disruptions are geared towards preventing pyruvate formation in the network. Thus, they have a similar effect to the disruption of PYRDC which limits pyruvate formation by preventing its utilization for acetaldehyde and subsequently, ethanol formation. Overall, these two disruptions cause a high flux through PPCK, ultimately leading to a growth-coupled acrylate yield of 1.61 moles per mole of glucose in the network. The strain is calculated to have a maximum growth rate of 0.19 per hour.
- Still another strain shown in Figure 18, has additional disruptions in fumarase (FUMm) and soluble fumarate reductase (FRDcm).
- FUMm fumarase
- FFDcm soluble fumarate reductase
- This strain can be grown in anaerobic conditions leading to acrylate production.
- the maximum theoretical acrylate yield of the strain is expected to be 1.65 moles per mole of glucose consumed at the maximum predicted growth rate of 0.18 per hour.
- the invention also provides a non-naturally occurring eukaryotic organism having a set of metabolic modifications coupling fumarate, malate, or acrylate production to growth of the organism, the set of metabolic modifications includes disruption of one or more genes selected from the set of genes encoding proteins that include: (a) glycerol-3-phosphate dehydrogenase (G3PD), (b) pyruvate decarboxylase (PYRDC), (c) soluble fumarate reductase (FRDcm) and (d) mitochondrial fumarase (FUMm).
- G3PD glycerol-3-phosphate dehydrogenase
- PYRDC pyruvate decarboxylase
- FUMm mitochondrial fumarase
- the set of metabolic modifications includes disruption of one or more genes selected from the set of genes encoding proteins that include: (a) malic enzyme (MEIm), (b) pyruvate kinase (PYK), (c) soluble fumarate reductase (FRDcm), and (d) mitochondrial fumarase (FUMm).
- MEIm malic enzyme
- PYK pyruvate kinase
- FUMm mitochondrial fumarase
- G3PD glycerol-3-phosphate dehydrogenase
- PYRDC pyruvate decarboxylase
- FUMm mitochondrial fumarase
- An alternative enzyme disruption set entails the removal of malic enzyme (MEIm), pyruvate kinase (PYK), soluble fumarate reductase (FRDcm) and mitochondrial fumarase (FUMm) for coupling fumarate production to growth.
- MEIm malic enzyme
- PYK pyruvate kinase
- FUMm mitochondrial fumarase
- Glycerol-3-phosphate dehydrogenase G3PD
- PYRDC pyruvate decarboxylase
- FUMm mitochondrial fumarase
- MEIm Malic enzyme
- PYK pyruvate kinase
- FUMm mitochondrial fumarase
- G3PD G3PD
- YLR044C YGR087C
- YLR134W isozymes for PYRDC
- YPL262W FUMm
- YEL047C FUMm
- YKL029C MEIm
- YOR347C YAL038W
- YPL262W FUMm
- YEL047C FUMm
- FUMm YEL047C
- G6PDH glucose-6- phosphate dehydrogenase
- ICDHy cytosolic NADP-dependent isocitrate dehydrogenase
- the enzyme disruption sets after introducing these auxiliary disruptions are: Glycerol- 3-phosphate dehydrogenase (G3PD), pyruvate decarboxylase (PYRDC), soluble fumarate reductase (FRDcm) and mitochondrial fumarase (FUMm), and glucose-6-phosphate dehydrogenase (G6PDH), or Malic enzyme (MEIm), pyruvate kinase (PYK), soluble fumarate reductase (FRDcm) and mitochondrial fumarase (FUMm), glucose-6-phosphate dehydrogenase (G6PDH) and cytosolic NADP-dependent isocitrate dehydrogenase (ICDHy).
- G3PD Glycerol- 3-phosphate dehydrogenase
- PYRDC pyruvate decarboxylase
- FUMm glucose-6-phosphate dehydrogenase
- MEIm Malic enzyme
- PYK pyru
- enzyme sets corresponds to the following gene disruption sets: YDL022W (G3PD), YLR044C, YGR087C, YLR134W (isozymes for PYRDC), YPL262W (FUMm), and YEL047C (FRDcm), YNL241C (G6PDH), or YKL029C (MEIm), YOR347C, YAL038W (isozymes for PYK), YPL262W (FUMm), and YEL047C (FRDcm), YNL241C (G6PDH), and YLR174W (ICDHy).
- the enzyme disruption sets can be augmented with the disruption of the cytosolic fumarase which is also encoded by YPL262W.
- YPL262W encodes for both the cytosolic and the mitochondrial fumarases. However, its localization is determined by the N- terminal mitochondrial targeting sequence and its conformation (Sass et al., J Biol Chem. 278:45109-45116 (2003)).
- the corresponding gene disruption sets are: YLR044C, YGR087C, YLR134W (PYRDC), or YKL029C (MEIm), YOR347C, YAL038W (PYK), or YOR347C, YAL038W (encode for PYK isozymes), YJR121W and YMR064W or any other combination of genes that eliminates mitochondrial synthase activity.
- auxiliary disruptions include but are not limited to mitochondrial fumarase, soluble fumarate reductase and glycerol- 3 -phosphate dehydrogenase.
- the corresponding gene disruptions are: YPL262W (FUMm), and YEL047C (FRDcm) and YDL022W (G3PD).
- the disruption of pyruvate decarboxylase is very similar to the disruption of alcohol dehydrogenase in that both are targeted to prevent ethanol formation in the network.
- the disruption of alcohol dehydrogenase activity can completely eliminate ethanol formation.
- PYRDC is included in the minimum enzyme disruption set.
- succinate dehydrogenase that oxidizes succinate to fumarate and is known to be active only under aerobic conditions may assume significant activity even under anaerobic conditions and may have to be knocked out.
- the list of gene disruption sets provided here serves as a starting point for construction of high- yielding growth-coupled malate, fumarate and acrylate production strains.
- the invention provides a method for producing fumaric acid malic acid, or acrylic acid that includes culturing a non- naturally occurring prokaryotic or eukaryotic microbial organism that includes one or more gene disruptions.
- the disruptions can occur in genes encoding an enzyme obligatory to coupling fumarate or malate production to growth of the microorganism when the gene disruption reduces an activity of the enzyme, such that the disruptions confer stable growth-coupled production of fumarate or malate onto the non- naturally occurring microorganism.
- the non-naturally occurring prokaryotic or eukaryotic organisms of the invention can be employed in the growth-coupled production of fumarate, malate, or acrylate.
- any quantity, including commercial quantities, can be synthesized using the growth-coupled fumarate, malate, or acrylate producers of the invention.
- the organisms of the invention obligatorily couple fumarate, malate, or acrylate to continuous growth or near-continuous growth processes are particularly useful for biosynthetic production of fumarate, malate, or acrylate.
- Such continuous and/or near continuous growth processes are described above and exemplified below in the Example I. Continuous and/or near-continuous microorganism growth processes also are well known in the art.
- continuous and/or near-continuous growth processes involve maintaining the microorganism in an exponential growth or logarithmic phase.
- Procedures include using apparatuses such as the EvolugatorTM evolution machine (Evolugate LLC, Gainesville, FL), fermentors and the like. Additionally, shake flask fermentation and grown under microaerobic conditions also can be employed. Given the teachings and guidance provided herein those skilled in the art will understand that the growth-coupled fumarate producing microorganisms can be employed in a variety of different settings under a variety of different conditions using a variety of different processes and/or apparatuses well known in the art.
- the continuous and/or near-continuous production of fumarate, malate, or acrylate will include culturing a non-naturally occurring growth-coupled fumarate, malate, or acrylate producing organism of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase.
- Continuous culture under such conditions can be grown, for example, for a day, 2, 3, 4, 5, 6 or 7 days or more.
- continuous cultures can include time durations of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods.
- culturing is conducted in a substantially anaerobic culture medium.
- Fumarate, malate, or acrylate can be harvested or isolated at any time point during the continuous and/or near-continuous culture period exemplified above. As exemplified below, the longer the microorganisms are maintained in a continuous and/or near-continuous growth phase, the proportionally greater amount of fumarate and malate can be produced.
- bioprocessing is whether to use a batch or continuous fermentation scheme.
- One difference between the two schemes that will influence the amount of product produced is the presence of a preparation, lag, and stationary phase for the batch scheme in addition to the exponential growth phase.
- continuous processes are kept in a state of constant exponential growth and, if properly operated, can run for many months at a time.
- productivities i.e., dilution rate times cell mass
- the invention provides a method for producing fumaric acid, malic acid, or acrylic acid that includes culturing a non-naturally occurring prokaryotic or eukaryotic organism that includes one or more gene disruptions as described above.
- the disruptions can occur in genes encoding an enzyme obligatory to coupling fumarate, malate, or acrylate production to growth of the microorganism when the gene disruption reduces an activity of the enzyme, such that the disruptions confer increased production of fumarate, malate, or acrylate onto the non-naturally prokaryotic or eukaryotic organism.
- the gene disruptions can also be non-growth coupled in other embodiments.
- the gene disruption can include a complete gene deletion.
- other means to disrupt a gene include, for example, frameshifting by omission or addition of oligonucleotides or by mutations that render the gene inoperable.
- frameshifting by omission or addition of oligonucleotides or by mutations that render the gene inoperable.
- gene disruptions are selected from the gene set that includes genes detailed herein above.
- strains can be constructed, evolved, and tested. Gene deletions are introduced into wild-type, haploid S. cerevisiae, for example, by homologous recombination of the gene interrupted by the KanMX cassette, flanked by loxP sites enabling removal and recycling of the resistance marker (Wach et al., PCR-based gene targeting in Saccharomyces cerevisiae, in Yeast Gene Analysis, M.F. Tuite, Editor. 1998, Academic Press: San Diego.).
- an artificial construct with this sequence flanked by fragments of the gene of interest can be created by PCR using primers containing both 45-50 bp target sequence followed by a region homologous to the above cassette.
- This linear DNA is transformed into wild-type S. cerevisiae, and recombinants are selected by geneticin resistance. Colonies can be purified and tested for correct double crossover by PCR.
- a plasmid containing the Cre recombinase and bleomycin resistance will be introduced, promoting recombination between the loxP sites (Gueldener, U., et al., A second set ofloxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast, in Nucleic Acids Res. 2002. p. e23.).
- the resulting strain can be cured of the Cre plasmid by successive culturing on media without any antibiotic present.
- the final strain will have a markerless gene deletion, and thus the same method can be used to introduce multiple deletions in the same strain.
- the engineered strains can be characterized by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. Cultures are grown overnight and used as inoculum for a fresh batch culture for which measurements are taken during exponential growth. The growth rate can be determined by measuring optical density using a spectrophotometer (A600). Concentrations of glucose and other organic acid byproducts in the culture supernatant are determined by HPLC using an HPX-87H column (BioRad), and used to calculate uptake and secretion rates. All experiments are performed with triplicate cultures.
- the disruption strains are initially expected to exhibit suboptimal growth rates until their metabolic networks have adjusted to their missing functionalities.
- the strains are adaptively evolved.
- cellular growth rate becomes the primary selection pressure and the mutant cells are compelled to reallocate their metabolic fluxes in order to enhance their rates of growth.
- This reprogramming of metabolism has been recently demonstrated for several E. coli mutants that had been adaptively evolved on various substrates to reach the growth rates predicted a priori by an in silico model (Fong and Palsson, Nat Genet, 36:1056-1058 (2004)). Should the OptKnock predictions prove successful; the growth improvements brought about by adaptive evolution will be accompanied by enhanced rates of fumarate, malate or acrylate production.
- the OptKnock- generated strains are adaptively evolved in triplicate (running in parallel) due to differences in the evolutionary patterns witnessed previously in E. coli ((Fong and Palsson, Nat Genet,
- the new strains are characterized again by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. These results will be compared to the OptKnock predictions by plotting actual growth and production yields along side the production envelopes in the above figures.
- the most successful OptKnock design/evolution combinations are chosen to pursue further, and are characterized in lab-scale batch and continuous fermentations.
- the growth-coupled biochemical production concept behind the OptKnock approach should also result in the generation of genetically stable overproducers.
- the cultures are maintained in continuous mode for one month to evaluate long-term stability. Periodic samples are taken to ensure that yield and productivity are maintained throughout the experiment.
- the present invention provides a non-naturally occurring microbial organism, that includes a microbial organism having an olefin pathway having at least one exogenous nucleic acid encoding an olefin pathway enzyme expressed in a sufficient amount to produce an olefin.
- the olefin pathway includes a decarboxylase.
- this exogenous nucleic acid is a heterologous nucleic acid.
- the microbial organism having this decarboxylase can be optionally cultured under substantially anaerobic conditions.
- the present disclosure provides non-naturally occurring microbial organisms having an acrylate pathway that includes at least one exogenous nucleic acid encoding an acrylate pathway enzyme expressed in a sufficient amount to produce acrylate.
- This acrylate pathway includes a decarboxylase as described herein below.
- the decarboxylase catalyzes fumarate decarboxylation to provide acrylate.
- Decarboxylases also known as carboxy lyases catalyze the loss of carbon dioxide from an organic compound or a cellular metabolite possessing a carboxylic acid function.
- Decarboxylases are prevalent in nature and can require either pyridoxal phosphate or pyruvate as a co-factor, although many require no bound co-factors. Over 50 decarboxylase enzymes have been reported and characterized by biochemical and/or analytical methods.
- FIGs 20 and 21 A show the decarboxylation of fumaric acid to acrylic acid.
- Numerous decarboxylase enzymes have been characterized and shown to decarboxylate structurally similar substrates to fumarate( Figures 21B-D). These enzymes are applicable for use in the present invention to decarboxylate fumarate and other unsaturated carboxylic acids, as shown in Figure 19.
- One enzyme with closely related function is aconitate decarboxylase ( Figure 21B). This enzyme catalyzes the final step in itaconate biosynthesis in a strain of Candida and also in the filamentous fungus Aspergillus terreus. (Bonnarme et al. /. Bacteriol.
- Aconitate decarboxylase has been purified and characterized from Aspergillus terreus (Dwiarti et al. J. Biosci. Bioeng., 94(1): 29-33 (2002).
- the gene and protein sequence for the cis-aconitic acid decarboxylase (CAD) enzyme are described in EP2017344 and WO 2009/014437. The protein sequence is listed below along with several close homologs described in EP2017344 and WO2009/014437.
- FIG. 21C Another enzyme type with similar function is 4-oxalocrotonate decarboxylase (Figure 21C).
- This enzyme has been isolated from numerous organisms and characterized. Genes encoding this enzyme include dmpH and dmpE in Pseudomonas sp. (strain 600) (Shingler et al. /. Bacteriol. 174:711-724 (1992)), xy/// and xyllll from Pseudomonas putida (Kato et al. Arch. Microbiol. 168:457-463 (1997); Stanley et al. Biochemistry 39:718-726 (2000); Lian et al. /. Am. Chem. Soc.
- each of the decarboxylases listed above represents a suitable enzyme for the transformation shown in Figures 20 and 21 A. If the desired activity or productivity of the enzyme is not observed in the conversion of fumarate to acrylate, or if acrylic acid production inhibits the decarboxylase enzymes, the decarboxylase enzymes can be evolved using known protein engineering methods to achieve the required performance. Importantly, it was shown through the use of chimeric enzymes that the C-terminal region of decarboxylases appears to be responsible for substrate specificity (Barthelmebs et al. (2001) supra). Accordingly, directed evolution experiments to broaden the specificity of decarboxylases in order to gain activity with fumarate can be focused on the C-terminal region of these enzymes.
- Some of the decarboxylases can exhibit higher activity on the cis-isomer of fumarate known as maleate.
- Fumarate can be converted to maleate by maleate cis-trans isomerase encoded by the maiA gene from Alcaligenes faecalis (Hatakeyama, et al., Biochem. Biophys. Research Comm. 239, 74-79 (1997)) or similar genes that can be identified by sequence homology including those from Geobacillus stearothermophilus and Ralstonia pickettii 12D.
- Additional maleate cis-trans isomerase enzymes are encoded by the enzymes whose amino acid sequences are described (SEQ ID NO:l-4) in U.S. Patent 6,133,014, which is incorporated by reference in its entirety.
- Useful GenBank information for some of these isomerases is shown below.
- the exogenous nucleic acid encoding the decarboxylase can come from another organism such as those described above, thus providing a heterologous nucleic acid.
- additional copies of the decarboxylase can be introduced to increase its expression.
- a non-naturally occurring microbial organism will have certain energy requirements for growth and maintenance as outlined below.
- This energetic limitation can be remedied by either supplying a limited amount of an external electron acceptor such as oxygen or nitrate to enable energy generation via respiration, or by at least two strain engineering strategies provided herein below.
- Either strain engineering method ensures that the pathway for production of acrylate via fumarate decarboxylase generates sufficient energy to support cell growth and maintenance under anaerobic or aerobic conditions.
- the non- naturally occurring microbial organism can be grown under aerobic or anaerobic conditions, a substantially anaerobic culture medium is preferred.
- the two exemplary designs described below can be implemented in order to generate the requisite energy for growth and maintenance under anaerobic conditions.
- a non-naturally occurring microbial organism can include an exogenous nucleic acid encoding at least one malic enzyme to supply the requisite energy for growth and maintenance.
- Malic enzymes for this purpose can include, without limitation, malic enzyme (NAD-dependent) and malic enzyme (NADP-dependent).
- NAD-dependent malic enzyme
- NADP-dependent malic enzyme
- one of the native E. coli malic enzymes (Takeo, K., J. Biochem. 66:379-387 (1969)) or a similar non- native enzyme with higher activity can be expressed to enable the conversion of pyruvate and CO 2 to malate.
- malic enzyme By fixing carbon to pyruvate as opposed to PEP, malic enzyme enables the high-energy phosphate bond from PEP to be conserved by pyruvate kinase whereby ATP is generated in the formation of pyruvate or by the phosphotransferase system for glucose transport.
- malic enzyme is typically assumed to operate in the direction of pyruvate formation from malate, overexpression of the NAD-dependent enzyme, encoded by maeA, has been demonstrated to increase succinate production in E.
- the non-naturally occurring microbial organism can include an exogenous nucleic acid providing a gene such as maeA.
- an exogenous nucleic acid providing a gene such as maeA.
- the non-naturally occurring microbial organism can include an exogenous nucleic acid providing a gene such as maeB.
- the relevant malic enzyme gene information is shown below.
- PPCK reversible phosphoenolpyruvate kinase
- the non-naturally occurring microbial organism can also include an exogenous nucleic acid encoding a phosphoenolpyruvate carboxykinase.
- PEP carboxykinase is known to produce oxaloacetate from PEP in rumen bacteria such as Mannheimia succiniciproducens (Hong et al., Nat Biotechnol 22:1275-1281 (2004))
- the role of PEP carboxykinase, encoded by pck, in producing oxaloacetate in E. coli is believed to be minor as compared to PEP carboxylase, possibly due to the higher K m for bicarbonate of PEP carboxykinase (Kim et al., Appl Environ Microbiol 70:1238-1241 (2004)) Nevertheless, activity of the native E.
- coli PEP carboxykinase from PEP towards oxaloacetate has been recently demonstrated in ppc mutants of E. coli K-12 (Kwon et al., /. Microbiol. Biotechnol. 16:1448-1452 (2006)). These strains exhibited no growth defects and had increased succinate production at high NaHCU3 concentrations.
- examples of non-native PEP carboxykinase genes that have been cloned and shown to function in E. coli include those from M. succiniciproducens ( Lee et al., Gene. Biotechnol. Bioprocess Eng.
- the formation of acrylate can also be optimized by modifying the non- naturally occurring microbial organism's metabolic production of fumarate.
- the non-naturally occurring microbial organism can include one or more gene disruptions in addition to the inserted nucleic acid sequences outline above. Gene disruptions can result from, for example, single nucleotide insertion or deletions, stable mutations, and complete gene deletions. Exemplary pathway designs are described below.
- the non-naturally occurring microbial organisms that synthesize acrylate can be produced by introducing expressible nucleic acids encoding one or more of the enzymes or proteins participating in one or more acrylate biosynthetic pathways.
- nucleic acids for some or all of a particular acrylate biosynthetic pathway can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression.
- a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as acrylate.
- the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed acrylate pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more acrylate biosynthetic pathways.
- acrylate biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid.
- exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins.
- exogenous expression of all enzymes or proteins in a pathway for production of acrylate can be included, such as a decarboxylase.
- a non-naturally occurring microbial organism of the invention can have one, two, three, four, up to all nucleic acids encoding the enzymes or proteins constituting an acrylate biosynthetic pathway disclosed herein.
- the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize acrylate biosynthesis or that confer other useful functions onto the host microbial organism.
- One such other functionality can include, for example, augmentation of the synthesis of one or more of the acrylate pathway precursors such as fumarate.
- a host microbial organism is selected such that it produces the precursor of an acrylate pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism.
- fumarate is produced naturally in a host organism such as E. coli.
- a host organism can be engineered to increase production of a precursor, as disclosed herein.
- a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of an acrylate pathway.
- a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize acrylate.
- it can be useful to increase the synthesis or accumulation of an acrylate pathway product to, for example, drive acrylate pathway reactions toward acrylate production.
- Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described acrylate pathway enzymes or proteins.
- Over expression the enzyme or enzymes and/or protein or proteins of the acrylate pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes.
- naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms of the invention, for example, producing acrylate, through overexpression of one, two, three, four, five, that is, up to all nucleic acids encoding acrylate biosynthetic pathway enzymes or proteins.
- a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the acrylate biosynthetic pathway.
- exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user.
- endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene' s promoter when linked to an inducible promoter or other regulatory element.
- an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time.
- an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.
- any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention.
- the nucleic acids can be introduced so as to confer, for example, an acrylate biosynthetic pathway onto the microbial organism.
- encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer acrylate biosynthetic capability.
- a non-naturally occurring microbial organism having an acrylate biosynthetic pathway can comprise at least one exogenous nucleic acids encoding desired enzymes or proteins, such as a decarboxylase, and the like.
- the non-naturally occurring microbial organisms and methods of the invention also can be utilized in various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes.
- one alternative to produce acrylate other than use of the acrylate producers is through addition of another microbial organism capable of converting an acrylate pathway intermediate to acrylate.
- One such procedure includes, for example, the fermentation of a microbial organism that produces an acrylate pathway intermediate.
- the acrylate pathway intermediate can then be used as a substrate for a second microbial organism that converts the acrylate pathway intermediate to acrylate.
- the acrylate pathway intermediate can be added directly to another culture of the second organism or the original culture of the acrylate pathway intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps.
- the non- naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, acrylate.
- biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product.
- the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized.
- the biosynthesis of acrylate can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product.
- acrylate also can be biosynthetically produced from microbial organisms through co-culture or co- fermentation using two organisms in the same vessel, where the first microbial organism produces a fumarate intermediate and the second microbial organism converts the intermediate to acrylate.
- Microorganisms capable of directly producing acrylate are constructed by introducing genes encoding decarboxylase enzymes into the strains engineered as described above for maximal fumarate production.
- the following example describes the creation of a microbial organism that can produce acrylic acid from renewable feedstocks such as glucose or sucrose.
- nucleic acids encoding the decarboxylase enzymes are cloned and expressed in E. coli capable of overproducing fumarate using well known molecular biology techniques and recombinant and detection methods well known in the art. Such methods are described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).
- An acrylate producing strain is constructed, by cloning the individual phenylacrylic acid decarboxylase genes padl (AB368798), pdc (U63827), pofK (AB330293), padC (AF017117), pad (AJ276891), and pad (AJ278683) into pZA33 or pZE13 vectors (Expressys, Ruelzheim, Germany) under the IPTG-titratable PAl/lacO promoter.
- the plasmids are transformed into the fumarate overproducing E. coli strain using standard methods such as electroporation.
- the resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). Expression of the decarboxylase genes are corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunob lotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce acrylic acid is confirmed using HPLC, gas chromatography- mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).
- GCMS gas chromatography- mass spectrometry
- LCMS liquid chromatography-mass spectrometry
- Microbial strains engineered to have a functional acrylic acid synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether exogenous genes are expressed at a rate limiting level. Flux analysis using 13 C-labeled glucose is performed to assess bottlenecks in the system. Expression is increased for enzymes produced at low levels and that limit the flux through the pathway by, for example, introduction of additional gene copy numbers or changes to the promoter and ribosome binding sites.
- metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway, as described above. Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of acrylic acid. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat Genet. 36:1056-1058 (2004)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the acrylic acid producer to further increase production.
- the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained in the optimum range by addition of acids such as H 2 SO 4 or bases such as NaOH or Na 2 CU 3 .
- the growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time.
- Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia MD), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).
- E. coli and other microorganisms are known to possess fatty acid and organic acid degradation pathways that could lead to acrylate degradation. While fermentative production of acrylic acid under anaerobic conditions should not be accompanied by degradation, should product degradation be observed, the pathways responsible for product degradation will be deleted.
- Sources of encoding nucleic acids for an acrylate pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction.
- Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human.
- Exemplary species for such sources include, for example, Escherichia coli, Candida albicans, Candida boidinii, Aspergillus terreus,
- Pseudomonas sp. CF600 Pseudomonas putida, Ralstonia eutropha JMPl 34, Saccharomyces cerevisae, Lactobacillus plantarum, Klebsiella oxytoca, Bacillus subtilis, Bacillus pumilus, Pedicoccus pentosaceus, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes.
- coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.
- acrylate biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms can differ.
- teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize acrylate.
- Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes.
- Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobio spirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.
- Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger and Pichiapastoris.
- E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering.
- Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae.
- Methods for constructing and testing the expression levels of a non-naturally occurring acrylate- producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New
- Exogenous nucleic acid sequences involved in a pathway for production of acrylate can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation.
- some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired.
- genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells.
- a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells.
- An expression vector or vectors can be constructed to include one or more acrylate biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism.
- Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome.
- the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media.
- Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art.
- both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors.
- the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art.
- Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
- nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA
- PCR polymerase chain reaction
- immunoblotting for expression of gene products
- a method for producing acrylate includes culturing a non-naturally occurring microbial organism having an acrylate pathway.
- the pathway includes at least one exogenous nucleic acid encoding an acrylate pathway enzyme expressed in a sufficient amount to produce acrylate under conditions and for a sufficient period of time to produce acrylate.
- the non-naturally occurring microbial organism is in a substantially anaerobic culture medium as described above.
- the acrylate pathway includes a decarboxylase gene introduced into an organism that is engineered to produce high levels of fumaric acid under anaerobic conditions from carbon substrates such as glucose or sucrose.
- a decarboxylase gene introduced into an organism that is engineered to produce high levels of fumaric acid under anaerobic conditions from carbon substrates such as glucose or sucrose.
- decarboxylation of fumaric acid occurs intracellularly and acrylate is produced directly and is secreted from the cell and recovered through standard methods employed for acid separation and purification.
- An alternative to this production mode is to engineer a decarboxylase enzyme so that it is secreted from the fumarate-producing cell, in which case acrylate production occurs in the same vessel as fumarate production.
- This approach is particularly effective if decarboxylase enzyme production and secretion are subject to inducible programming (e.g., using a temperature sensitive promoter) such that the enzyme is produced and secreted into the broth following completion of fumarate production.
- the present invention provides a method for producing acrylate, that includes culturing a first non-naturally occurring microbial organism having one or more gene disruptions.
- the one or more gene disruptions can occur in one or more genes encoding one or more enzymes obligatory to coupling fumarate production to growth of the microorganism when the disruptions reduce an activity of the enzymes such that the disruptions confer stable growth-coupled production of fumarate.
- a decarboxylase to the cultured first non-naturally occurring microbial organism, said decarboxylase catalyzing the decarboxylation of fumarate.
- the decarboxylase is expressed in a second non-naturally occurring microbial organism.
- the first and second non-naturally occurring microbial organisms can be co-cultured.
- the decarboxylase can also be secreted by a second non-naturally occurring microbial organism which still allows for the first and second microbial organisms to be co-cultured.
- Suitable purification and/or assays to test for the production of acrylate can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid
- the acrylate can be separated from other components in the culture using a variety of methods well known in the art.
- separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.
- any of the non- naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention.
- the acrylate producers can be cultured for the biosynthetic production of acrylate.
- the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United States Patent application serial No. 11/891,602, filed August 10, 2007. Fermentations can be performed in a batch, fed- batch or continuous manner, as disclosed herein.
- the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH.
- the growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.
- the growth medium can be, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism.
- Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
- Other sources of carbohydrate include, for example, renewable feedstocks and biomass.
- Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
- carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
- the acrylate microbial organisms of the invention also can be modified for growth on syngas as its source of carbon.
- one or more proteins or enzymes are expressed in the acrylate producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.
- Synthesis gas also known as syngas or producer gas
- syngas is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues.
- Syngas is a mixture primarily of H 2 and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H 2 and CO, syngas can also include CO 2 and other gases in smaller quantities.
- synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, CO 2 .
- the Wood-Ljungdahl pathway catalyzes the conversion of CO and H 2 to acetyl-CoA and other products such as acetate.
- Organisms capable of utilizing CO and syngas also generally have the capability of utilizing CO 2 and CO 2 /H 2 mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway.
- H 2 -dependent conversion of CO 2 to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved.
- non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize CO 2 and H 2 mixtures as well for the production of acetyl-CoA and other desired products.
- the Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch.
- the methyl branch converts syngas to methyl-tetrahydrofolate (methyl- THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA.
- the reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase.
- the reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: cobalamide corrinoid/iron-sulfur protein, methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase, acetyl-CoA synthase disulfide reductase and hydro genase.
- cobalamide corrinoid/iron-sulfur protein methyltransferase
- carbon monoxide dehydrogenase carbon monoxide dehydrogenase
- acetyl-CoA synthase acetyl-CoA synthase disulfide reductase
- hydro genase hydro genase
- a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds of the invention when grown on a carbon source such as a carbohydrate.
- Such compounds include, for example, acrylate and any of the intermediate metabolites in the acrylate pathway. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the acrylate biosynthetic pathways.
- the invention provides a non-naturally occurring microbial organism that produces and/or secretes acrylate when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the acrylate pathway when grown on a carbohydrate or other carbon source.
- the acrylate producing microbial organisms of the invention can initiate synthesis from an intermediate, for example, fumarate.
- the non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding an acrylate pathway enzyme or protein in sufficient amounts to produce acrylate. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce acrylate. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of acrylate resulting in intracellular concentrations between about 0.1-200 mM or more.
- the intracellular concentration of acrylate is between about 3-150 mM, particularly between about 5-125 mM and more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non-naturally occurring microbial organisms of the invention.
- the fumarate, malate, or acrylate can be separated from other components in the culture using a variety of methods well known in the art.
- separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.
- any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention.
- the fumarate, malate, or acrylate producers can be cultured for the biosynthetic production of fumarate, malate, or acrylate.
- the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United States Patent application serial No. 11/891,602, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein.
- the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH.
- the growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.
- the growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism.
- Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
- Other sources of carbohydrate include, for example, renewable feedstocks and biomass.
- Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks.
- Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
- carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
- the fumarate, malate, or acrylate microbial organisms of the invention also can be modified for growth on syngas as its source of carbon.
- one or more proteins or enzymes are expressed in the fumarate, malate, or acrylate producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.
- Synthesis gas also known as syngas or producer gas
- syngas is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues.
- Syngas is a mixture primarily of H 2 and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H 2 and CO, syngas can also include CO 2 and other gases in smaller quantities.
- synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, CO 2 .
- the Wood-Ljungdahl pathway catalyzes the conversion of CO and H 2 to acetyl-CoA and other products such as acetate.
- Organisms capable of utilizing CO and syngas also generally have the capability of utilizing CO 2 and CO 2 /H 2 mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. H 2 -dependent conversion of CO 2 to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of CO 2 and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation:
- non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize CO 2 and H 2 mixtures as well for the production of acetyl-CoA and other desired products.
- the Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch.
- the methyl branch converts syngas to methyl-tetrahydrofolate (methyl- THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA.
- the reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase.
- the reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: methyltetrahydrofolate:corrinoid protein methyltransferase (for example, AcsE), corrinoid iron- sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC).
- methyltetrahydrofolate corrinoid protein methyltransferase
- corrinoid iron- sulfur protein for example, corrinoid iron- sulfur protein
- nickel-protein assembly protein for example, AcsF
- ferredoxin ferredoxin
- acetyl-CoA synthase carbon monoxide dehydrogenase
- nickel-protein assembly protein for example, CooC
- a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds of the invention when grown on a carbon source such as a carbohydrate.
- Such compounds include, for example, fumarate, malate, or acrylate and any of the intermediate metabolites in the fumarate, malate, or acrylate pathway. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the fumarate, malate, or acrylate biosynthetic pathways.
- the invention provides a non- naturally occurring microbial organism that produces and/or secretes fumarate, malate, or acrylate when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the fumarate, malate, or acrylate pathway when grown on a carbohydrate or other carbon source.
- the fumarate, malate, or acrylate producing microbial organisms of the invention can initiate synthesis from any of the aforementioned intermediates.
- the non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a fumarate, malate, or acrylate pathway enzyme or protein in sufficient amounts to produce fumarate, malate, or acrylate. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce fumarate, malate, or acrylate. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of fumarate, malate, or acrylate resulting in intracellular concentrations between about 0.1-200 mM or more.
- the intracellular concentration of fumarate, malate, or acrylate is between about 3-200 mM, particularly between about 10-175 mM and more particularly between about 50-150 mM, including about 50 mM, 75 mM, 100 mM, 125 mM, or more. Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non-naturally occurring microbial organisms of the invention.
- culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions.
- Exemplary anaerobic conditions have been described previously and are well known in the art.
- Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. patent application serial No. 11/891,602, filed August 10, 2007. Any of these conditions can be employed with the non-naturally occurring microbial organisms as well as other anaerobic conditions well known in the art.
- the fumarate, malate, or acrylate producers can synthesize fumarate, malate, or acrylate at intracellular concentrations of 5-10 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, fumarate, malate, or acrylate producing microbial organisms can produce fumarate, malate, or acrylate intracellularly and/or secrete the product into the culture medium.
- the culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.
- one exemplary growth condition for achieving biosynthesis of fumarate, malate, or acrylate includes anaerobic culture or fermentation conditions.
- the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions.
- anaerobic conditions refers to an environment devoid of oxygen.
- substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation.
- Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N 2 /CO 2 mixture or other suitable non-oxygen gas or gases.
- the culture conditions described herein can be scaled up and grown continuously for manufacturing of fumarate, malate, or acrylate.
- Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of fumarate, malate, or acrylate.
- the continuous and/or near-continuous production of fumarate, malate, or acrylate can include culturing a non-naturally occurring fumarate, malate, or acrylate producing organism of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase.
- Continuous culture under such conditions can include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms of the invention can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.
- Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of fumarate, malate, or acrylate can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.
- the fumarate, malate, or acrylate producers of the invention for continuous production of substantial quantities of fumarate, malate, or acrylate
- the fumarate, malate, or acrylate producers also can be, for example, simultaneously subjected to chemical synthesis procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical conversion to convert the product to other compounds, if desired.
- Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (e.g., >10 4 ). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened.
- Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example, selectivity/specificity - for conversion of non-natural substrates; temperature stability - for robust high temperature processing; pH stability - for bioprocessing under lower or higher pH conditions; substrate or product tolerance - so that high product titers can be achieved; binding (K m ) - broadens substrate binding to include non-natural substrates; inhibition (K 1 ) - to remove inhibition by products, substrates, or key intermediates; activity (kcat) - increases enzymatic reaction rates to achieve desired flux; expression levels - increases protein yields and overall pathway flux; oxygen stability - for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity - for operation of an aerobic enzyme in the absence of oxygen.
- EpPCR (Pritchard et al., J Theor. Biol. 234:497-509 (2005).) introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions by the addition of Mn 2+ ions, by biasing dNTP concentrations, or by other conditional variations.
- the five step cloning process to confine the mutagenesis to the target gene of interest involves: 1) error-prone PCR amplification of the gene of interest; 2) restriction enzyme digestion; 3) gel purification of the desired DNA fragment; 4) ligation into a vector; 5) transformation of the gene variants into a suitable host and screening of the library for improved performance.
- This method can generate multiple mutations in a single gene simultaneously, which can be useful.
- a high number of mutants can be generated by EpPCR, so a high-throughput screening assay or a selection method (especially using robotics) is useful to identify those with desirable characteristics.
- Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., Nucl. Acids Res 32:el45 (2004); and Fujii et al., Nat. Protoc. 1 :2493-2497 (2006).) has many of the same elements as epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats.
- Adjusting the Mn + concentration can vary the mutation rate somewhat.
- This technique uses a simple error-prone, single-step method to create a full copy of the plasmid with 3 - 4 mutations/kbp. No restriction enzyme digestion or specific primers are required. Additionally, this method is typically available as a kit. DNA or Family Shuffling (Stemmer, W. P., Proc Natl Acad Sci U.S.A. 91 :10747-10751 (1994);and Stemmer, W.
- Staggered Extension (StEP) (Zhao et al., Nat. Bio techno 1 16:258-261 (1998).) entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec). Growing fragments anneal to different templates and extend further, which is repeated until full-length sequences are made. Template switching means most resulting fragments have multiple parents. Combinations of low-fidelity polymerases (Taq and Mutazyme) reduce error-prone biases because of opposite mutational spectra.
- Random Priming Recombination random sequence primers are used to generate many short DNA fragments complementary to different segments of the template.
- Base misincorporation and mispriming via epPCR give point mutations. Short DNA fragments prime one another based on homology and are recombined and reassembled into full-length by repeated thermocycling. Removal of templates prior to this step assures low parental recombinants. This method, like most others, can be performed over multiple iterations to evolve distinct properties. This technology avoids sequence bias, is independent of gene length, and requires very little parent DNA for the application.
- Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al., Nat. Biotechnol 19:354-359 (2001).) employs Dnase I fragmentation and size fractionation of ssDNA. Homologous fragments are hybridized in the absence of polymerase to a complementary ssDNA scaffold. Any overlapping unhybridized fragment ends are trimmed down by an exonuclease. Gaps between fragments are filled in, and then ligated to give a pool of full-length diverse strands hybridized to the scaffold (that contains U to preclude amplification). The scaffold then is destroyed and is replaced by a new strand complementary to the diverse strand by PCR amplification.
- the method involves one strand (scaffold) that is from only one parent while the priming fragments derive from other genes; the parent scaffold is selected against. Thus, no reannealing with parental fragments occurs. Overlapping fragments are trimmed with an exonuclease. Otherwise, this is conceptually similar to DNA shuffling and StEP. Therefore, there should be no siblings, few inactives, and no unshuffled parentals. This technique has advantages in that few or no parental genes are created and many more crossovers can result relative to standard DNA shuffling.
- Recombined Extension on Truncated templates entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates.
- No DNA endonucleases are used. Unidirectional ssDNA is made by by DNA polymerase with random primers or serial deletion with exonuclease. Unidirectional ssDNA are only templates and not primers. Random priming and exonucleases don't introduce sequence bias as true of enzymatic cleavage of DNA shuffling/RACHITT. RETT can be easier to optimize than StEP because it uses normal PCR conditions instead of very short extensions. Recombination occurs as a component of the PCR steps— no direct shuffling. This method can also be more random than StEP due to the absence of pauses.
- DOGS Oligonucleotide Gene Shuffling
- Incremental Truncation for the Creation of Hybrid Enzymes creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest. (Ostermeier et al., Proc Natl Acad Sci U SA 96:3562-3567 (1999); Ostermeier et Ia., Nat.Biotechnol 17:1205-1209 (1999).) Truncations are introduced in opposite direction on pieces of 2 different genes. These are ligated together and the fusions are cloned.
- THIO-ITCHY Thio-Incremental Truncation for the Creation of Hybrid Enzymes
- ITCHY Thio-Incremental Truncation for the Creation of Hybrid Enzymes
- SCRATCHY - ITCHY combined with DNA shuffling is a combination of DNA shuffling and ITCHY; therefore, allowing multiple crossovers.
- SCRATCHY combines the best features of ITCHY and DNA shuffling. Computational predictions can be used in optimization. SCRATCHY is more effective than DNA shuffling when sequence identity is below 80%.
- RNDM Random Drift Mutagenesis
- Sequence Saturation Mutagenesis is a random mutagenesis method that: 1) generates pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage; this pool is used as a template to 2) extend in the presence of "universal" bases such as inosine; 3) replication of a inosine-containing complement gives random base incorporation and, consequently, mutagenesis.
- “universal" bases such as inosine
- 3) replication of a inosine-containing complement gives random base incorporation and, consequently, mutagenesis.
- overlapping oligonucleotides are designed to encode "all genetic diversity in targets" and allow a very high diversity for the shuffled progeny.
- Nat.Biotechnol 20:1251-1255 (2002). In this technique, one can design the fragments to be shuffled. This aids in increaseing the resulting diversity of the progeny.
- sequence/codon biases to make more distantly related sequences recombine at rates approaching more closely related sequences and it doesn't require possessing the template genes physically.
- Nucleotide Exchange and Excision Technology NexT exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation.
- the gene is reassembled using internal PCR primer extension with proofreading polymerase.
- the sizes for shuffling are directly controllable using varying dUPT::dTTP ratios. This is an end point reaction using simple methods for uracil incorporation and cleavage.
- One can use other nucleotide analogs such as 8-oxo-guanine with this method. Additionally, the technique works well with very short fragments (86 bp) and has a low error rate. Chemical cleavage of DNA means very few unshuffled clones.
- SHIPREC Sequence Homology-Independent Protein Recombination
- GSSM Gene Site Saturation Mutagenesis
- Primers carry the mutation of interest and anneal to the same sequence on opposite strands of DNA; mutation in the middle of the primer and -20 nucleotides of correct sequence flanking on each side.
- Dpnl is used to digest dam-methylated DNA to eliminate the wild-type template.
- This technique explores all possible amino acid substitutions at a given locus (i.e., one codon).
- the technique facilitates the generation of all possible replacements at one site with no nonsense codons and equal or near-equal representation of most possible alleles. It does not require prior knowledge of structure, mechanism, or domains of the target enzyme. If followed by shuffling or Gene Reassembly, this technology creates a diverse library of recombinants containing all possible combinations of single- site up-mutations. The utility of this technology combination has been demonstrated for the successful evolution of over 50 different enzymes, and also for more than one property in a given enzyme.
- Combinatorial Cassette Mutagenesis involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations.
- Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al., Science 241 :53-57 (1988).
- Simultaneous substitutions at 2 or 3 sites are possible using this technique.
- the method tests a large multiplicity of possible sequence changes at a limited range of sites. It has been used to explore the information content of lambda repressor DNA-binding domain.
- CMCM Combinatorial Multiple Cassette Mutagenesis
- conditional ts mutator plasmids allow increases of 20- to 4000- X in random and natural mutation frequency during selection and to block accumulation of deleterious mutations when selection is not required.
- This technology is based on a plasmid-derived mutD5 gene, which encodes a mutant subunit of DNA polymerase III. This subunit binds to endogenous DNA polymerase III and compromises the proofreading ability of polymerase III in any of the strain that harbors the plasmid. A broad-spectrum of base substitutions and frameshift mutations occur.
- the mutator plasmid should be removed once the desired phenotype is achieved; this is accomplished through a temperature sensitive origin of replication, which allows plasmid curing at 41 0 C. It should be noted that mutator strains have been explored for quite some time (e.g., see Winter and coworkers, /. MoI. Biol. 260:359-3680 (1996). In this technique very high spontaneous mutation rates are observed. The conditional property minimizes non-desired background mutations. This technology could be combined with adaptive evolution to enhance mutagenesis rates and more rapidly achieve desired phenotypes.
- LTM Look- Through Mutagenesis
- Gene Reassembly is a DNA shuffling method that can be applied to multiple genes at one time or to creating a large library of chimeras (multiple mutations) of a single gene, (on the worldwide web at verenium.com/Pages/Technology/EnzymeTech/TechEnzyTGR.html)
- this technology is used in combination with ultra-high- throughput screening to query the represented sequence space for desired improvements.
- This technique allows multiple gene recombination independent of homology.
- the exact number and position of cross-over events can be predetermined using fragments designed via bioinformatic analysis. This technology leads to a very high level of diversity with virtually no parental gene reformation and a low level of inactive genes.
- GSSM GSSM
- a large range of mutations can be tested for improved activity.
- the method allows "blending" and “fine tuning” of DNA shuffling, e.g. codon usage can be optimized.
- Silico Protein Design Automation PDA is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics. (Hayes et al.,. Proc Natl Acad Sci U. S.A. 99:15926-15931 (2002).) This technology allows in silico structure-based entropy predictions in order to search for structural tolerance toward protein amino acid variations.
- ISM Iterative Saturation Mutagenesis involves 1) Use knowledge of structure/function to choose a likely site for enzyme improvement. 2) Saturation mutagenesis at chosen site using Stratagene QuikChange (or other suitable means). 3) Screen/select for desired properties. 4) With improved clone(s), start over at another site and continue repeating. (Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew.Chem.Int.Ed Engl. 45:7745-7751 (2006).) This is a proven methodology assures all possible replacements at a given position are made for screening/selection.
- Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques.
- Modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene disruptions that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of fumarate, malate, or acrylate.
- SimPheny® Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®.
- This computational method and system is described in, for example, U.S. publication 2003/0233218, filed June 14, 2002, and in International Patent Application No. PCT/US03/18838, filed June 13, 2003.
- SimPheny® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system.
- constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions.
- the space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.
- metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock.
- SimPheny® and OptKnock For illustration of the invention, some methods are described herein with reference to the OptKnock computation framework for modeling and simulation.
- OptKnock computation framework for modeling and simulation.
- Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art.
- the methods described above will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes.
- the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set.
- One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene.
- These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur.
- an optimization method termed integer cuts. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions.
- the integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®.
- the methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny®.
- the set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.
- This Example describes the construction in silico designed strains for the increased production of fumarate in E. CoIi.
- Escherichia coli K-12 MG1655 serves as the wild-type strain into which the deletions are introduced.
- the strains are constructed by incorporating in-frame deletions using homologous recombination via the ⁇ Red recombinase system of Datsenko and Wanner. (Datsenko and Wanner, Pro c Natl Acad S ci U.S.A., 97(12):6640-5 (2000).)
- the approach involves replacing a chromosomal sequence (i.e., the gene targeted for removal) with a selectable antibiotic resistance gene, which itself is later removed. Knockouts are integrated one by one into the recipient strain. No antibiotic resistance markers will remain after each deletion allowing accumulation of multiple mutations in each target strain.
- the deletion technology completely removes the gene targeted for removal so as to substantially reduce the possibility of the constructed mutants reverting back to the wild-type.
- one exemplary growth condition for achieving biosynthesis of fumarate/malate includes anaerobic culture or fermentation conditions.
- the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions.
- anaerobic conditions refers to an environment devoid of oxygen.
- substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation.
- Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1 % oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N 2 /CO 2 mixture or other suitable non-oxygen gas or gases.
- the engineered strains are characterized by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. Cultures are grown overnight and used as inoculum for a fresh batch culture for which measurements are taken during exponential growth. The growth rate is determined by measuring optical density using a spectrophotometer (A600). Concentrations of glucose, fumarate, malate, and other organic acid byproducts in the culture supernatant are determined by HPLC using an HPX-87H column (BioRad), and are used to calculate uptake and secretion rates. All experiments are performed with triplicate cultures.
- the knockout strains can exhibit suboptimal growth rates until their metabolic networks have adjusted to their missing functionalities.
- the strains are adaptively evolved. By subjecting the strains to adaptive evolution, cellular growth rate becomes the primary selection pressure and the mutant cells are compelled to reallocate their metabolic fluxes in order to enhance their rates of growth. This reprogramming of metabolism has been recently demonstrated for several E. coli mutants that had been adaptively evolved on various substrates to reach the growth rates predicted a priori by an in silico model. (Fong and Palsson, Nat Genet, 36(10): 1056-8 (2004).)
- the OptKnock-generated strains are adaptively evolved in triplicate (running in parallel) due to differences in the evolutionary patterns witnessed previously in E. coli (Fong and Palsson, Nat Genet, 36(10):1056-8 (2004); Fong et al., J Bacteriol, 185(21):6400-6408 (2003); Ibarra et al., Nature 420:186-189 (2002)) that could potentially result in one strain having superior production qualities over the others. Evolutions are run for a period of 2-6 weeks, depending upon the rate of growth improvement attained. In general, evolutions are stopped once a stable phenotype is obtained.
- the evolutions are performed in triplicate (i.e., 18 evolutions total) due to differences in the evolutionary patterns witnessed previously Donnelly et al., Appl Biochem Biotechnol 70-72: 187-98 (1998); Vemuri et al., Appl Environ Microbiol 68:1715-27 (2002), that could potentially result in one strain having superior production qualities over the others.
- the adaptive evolution step can take up to about two months or more.
- the adaptive evolution step also can be less than two months depending on the strain design, for example.
- EvolugatorTM evolution machine which results in significant time and effort savings over non- automated evolution techniques.
- Cells are maintained in prolonged exponential growth by the serial passage of batch cultures into fresh medium before the stationary phase is attained.
- the Evolugator can perform serial transfer at high rates using large culture volumes, thus approaching the efficiency of a chemostat for evolution of cell fitness 25 .
- the machine operates by moving from one "reactor” to the next in subdivided regions of a spool of tubing, thus eliminating any selection for wall-growth. Culture samples are taken, frozen with liquid nitrogen, and the optical culture density recorded each day throughout the course of the evolutions.
- the Evolugator is used for each strain until a stable growth rate is achieved. Growth rate improvements of nearly 50% have been observed in two weeks using this device.
- the above-described strains are adaptively evolved in triplicate (running in parallel). At ten day intervals, culture samples are taken from the Evolugator, purified on agar plates, and cultured in triplicate as discussed above to assess strain physiology.
- the new strains are again characterized by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. These results are compared to the OptKnock predictions by plotting actual growth and production yields along side the production envelopes.
- the most successful OptKnock design/evolution combinations are chosen to pursue further, and is characterized in lab-scale batch and continuous fermentations.
- the growth-coupled biochemical production concept behind the OptKnock approach should also result in the generation of genetically stable overproducers.
- the cultures can be maintained in continuous mode for one month to evaluate long-term stability. Periodic samples will be taken to ensure that yield and productivity are maintained throughout the experiment.
- This Example describes the construction in silico designed strains for the increased production of fumarate in S. cerevisiae.
- This linear DNA will be transformed into wild-type S. cerevisiae, and recombinants will be selected by geneticin resistance. Colonies will be purified and tested for correct double crossover by PCR. To remove the KanMX marker, a plasmid containing the Cre recombinase and bleomycin resistance will be introduced, promoting recombination between the loxP sites (Gueldener et al., Nucleic Acids Res. 30:e23 (2002)). Finally, the resulting strain can be cured of the Cre plasmid by successive culturing on media without any antibiotic present. The final strain will have a markerless gene deletion, and thus the same method can be used to introduce multiple deletions in the same strain.
- one exemplary growth condition for achieving biosynthesis of fumarate includes anaerobic culture or fermentation conditions.
- the non-naturally occurring eukaryotic organism of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions.
- anaerobic conditions refer to an environment devoid of oxygen.
- substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation.
- substantially anaerobic conditions include microaerobic conditions.
- Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1 % oxygen.
- the percent of oxygen can be maintained by, for example, sparging the culture with an N 2 /CO 2 mixture or other suitable non-oxygen gas or gases.
- the engineered strains are characterized by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. Cultures are grown overnight and used as inoculum for a fresh batch culture for which measurements are taken during exponential growth. The growth rate is determined by measuring optical density using a spectrophotometer (A600). Concentrations of glucose, fumarate, malate, and other organic acid byproducts in the culture supernatant are determined by HPLC using an HPX-87H column (BioRad), and are used to calculate uptake and secretion rates. All experiments are performed with triplicate cultures.
- the knockout strains can exhibit suboptimal growth rates until their metabolic networks have adjusted to their missing functionalities.
- the strains are adaptively evolved. By subjecting the strains to adaptive evolution, cellular growth rate becomes the primary selection pressure and the mutant cells are compelled to reallocate their metabolic fluxes in order to enhance their rates of growth.
- This reprogramming of metabolism has been recently demonstrated for several E. coli mutants that had been adaptively evolved on various substrates to reach the growth rates predicted a priori by an in silico model. (Fong and Palsson, Nat Genet, 36:1056-1058 (2004).) These teachings can be applied to S. cerevisiae.
- the evolutions are performed in triplicate due to differences in the evolutionary patterns witnessed previously Donnelly et al., Appl Biochem Biotechnol 70-72: 187-98 (1998); Vemuri et al., Appl Environ Microbiol 68:1715-27 (2002), that could potentially result in one strain having superior production qualities over the others.
- the adaptive evolution step can take up to about two months or more.
- the adaptive evolution step also can be less than two months depending on the strain design, for example.
- Evolugate, LLC Gainesville, FL
- the procedure employs the EvolugatorTM evolution machine which results in significant time and effort savings over non- automated evolution techniques.
- Cells are maintained in prolonged exponential growth by the serial passage of batch cultures into fresh medium before the stationary phase is attained.
- automating optical density measurement and liquid handling the Evolugator can perform serial transfer at high rates using large culture volumes, thus approaching the efficiency of a chemostat for evolution of cell fitness.
- the machine operates by moving from one "reactor” to the next in subdivided regions of a spool of tubing, thus eliminating any selection for wall-growth.
- Culture samples are taken, frozen with liquid nitrogen, and the optical culture density recorded each day throughout the course of the evolutions. The Evolugator is used for each strain until a stable growth rate is achieved. Growth rate improvements of nearly 50% have been observed in two weeks using this device.
- the above-described strains are adaptively evolved in triplicate (running in parallel). At ten day intervals, culture samples are taken from the Evolugator, purified on agar plates, and cultured in triplicate as discussed above to assess strain physiology. Following the adaptive evolution process, the new strains are again characterized by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. These results are compared to the OptKnock predictions by plotting actual growth and production yields along side the production envelopes.
- OptKnock design/evolution combinations are chosen to pursue further, and is characterized in lab-scale batch and continuous fermentations.
- the growth-coupled biochemical production concept behind the OptKnock approach should also result in the generation of genetically stable overproducers.
- the cultures can be maintained in continuous mode for one month to evaluate long-term stability. Periodic samples will be taken to ensure that yield and productivity are maintained throughout the experiment.
- This Example describes the generation of a microbial organism capable of producing acrylate using a decarboxylase metabolic pathway.
- Escherichia coli is used as a target organism to engineer a decarboxylase pathway ( Figure 1), and testing growth and acrylate production from glucose.
- E. coli provides a good model for developing a non- naturally occurring microorganism capable of producing acrylate, from glucose since it is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, effectively under anaerobic conditions from glucose.
- nucleic acids encoding proteins and enzymes required for the acrylate production pathway via fumarate decarboxylation as described above are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel supra, 1999; Roberts et al., supra, 1989).
- the padl gene (AB368798) encoding a decarboxylase under anaerobic conditions, are cloned into the pZE13 vector under the PAl/lacO promoter.
- the of plasmid is transformed into E. coli strain MG1655 to express the enzyme aconitate decarboxylase required for decarboxylation of fumarate to acrylate.
- the engineered production organism containing a decarboxylase enzyme is grown in a 1OL bioreactor sparged with an N 2 /CO 2 mixture, using 5 L broth containing 5 g/L potassium phosphate, 2.5 g/L ammonium chloride, 0.5 g/L magnesium sulfate, and 30 g/L corn steep liquor, and an initial glucose concentration of 20 g/L.
- additional 70% glucose is fed into the bioreactor at a rate approximately balancing glucose consumption.
- the temperature of the bioreactor is maintained at 30 degrees C. Growth continues for approximately 24 hours, until acrylate reaches a concentration of between 10-200 g/L, with the cell density being between 5 and 50 g/L.
- the fermenter contents are passed through a cell separation unit (e.g., centrifuge) to remove cells and cell debris, and the fermentation broth and acrylate is separated from the broth and purified by standard methods for organic acid recovery.
- a cell separation unit e.g., centrifuge
- Escherichia coli K-12 MG1655 is used as one reference wild-type strain into which the deletions are introduced.
- the knockouts are integrated, for example, one-by-one into the recipient strain allowing the accumulation of several deletions.
- the deletion methodology completely removes the gene targeted for removal so as to avoid the possibility of the constructed mutants reverting back to their wild-type.
- the strains are constructed by incorporating in- frame deletions using homologous recombination by well known methods such as the ⁇ Red recombinase system (Datsenko and Wanner, Proc Natl Acad Sci U.S.A., 97:6640-6645 (2000)).
- the approach involves replacing a chromosomal sequence (i.e., the gene targeted for removal) with a selectable antibiotic resistance gene, which itself is later removed.
- the knockouts are integrated sequentially into the recipient strain. Antibiotic resistance markers are removed after each deletion, thus allowing accumulation of multiple mutations in each target strain.
- An organism engineered for high level fumarate production is grown in a 1OL bioreactor sparged with an N 2 /CO 2 mixture, using 5 L broth containing 5 g/L potassium phosphate, 2.5 g/L ammonium chloride, 0.5 g/L magnesium sulfate, and 30 g/L corn steep liquor, and an initial glucose concentration of 20 g/L.
- additional 70% glucose is fed into the bioreactor at a rate approximately balancing glucose consumption.
- the temperature of the bioreactor is maintained at 30 degrees C. Growth continues for approximately 24 hours, until fumarate reaches a concentration of between 10-200 g/L, with the cell density being between 5 and 50 g/L.
- a decarboxylase enzyme is added either directly to the fermenter or after initial removal of cells and cell debris. After agitating for the required length of time required for complete conversion of fumarate to acrylate, the acrylate is recovered as described above.
- Table 1 The list of all strains identified by OptKnock that are most likely to provide increased fumarate yields in E. CoIi.
- ACKr ADHEr GLCpts GLUDy LDH_D ME2 SUCD4 27.
- ADHEr ALAR CBMK2 GLUDy LDH_D PROIz SUCD4 93.
- ADHEr ALAR GLU5K LDH_D ME2 PROIz SUCD4 98.
- ADHEr ALAR GLUDy LDH_D PROIz SUCD4 100.
- ADHEr ASNS2 CBMK2 FRD2 GLU5K LDH_D ME2 109.
- ADHEr CBMK2 FRD2 G5SD LDH_D ME2 149.
- ADHEr CBMK2 FRD2 GLUDy LDH_D ME2 THD2 154.
- ADHEr CBMK2 FRD2 GLUDy LDH_D ME2 THD2 155.
- ADHEr FRD2 G5SD GLCpts LDH_D ME2 180.
- ADHEr FRD2 GLUDy HEXl LDH_D ME2 THD2 190.
- ADHEr FRD2 GLUDy LDH_D ME2 THD2 195.
- ADHEr GLUDy LDH_D ME2 PROIz SUCD4 211.
- ASNS2 ATPS4r PTAr RPE SUCOAS 250.
- ASNS2 ATPS4r PTAr RPE SUCOAS 251.
- ATPS4r CBMK2 PTAr SUCOAS TKTl 261.
- ATPS4r PTAr SUCOAS TKTl ATPS4r PTAr SUCOAS TKT2
- FRD2 MEIx ME2 PYK RPE 328 FRD2 MEIx ME2 PYK TKT2
- ATPS4r PDH PFLi TAL 52 ATPS4r PDH PFLi TKTl
- ASPT ATPS4r FUM PFK PFLi Table 3 A list of all the reaction stoichiometries and the associated genes known to be associated with the reactions identified for deletion in the strains listed in Tables 1 and 2.
- Table 4 List of the metabolite abbreviations, the corresponding names and locations of all the metabolites that participate in the reactions listed in Table 3.
- Table 5 The list of all strains identified by OptKnock that are most likely to provide increased fumarate yields in S. Cerevisiae under microaerobic culture conditions. These same designs can be used for malate production if the cytosolic fumarase (FUM) is deleted additionally.
- FUM cytosolic fumarase
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CA2725549A CA2725549A1 (en) | 2008-06-17 | 2009-06-17 | Microorganisms and methods for the biosynthesis of fumarate, malate, and acrylate |
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Also Published As
Publication number | Publication date |
---|---|
EP2304039A1 (en) | 2011-04-06 |
US8129154B2 (en) | 2012-03-06 |
US9689006B2 (en) | 2017-06-27 |
US20100009419A1 (en) | 2010-01-14 |
BRPI0913901A2 (en) | 2016-12-13 |
US20160017382A1 (en) | 2016-01-21 |
EP2304039A4 (en) | 2012-11-07 |
CA2725549A1 (en) | 2009-12-23 |
US20180363012A1 (en) | 2018-12-20 |
EP2304039B1 (en) | 2019-08-21 |
US11525149B2 (en) | 2022-12-13 |
US9062330B2 (en) | 2015-06-23 |
US20120237990A1 (en) | 2012-09-20 |
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