Patent Application
20200157579
Energy conversion, utilization and access underlie many of the great challenges of our time, including those associated with sustainability, environmental quality, security, and quality of life. New applications of emerging technologies are required to respond to these challenges. Biotechnology, one of the most powerful of the emerging technologies, can give rise to important new energy conversion processes. Plant biomass and derivatives thereof are a resource for the biological conversion of energy to forms useful to humanity.
Among forms of plant biomass, both grain-based biomass and lignocellulosic biomass (collectively “biomass”) are well-suited for energy applications. Each feedstock has advantages and disadvantages. For example, because of its large-scale availability, low cost, and environmentally benign production lignocellulosic biomass has gained attention as a viable feed source for biofuel production. In particular, many energy production and utilization cycles based on cellulosic biomass have near-zero greenhouse gas emissions on a life-cycle basis.
However, grain-based feed stocks are more readily converted to fuels by existing microorganisms, although grain-based feed stock is more expensive than lignocellulosic feed stock and conversion to fuel competes with alternative uses for the grain.
Biomass processing schemes involving enzymatic or microbial hydrolysis commonly involve four biologically mediated transformations: (1) the production of saccharolytic enzymes (cellulases and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose, and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose). These four transformations can occur in a single step in a process configuration called consolidated bioprocessing (“CBP”), which is distinguished from other less highly integrated configurations in that it does not involve a dedicated process step for cellulase and/or hemicellulase production.
CBP offers the potential for lower cost and higher efficiency than processes featuring dedicated cellulase production. The benefits result in part from avoided capital costs, substrate and other raw materials, and utilities associated with cellulase production. In addition, several factors support the realization of higher rates of hydrolysis, and hence reduced reactor volume and capital investment using CBP, including enzyme-microbe synergy and the use of thermophilic organisms and/or complexed cellulase systems. Moreover, cellulose-adherent cellulolytic microorganisms are likely to compete successfully for products of cellulose hydrolysis with non-adhered microbes, e.g., contaminants. Successful competition of desirable microbes increases the stability of industrial processes based on microbial cellulose utilization. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer; and engineering non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase and hemicellulase system enabling cellulose and hemicellulose utilization.
One way to meet the demand for ethanol production is to convert sugars found in biomass, i.e., materials such as agricultural wastes, corn hulls, corncobs, cellulosic materials, and the like to produce ethanol. Efficient biomass conversion in large-scale industrial applications requires a microorganism that is able to tolerate high concentrations of sugar and ethanol, and which is able to ferment more than one sugar simultaneously.
Bakers' yeast (Saccharomyces cerevisiae) is the preferred microorganism for the production of ethanol (Hahn-Hägerdal, B., et al., Adv. Biochem. Eng. Biotechnol. 73: 53-84 (2001)). Pathways for ethanol production in S. cerevisiae can be seen in
However, glycerol is a required metabolic end-product of native yeast ethanolic fermentation (
There is a strong commercial incentive to not produce glycerol, as it represents lost ethanol yield. In industrial corn ethanol fermentations, this yield loss can be up to 6% of theoretical, for a market of ˜14 billion gallons/yr. At selling price of $2.50/gal, this is a total market value of $2 B/yr.
Strategies from the literature to address this problem include decreasing glycerol formation by engineering ammonia fixation to function with NADH instead of NADPH via up-regulation of GLN1, encoding glutamine synthetase, or GLT1, encoding glutamate synthase with deletion of GDH1, encoding the NADPH-dependent glutamate dehydrogenase. (Nissen, T. L., et al., Metabolic Engineering 2: 69-77 (2000)). Another strategy engineering cells to produce excess NADPH during glycolysis via expression of a NADPH linked glyceraldehyde-3-phosphate dehydrogenase. (Bro, C., et al., Metabolic Engineering 8: 102-111 (2006)).
However, most glycerol reduction strategies either only partially reduce the requirement for glycerol formation, or create a by-product other than ethanol. The present invention overcomes the shortcomings of these other strategies by redirecting electrons typically placed on glycerol into ethanol formation, thereby reducing glycerol production and increasing ethanol production (
Aspects of the invention are directed to a recombinant microorganism comprising a heterologous nucleic acid encoding a phosphoketolase; at least one heterologous nucleic acid encoding an enzyme in an acetyl-CoA production pathway; a heterologous nucleic acid encoding a bifunctional acetaldehyde-alcohol dehydrogenase; and, at least one genetic modification that leads to the down-regulation of an enzyme in a glycerol-production pathway. In some embodiments, the phosphoketolase is a single-specificity phosphoketolase with the Enzyme Commission Number 4.1.2.9. In some embodiments, the phosphoketolase is dual-specificity phosphoketolase with the Enzyme Commission Number 4.1.2.22. Aspects of the invention are directed to a recombinant microorganism comprising at least one heterologous nucleic acid encoding an enzyme in an acetyl-CoA production pathway; a heterologous nucleic acid encoding a bifunctional acetaldehyde-alcohol dehydrogenase; and, optionally, at least one genetic modification that leads to the down-regulation of an enzyme in a glycerol-production pathway. In some embodiments, the enzyme in the acetyl-CoA production pathway is phosphotransacetylase with the Enzyme Commission Number 2.3.1.8. In some embodiments, the enzyme in the acetyl-CoA production pathway is acetate kinase with the Enzyme Commission Number 2.7.2.12. In some embodiments, the bifunctional acetaldehyde-alcohol dehydrogenase is selected from a group of enzymes having both of the following Enzyme Commission Numbers: EC 1.2.1.10 and 1.1.1.1. In some embodiments, the bifunctional acetaldehyde-alcohol dehydrogenase is an NADPH dependent bifunctional acetaldehyde-alcohol dehydrogenase selected from a group of enzymes having the following Enzyme Commission Numbers: EC 1.2.1.10 and 1.1.1.2. In some embodiments, the enzyme in the glycerol-production pathway is glycerol-3-phosphate dehydrogenase with the Enzyme Commission Number 1.1.1.8. In some embodiments, the enzyme in the glycerol-production pathway is glycerol-3-phosphate phosphatase with the Enzyme Commission Number 3.1.3.21.
In some embodiments, the microorganism further comprises at least one additional up-regulated enzyme. In some embodiments, transaldolase with the Enzyme Commission Number 2.2.1.2 is up-regulated. In some embodiments, transketolase with the Enzyme Commission Number 2.2.1.1 is up-regulated. In some embodiments, ribose-5-P isomerase with the Enzyme Commission Number 5.3.1.6 is up-regulated. In some embodiments, ribulose-5-P 3-epimerase with the Enzyme Commission Number 5.1.3.1 is up-regulated.
In some embodiments, at least one enzyme in a glycolysis pathway is up-regulated in the microorganism. In some embodiments, the enzyme in the glycolysis pathway is pyruvate decarboxylase with the Enzyme Commission Number 4.1.1.1. In some embodiments, the enzyme in the glycolysis pathway is alcohol dehydrogenase selected from a group of enzymes having the following Enzyme Commission Numbers: 1.1.1.1 and 1.1.1.2.
In some embodiments, the microorganism additionally comprises at least one genetic modification that leads to the down-regulation of aldehyde dehydrogenase selected from a group of enzymes having the following Enzyme Commission Numbers: 1.2.1.3, 1.2.1.4 and 1.2.1.10. In some embodiments, the microorganism additionally comprises at least one genetic modification that leads to the up-regulation of aldehyde dehydrogenase selected from a group of enzymes having the following Enzyme Commission Numbers: 1.2.1.3, 1.2.1.4 and 1.2.1.10. In some embodiments, the aldehyde dehydrogenase is acetaldehyde dehydrogenase.
In some embodiments, the microorganism additionally comprises at least one genetic modification that leads to the down-regulation of formate dehydrogenase selected from a group of enzymes having the following Enzyme Commission Numbers: 1.2.1.43 and 1.2.1.2.
In some embodiments, the microorganism additionally comprises at least one genetic modification that leads to the up-regulation of pyruvate formate lyase with the Enzyme Commission Number 2.3.1.54. In some embodiments, the microorganism additionally comprises at least one genetic modification that leads to the up-regulation of pyruvate formate lyase activating enzyme with the Enzyme Commission Number 1.97.1.4.
In some embodiments, the microorganism is yeast. In some embodiments, the microorganism is from the genus Saccharomyces. In some embodiments, the microorganism is Saccharomyces cerevisiae.
In some embodiments, the recombinant microorganisms described herein produce ethanol at a higher yield than an otherwise identical microorganism lacking said genetic modifications. In some embodiments, the microorganism produces an ethanol titer 1%-10% more than an otherwise identical microorganism lacking said genetic modifications.
In some embodiments, the recombinant microorganisms described herein produce glycerol at a lower yield than an otherwise identical microorganism lacking said genetic modifications. In some embodiments, the microorganism produces a glycerol titer of 10-100% less than an otherwise identical microorganism lacking said genetic modifications.
In some embodiments, the invention is directed to the host cells described herein and a carbon-containing feedstock. In some embodiments, the feedstock is selected from the group consisting of woody biomass, grasses, sugar-processing residues, municipal waste, agricultural wastes or any combination thereof. In some embodiments, the feedstock comprises recycled wood pulp fiber, sawdust, hardwood, softwood, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, succulents, agave, cane bagasse, switchgrass, miscanthus, paper sludge, municipal waste or any combination thereof.
In some embodiments, the invention is directed to a method of producing a fermentation product using a composition described herein, wherein the host cell of the composition is capable of fermenting the carbon containing feedstock to yield the fermentation product.
In some embodiments, the invention is directed to a method of producing ethanol comprising providing any host cell described herein; culturing the host cell in the presence of a carbon containing feedstock for sufficient time to produce ethanol; and, optionally, extracting the ethanol.
In some embodiments, the invention is directed to a method of reducing glycerol production comprising providing any host cell described herein, wherein said glycerol production is 10-100% less than an otherwise identical microorganism lacking said genetic modifications. In some embodiments, the glycerol production of the host cell is reduced as compared to an otherwise identical microorganism lacking said genetic modifications, and wherein the ethanol titer increased by at least 1-10% when the host cell is cultured in the presence of a carbon containing feedstock for a sufficient time to produce ethanol.
In some embodiments, the invention is directed to a co-culture comprising at least two host cells wherein one of the host cells comprises a host cell described herein and another host cell that is genetically distinct from the host cell of the invention. In some embodiments, the genetically distinct host cell of the co-culture is a yeast or bacterium. In some embodiments, the genetically distinct host cell is any organism from the genus Saccharomyces, Issatchenkia, Pichia, Clavispora, Candida, Hansenula, Kluyveromyces, Trichoderma, Thermoascus, Escherichia, Clostridium, Caldicellulosiruptor, Zymomonas, Thermoanaerobacter and Thermoanaerobacterium.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art of microbial metabolic engineering. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods, devices and materials are described herein.
The embodiments described, and references in the specification to “one embodiment”, “an embodiment”, “an example embodiment”, etc., indicate that the embodiments described can include a particular feature, structure, or characteristic, but every embodiment does not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is understood that it is within the knowledge of one skilled in the art to effect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.
The description of “a” or “an” item herein may refer to a single item or multiple items. It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided. Thus, for example, reference to “a polynucleotide” includes a plurality of such polynucleotides and reference to “the microorganism” includes reference to one or more microorganisms, and so forth.
The term “heterologous” is used in reference to a polynucleotide or a gene not normally found in the host organism. “Heterologous” includes up-regulated or down-regulated endogenous genes. “Heterologous” also includes a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene, e.g., not in its natural location in the organism's genome. “Heterologous” also includes any gene that has been modified and placed into an organism. A heterologous gene may include a native coding region that is a portion of a chimeric gene including a non-native regulatory region that is reintroduced into the native host or modifications to the native regulatory sequences that affect the expression level of the gene. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A heterologous polynucleotide, gene, polypeptide, or an enzyme may be derived or isolated from any source, e.g., eukaryotes, prokaryotes, viruses, or synthetic polynucleotide fragments, and includes up-regulated endogenous genes.
The terms “gene(s)” or “polynucleotide” or “nucleic acid” or “polynucleotide sequence(s)” are intended to include nucleic acid molecules, e.g., polynucleotides which include an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences, and introns. In addition, the terms are intended to include one or more genes that map to a functional locus. Also, the terms are intended to include a specific gene for a selected purpose. The gene may be endogenous to the host cell or may be recombinantly introduced into the host cell, e.g., as a plasmid maintained episomally or a plasmid (or fragment thereof) that is stably integrated into the genome. In addition to the plasmid form, a gene may, for example, be in the form of linear DNA or RNA. The term “gene” is also intended to cover multiple copies of a particular gene, e.g., all of the DNA sequences in a cell encoding a particular gene product.
The term “expression” is intended to include the expression of a gene at least at the level of mRNA production, generally subsequently translated into a protein product.
As used herein, an “expression vector” is a vector capable of directing the expression of genes to which it is operably linked.
In some embodiments, the microorganisms contain enzymes involved in cellulose digestion, metabolism and/or hydrolysis. A “cellulolytic enzyme” can be any enzyme involved in cellulose digestion, metabolism, and/or hydrolysis. The term “cellulase” refers to a class of enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis (i.e. the hydrolysis) of cellulose. However, there are also cellulases produced by other types of organisms such as plants and animals. Several different kinds of cellulases are known, which differ structurally and mechanistically. There are general types of cellulases based on the type of reaction catalyzed: endocellulase breaks internal bonds to disrupt the crystalline structure of cellulose and expose individual cellulose polysaccharide chains; exocellulase cleaves 2-4 units from the ends of the exposed chains produced by endocellulase, resulting in the tetrasaccharides or disaccharide such as cellobiose. There are two main types of exocellulases (or cellobiohydrolases, abbreviated CBH)—one type working processively from the reducing end, and one type working processively from the non-reducing end of cellulose; cellobiase or beta-glucosidase hydrolyses the exocellulase product into individual monosaccharides; oxidative cellulases that depolymerize cellulose by radical reactions, as for instance cellobiose dehydrogenase (acceptor); cellulose phosphorylases that depolymerize cellulose using phosphates instead of water. In the most familiar case of cellulase activity, the enzyme complex breaks down cellulose to beta-glucose. A “cellulase” can be any enzyme involved in cellulose digestion, metabolism and/or hydrolysis, including, for example, an endoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase, arabinofuranosidase, galactosidase, cellobiose phosphorylase, cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase, endoxylanase, glucuronidase, acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin, pectinase, and feruoyl esterase protein.
A “plasmid” or “vector” refers to an extrachromosomal element often carrying one or more genes, and is usually in the form of a circular double-stranded DNA molecule. Plasmids and vectors may also contain additional genetic elements such as autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences. They may also be linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source. Plasmids and vectors may be constructed by known techniques in which a number of nucleotide sequences have been joined or recombined into a unique construction. Plasmids and vectors generally also include a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence. Generally, the plasmids of the present invention are stable and self-replicating.
As used herein, the term “anaerobic” refers to an organism, biochemical reaction or process that is active or occurs under conditions of an absence of gaseous O2.
“Anaerobic conditions” are defined as conditions under which the oxygen concentration in the fermentation medium is too low for the microorganism to use as a terminal electron acceptor. Anaerobic conditions may be achieved by sparging a fermentation medium with an inert gas such as nitrogen until oxygen is no longer available to the microorganism as a terminal electron acceptor. Alternatively, anaerobic conditions may be achieved by the microorganism consuming the available oxygen of fermentation until oxygen is unavailable to the microorganism as a terminal electron acceptor.
“Aerobic metabolism” refers to a biochemical process in which oxygen is used as a terminal electron acceptor to convert energy, typically in the form of ATP, from carbohydrates. Aerobic metabolism typically occurs, for example, via the electron transport chain in mitochondria in eukaryotes, wherein a single glucose molecule is metabolized completely into carbon dioxide in the presence of oxygen.
In contrast, “anaerobic metabolism” refers to a biochemical process in which oxygen is not the final acceptor of electrons generated. Anaerobic metabolism can be divided into anaerobic respiration, in which compounds other than oxygen serve as the terminal electron acceptor, and substrate level phosphorylation, in which no exogenous electron acceptor is used and products of an intermediate oxidation state are generated via a “fermentative pathway.”
In “fermentative pathways”, the amount of NAD(P)H generated by glycolysis is balanced by the consumption of the same amount of NAD(P)H in subsequent steps. For example, in one of the fermentative pathways of certain yeast strains, NAD(P)H generated through glycolysis donates its electrons to acetaldehyde, yielding ethanol. Fermentative pathways are usually active under anaerobic conditions but may also occur under aerobic conditions, under conditions where NADH is not fully oxidized via the respiratory chain.
As used herein, the term “end-product” refers to a chemical compound that is not or cannot be used by a cell, and so is excreted or allowed to diffuse into the extracellular environment. Common examples of end-products from anaerobic fermentation include, but are not limited to, ethanol, acetic acid, formic acid, lactic acid, hydrogen and carbon dioxide.
As used herein, “cofactors” are compounds involved in biochemical reactions that are recycled within the cells and remain at approximately steady state levels. Common examples of cofactors involved in anaerobic fermentation include, but are not limited to, NAD+ and NADP+. In metabolism, a cofactor can act in oxidation-reduction reactions to accept or donate electrons. When organic compounds are broken down by oxidation in metabolism, their energy can be transferred to NAD+ by its reduction to NADH, to NADP− by its reduction to NADPH, or to another cofactor, FAD+, by its reduction to FADH2. The reduced cofactors can then be used as a substrate for a reductase.
As used herein, a “pathway” is a group of biochemical reactions that together can convert one compound into another compound in a step-wise process. A product of the first step in a pathway may be a substrate for the second step, and a product of the second step may be a substrate for the third, and so on. Pathways of the present invention include, but are not limited to, the pyruvate metabolism pathway the lactate production pathway, the ethanol production pathway, and the glycerol-production pathway.
The term “recombination” or “recombinant” refers to the physical exchange of DNA between two identical (homologous), or nearly identical, DNA molecules. Recombination can be used for targeted gene deletion or to modify the sequence of a gene. The term “recombinant microorganism” and “recombinant host cell” are used interchangeably herein and refer to microorganisms that have been genetically modified to express or over-express endogenous polynucleotides, or to express heterologous polynucleotides, such as those included in a vector, or which have a modification in expression of an endogenous gene.
By “expression modification” it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down-regulated, such that expression, level, or activity, is greater than or less than that observed in the absence of the modification.
In one aspect of the invention, genes or particular polynucleotide sequences are partially, substantially, or completely deleted, silenced, inactivated, or down-regulated in order to inactivate the enzymatic activity they encode. Complete deletions provide maximum stability because there is no opportunity for a reverse mutation to restore function. Alternatively, genes can be partially, substantially, or completely deleted, silenced, inactivated, or down-regulated by insertion, deletion, removal or substitution of nucleic acid sequences that disrupt the function and/or expression of the gene.
As used herein, the term “down-regulate” includes the deletion or mutation of a genetic sequence, or insertion of a disrupting genetic element, coding or non-coding, such that the production of a gene product is lessened by the deletion, mutation, or insertion. It includes a decrease in the expression level (i.e., molecular quantity) of a mRNA or protein. “Delete” or “deletion” as used herein refers to a removal of a genetic element such that a corresponding gene is completely prevented from being expressed. In some embodiments, deletion refers to a complete gene deletion. Down-regulation can also occur by causing the repression of genetic elements by chemical or other environmental means, for example by engineering a chemically-responsive promoter element (or other type of conditional promoter) to control the expression of a desired gene product. Down-regulation can also occur through use of a weak promoter.
As used herein, the term “up-regulate” includes the insertion, reintroduction, mutation, or increased expression of a genetic sequence, such that the production of a gene product is increased by the insertion, reintroduction, or mutation. “Insert” or “insertion” as used herein refers to an introduction of a genetic element such that a corresponding gene is expressed. Up-regulation can also occur by causing the increased expression of genetic elements through an alteration of the associated regulatory sequence.
As used herein, the term “glycerol-production pathway” refers to the collection of biochemical pathways that produce glycerol from DHAP. Components of the pathway consist of all substrates, cofactors, byproducts, intermediates, end-products, and enzymes in the pathway.
As used herein, the term “ethanol production pathway” refers the collection of biochemical pathways that produce ethanol from pyruvate. Components of the pathway consist of all substrates, cofactors, byproducts, intermediates, end-products, and enzymes in the pathway.
As used herein, the term “acetyl-CoA production pathway” refers to the collection of biochemical pathways that produce acetyl-CoA from acetyl-phosphate. Components of the pathway consist of all substrates, cofactors, byproducts, intermediates, end-products, and enzymes in the pathway.
As used herein, the term “pyruvate metabolism pathway” refers to the collection of biochemical pathways that convert pyruvate into any product, including, but not limited to, ethanol, lactic acid, acetic acid and formate. It also includes the collection of pathways that result in the production of pyruvate, such as glycolysis. Components of the pathway consist of all substrates, cofactors, byproducts, intermediates, end-products, and enzymes in the pathway.
As used herein, the term “glycolysis” or “glycolytic pathway” refers to the canonical pathway of basic metabolism in which a sugar such as glucose is broken down into more oxidized products, converting energy and compounds required for cell growth. Components of the pathway consist of all substrates, cofactors, byproducts, intermediates end-products, and enzymes in the pathway.
As used herein, the term “phosphoketolase”, “single-specificity phosphoketolase” or “dual-specificity phosphoketolase” is intended to include the enzymes that catalyze the conversion of D-xylulose 5-phosphate to D-glyceraldehyde 3-phosphate. Dual specificity phosphoketolase additionally includes the enzymes that catalyze the conversion of D-fructose 6-phosphate to D-erythrose 4-phosphate. Phosphoketolase, single-specificity phosphoketolase and dual-specificity phosphoketolase are referred to collectively as “PHKs” or “phosphoketolase” (
As used herein, the term “alcohol dehydrogenase” or “ADH” is intended to include the enzymes that catalyze the conversion of ethanol into acetylaldehyde. Very commonly, the same enzyme catalyzes the reverse reaction from acetaldehyde to ethanol, which is the direction more relevant to fermentation. Alcohol dehydrogenase includes those enzymes that correspond to EC 1.1.1.1 and 1.1.1.2 and exemplified by the enzymes disclosed in GenBank Accession No. U49975.
As used herein, the term “aldehyde dehydrogenase”, “ALD” or “ALDH” is intended to include the enzymes that catalyze the oxidation of aldehydes. Aldehyde dehydrogenase enzymes include “acetaldehyde dehydrogenase”, which catalyzes the conversion of acetaldehyde into acetyl-CoA. Very commonly, the same enzyme catalyzes the reverse reaction from acetyl-CoA to acetaldehyde, which is the direction more relevant to fermentation. Aldehyde dehydrogenase includes those enzymes that correspond to EC 1.2.1.3, 1.2.1.4 and 1.2.1.10. In some embodiments, acetaldehyde dehydrogenase is from S. cerevisiae (ALD2: SEQ ID NO: 17 and 18; ALD3: SEQ ID NO: 19 and 20; ALD4: SEQ ID NO: 21 and 22; ALD5: SEQ ID NO: 23 and 24; or ALD6 SEQ ID NO: 25 and 26).
As used herein, the term “phosphotransacetylase” or “PTA” is intended to include the enzymes capable of converting acetyl-CoA to acetylphosphate. PTA includes those enzymes that correspond to EC 2.3.1.8. In some embodiments, PTA is from B. adolescentis (SEQ ID NOs: 1 and 10), C. cellulolyticum (SEQ ID NOs: 79 and 80), C. phytofermentans (SEQ ID NOs: 81 and 82), B. bifidum (SEQ ID NOs: 83 and 84), B. animalis (SEQ ID NOs: 85 and 86), L. mesenteroides (SEQ ID NOs: 95 and 96), or O. oenii (SEQ ID NOs: 103 and 34).
As used herein, the term “acetate kinase” or “ACK” is intended to include the enzymes capable of converting acetylphosphate to acetate. ACK includes those enzymes that correspond to EC 2.7.2.12. In some embodiments, ACK is from B. adolescentis (SEQ ID NOs: 2 and 16), C. cellulolyticum (SEQ ID NOs: 87 and 88), C. phytofermentans (SEQ ID NOs: 75 and 76), L. mesenteroides (SEQ ID NOs: 91 and 92), or O. oenii (SEQ ID NOs: 99 and 100).
As used herein, the term “glycerol-3-phosphate dehydrogenase” or “GPD” is intended to include those enzymes capable of converting dihydroxyacetone phosphate to glycerol-3-phosphate. GPD includes those enzymes that correspond to EC 1.1.1.8. In some embodiments, the GPD is GPD1 and/or GPD2 from S. cerevisiae (GDP1: SEQ ID NO: 27 and 28, GDP2: SEQ ID NO: 29 and 30).
As used herein, the term “glycerol-3-phosphate phosphatase” is intended to include those enzymes capable of converting glycerol-1-phosphate to glycerol. Glycerol-3-phosphate is intended to include those enzymes that correspond to EC 3.1.3.21.
As used herein, the term “formate dehydrogenase” or “FDH” is intended to include those enzymes capable of converting formate to bicarbonate (carbon dioxide). Formate dehydrogenase includes those enzymes that correspond to EC 1.2.1.43 and EC 1.2.1.2. In some embodiments, the FDH is from S. cerevisiae (FDH1: SEQ ID NO: 31, and 32, FDH2: SEQ ID NO: 33).
As used herein, the term “transaldolase” is intended to include those enzymes capable of converting glyceraldehyde-3-phosphate to fructose-6-phosphate. Transaldolase is intended to include those enzymes that correspond to EC 2.2.1.2. In some embodiments, the transaldolase is from S. cerevisiae (TAL1: SEQ ID NO: 35 and 36).
As used herein, the term “transketolase” is intended to include those enzymes capable of converting sedoheptulose-7-P and glyceraldehyde-3-P to D-ribose-5-P and D-xylulose-5-P and those enzymes capable of converting fructose 6-phosphate and glyceraldehyde-3-P to D-xylulose-5-P and aldose erythrose-4-phosphate. Transketolase is intended to include those enzymes that correspond to EC 2.2.1.1. In some embodiments, the transketolase is from S. cerevisiae (TKL1: SEQ ID NO: 37 and 38).
As used herein, the term “ribose-5-P isomerase” or “ribose-5-phosphate isomerase” is intended to include those enzymes capable of converting ribose-5-phosphate to ribulose-5-phosphate. Ribose-5-P isomerase is intended to include those enzymes that correspond to EC 5.3.1.6. In some embodiments, the ribose-5-P isomerase is from S. cerevisiae (RKI1: SEQ ID NO: 39 and 40).
As used herein, the term “ribulose-S-P 3 epimerase”, “ribulose-5-phosphate 3 epimerase” or “ribulose-phosphate 3-epimerase” is intended to include those enzymes capable of converting D-ribulose 5-phosphate to D-xylulose 5-phosphate. Ribulose-5-P 3 epimerase is intended to include those enzymes that correspond to EC 5.1.3.1. In some embodiments, the ribulose-5-P 3 epimerase is from S. cerevisiae (RPE1: SEQ ID NO: 41 and 42).
As used herein, the term “pyruvate decarboxylase” or “PDC” is intended to include those enzymes capable of converting pyruvic acid to acetaldehyde. PDC is intended to include those enzymes that correspond to EC 4.1.1.1.
As used herein, the term “bifunctional” is intended to include enzymes that catalyze more than one biochemical reaction step. A specific example of a bifunctional enzyme used herein is an enzyme (adhE) that catalyzes both the alcohol dehydrogenase and acetaldehyde dehydrogenase reactions, and includes those enzymes that correspond to EC 1.2.1.10 and 1.1.1.1. In some embodiments, the bifunctional acetaldehyde-alcohol dehydrogenase is from B. adolescentis (adhE: SEQ ID NO: 43 and 44). In some embodiments, the bifunctional enzyme is a NADPH specific bifunctional acetaldehyde-alcohol dehydrogenase, and includes those enzymes that correspond to EC 1.2.1.10 and 1.1.1.2. In some embodiments, the NADPH specific bifunctional acetaldehyde-alcohol dehydrogenase is from L. mesenteroides (SEQ ID NO: 89 and 90) or Oenococcus oenii (SEQ ID NO: 97 and 98).
As used herein, the term “pyruvate formate lyase” or “PFL” is intended to include the enzymes capable of converting pyruvate to formate and acetyl-CoA. PFL includes those enzymes that correspond to EC 2.3.1.54 and exemplified by SEQ ID NO: 47 and SEQ ID NO: 48.
As used herein, the term “PFL-activating enzymes” is intended to include those enzymes capable of aiding in the activation of PFL. PFL-activating enzymes include those enzymes that correspond to EC 1.97.1.4 and exemplified by SEQ ID NO: 45 and SEQ ID NO: 46.
The term “feedstock” is defined as a raw material or mixture of raw materials supplied to a microorganism or fermentation process from which other products can be made. For example, a carbon source, such as biomass or the carbon compounds derived from biomass are a feedstock for a microorganism that produces a product in a fermentation process. A feedstock can contain nutrients other than a carbon source.
Biomass can include any type of biomass known in the art or described herein. The terms “lignocellulosic material,” “lignocellulosic substrate” and “cellulosic biomass” mean any type of carbon containing feed stock including woody biomass, such as recycled wood pulp fiber, sawdust, hardwood, softwood, grasses, sugar-processing residues, agricultural wastes, such as, but not limited to, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, succulents, agave, or any combination thereof.
The term “yield” is defined as the amount of product obtained per unit weight of raw material and may be expressed as gram product per gram substrate (g/g). Yield may be expressed as a percentage of the theoretical yield. “Theoretical yield” is defined as the maximum amount of product that can be generated per a given amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to ethanol is 0.51 g EtOH per 1 g glucose. As such, a yield of 4.8 g ethanol from 10 g of glucose would be expressed as 94% of theoretical or 94% theoretical yield.
The term “titer” is defined as the strength of a solution or the concentration of a substance in solution. For example, the titer of a product in a fermentation broth is described as gram of product in solution per liter of fermentation broth (g/L) or as g/kg broth.
As used herein, the term “flux” is the rate of flow of molecules through a metabolic pathway, akin to the flow of material in a process.
“Bacteria”, or “eubacteria”, refers to a domain of prokaryotic organisms. Bacteria include gram-positive (gram+) bacteria and gram-negative (gram−) bacteria.
“Yeast” refers to a domain of eukaryotic organisms that are unicellular fungi.
The terms “derivative” and “analog” refer to a polypeptide differing from the enzymes of the invention, but retaining essential properties thereof. Generally, derivatives and analogs are overall closely similar, and, in many regions, identical to the enzymes of the invention. The terms “derived from”, “derivative” and “analog” when referring to enzymes of the invention include any polypeptides which retain at least some of the activity of the corresponding native polypeptide or the activity of its catalytic domain.
Derivatives of enzymes disclosed herein are polypeptides which may have been altered so as to exhibit features not found on the native polypeptide. Derivatives can be covalently modified by substitution (e.g. amino acid substitution), chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (e.g., a detectable moiety such as an enzyme or radioisotope). Examples of derivatives include fusion proteins, or proteins which are based on a naturally occurring protein sequence, but which have been altered. For example, proteins can be designed by knowledge of a particular amino acid sequence, and/or a particular secondary, tertiary, and quaternary structure. Derivatives include proteins that are modified based on the knowledge of a previous sequence, natural or synthetic, which is then optionally modified, often, but not necessarily to confer some improved function. These sequences, or proteins, are then said to be derived from a particular protein or amino acid sequence. In some embodiments of the invention, a derivative must retain at least about 50% identity, at least about 60% identity, at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 97% identity, or at least about 99% identity to the sequence the derivative is “derived from. ” In some embodiments of the invention, an enzyme is said to be derived from an enzyme naturally found in a particular species if, using molecular genetic techniques, the DNA sequence for part or all of the enzyme is amplified and placed into a new host cell.
The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
As known in the art, “similarity” between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide.
“Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
Suitable nucleic acid sequences or fragments thereof (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% to 75% identical to the amino acid sequences disclosed herein, at least about 80%, at least about 85%, or at least about 90% identical to the amino acid sequences disclosed herein, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequences disclosed herein. Suitable nucleic acid fragments are at least about 70%, at least about 75%, or at least about 80% identical to the nucleic acid sequences disclosed herein, at least about 80%, at least about 85%, or at least about 90% identical to the nucleic acid sequences disclosed herein, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleic acid sequences disclosed herein. Suitable nucleic acid fragments not only have the above identities/similarities but typically encode a polypeptide having at least about 50 amino acids, at least about 100 amino acids, at least about 150 amino acids, at least about 200 amino acids, or at least about 250 amino acids.
In some embodiments of the present invention, exogenous genes may be codon-optimized in order to express the polypeptide they encode most efficiently in the host cell. Methods of codon optimization are well known in the art. (See, e.g. Welch et al. “Designing genes for successful protein expression.” Methods Enzymol. 2011. 498:43-66.)
In general, highly expressed genes in an organism are biased towards codons that are recognized by the most abundant tRNA species in that organism. One measure of this bias is the “codon adaptation index” or “CAI,” which measures the extent to which the codons used to encode each amino acid in a particular gene are those which occur most frequently in a reference set of highly expressed genes from an organism. The Codon Adaptation Index is described in more detail in Sharp et al., “The Codon Adaptation Index: a Measure of Directional Synonymous Codon Usage Bias, and Its Potential Applications.” Nucleic Acids Research 1987. 15: 1281-1295, which is incorporated by reference herein in its entirety.
A codon optimized sequence may be further modified for expression in a particular organism, depending on that organism's biological constraints. For example, large runs of “As” or “Ts” (e.g., runs greater than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive bases) can effect transcription negatively. Therefore, it can be useful to remove a run by, for example, replacing at least one nucleotide in the run with another nucleotide. Furthermore, specific restriction enzyme sites may be removed for molecular cloning purposes by replacing at least one nucleotide in the restriction site with another nucleotide. Examples of such restriction enzyme sites include PacI, Ascl, BamHI, BglII, EcoRI and XhoI. Additionally, the DNA sequence can be checked for direct repeats, inverted repeats and mirror repeats with lengths of about 5, 6, 7, 8, 9 or 10 bases or longer. Runs of “As” or “Ts”, restriction sites and/or repeats can be modified by replacing at least one codon within the sequence with the “second best” codons, i.e., the codon that occurs at the second highest frequency for a particular amino acid within the particular organism for which the sequence is being optimized.
Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The “genetic code” which shows which codons encode which amino acids is reproduced herein as Table 1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six triplets each, whereas tryptophan and methionine are coded for by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.
ATG Met (M)
Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
In some embodiments of the invention, the host cell is a eukaryotic microorganism. In some embodiments, the host cell is a yeast. In some embodiments, the host cell is able to digest and ferment cellulose. In some embodiments, the host cell is from the genus Saccharomyces. In some embodiments, the host cell is Saccharomyces cerevisiae.
In some embodiments, the host cells of the invention are cultured at a temperature above about 20° C., above about 25° C., above about 27° C., above about 30° C., above about 33° C., above about 35° C., above about 37° C., above about 40° C., above about 43° C., above about 45° C., or above about 47° C.
In some embodiments, the host cells of the invention contain genetic constructs that lead to the down-regulation of one or more genes encoding a polypeptide at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to one or more of the polypeptides encompassed by EC 1.1.1.8, 3.1.3.21, 1.2.1.43, 1.2.1.10 and/or EC 1.2.1.2.
In some embodiments, the host cells of the invention contain genetic constructs that lead to the expression or up-regulation of one or more genes encoding a polypeptide at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to one or more of the polypeptides encoded by SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 34, 36, 38, 40, 42, 44, 46, 48, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, or 102. In some embodiments, the host cells of the invention contain genetic constructs that leads to the expression or up-regulation of a polypeptide encoding the activity associated with EC Nos.: 4.1.2.9, 4.1.2.22, 2.3.1.8, 1.2.1.10, 1.2.1.3, 1.2.1.4, 1.1.1.1, 1.1.1.2, 2.7.2.12, 2.2.1.2, 2.2.1.1, 5.3.1.6, 5.1.3.1, 2.3.1.54, 1.97.1.4, 1.2.1.3, 1.2.1.4, or 4.1.1.1.
In some embodiments, PHK is up-regulated. In some embodiments, single-specificity phosphoketolase is up-regulated. In some embodiments, dual-specificity phosphoketolase is up-regulated. In some embodiments, the up-regulated PHK is from an enzyme that corresponds to an EC number selected from the group consisting of: EC 4.1.2.9 and 4.1.2.22. In some embodiments, the PHK is from A. niger. In some embodiments, the PHK is from N. crassa. In some embodiments, the PHK is from L. casei. In some embodiments, the PHK is from L. plantarum. In some embodiments, the PHK is from B. adolescentis. In some embodiments, the PHK is derived from a genus selected from the group consisting of Aspergillus, Neurospora, Lactobacillus, Bifidobacterium, and Penicillium. In some embodiments, the phosphoketolase corresponds to a polypeptide selected from a group consisting of SEQ ID NOs: 9, 11, 12, 13, 14, 15, 62, 64, 66, 68, 70, 72, 74, 78, 94, or 102.
In some embodiments, phosphotransacetylase is up-regulated. In some embodiments, the up-regulated phosphotransacetylase is from an enzyme that corresponds to EC 2.3.1.8. In some embodiments, the phosphotransacetylase is from B. adolescentis. In some embodiments, the phosphotransacetylase is derived from a genus selected from the group consisting of Bifidobacterium, Lactobacillus, or Clostridium. In some embodiments, the phosphotransacetylase corresponds to a polypeptide selected from a group consisting of SEQ ID NOs: 10, 34, 80, 82, 84, 86, or 94.
In some embodiments of the invention, acetate kinase is up-regulated. In some embodiments, the up-regulated acetate kinase is from an enzyme that corresponds to EC 2.7.2.12. In some embodiments, the acetate kinase is from B. adolescentis. In some embodiments, the acetate kinase is derived from a genus selected from the group consisting of Bifidobacterium, Lactobacillus, or Clostridium. In some embodiments, the acetate kinase corresponds to a polypeptide selected from a group consisting of SEQ ID NOs: 16, 88, 76, 92, or 100.
In some embodiments, bifunctional acetaldehyde-alcohol dehydrogenase is up-regulated. In some embodiments, the up-regulated bifunctional acetaldehyde-alcohol dehydrogenase is from an enzyme that corresponds to an EC number selected from the group consisting of: EC 1.2.1.0 and 1.1.1.1. In some embodiments, the bifunctional acetaldehyde-alcohol dehydrogenase is a NADPH dependent bifunctional acetaldehyde-alcohol dehydrogenase selected from a group of enzymes having the following Enzyme Commission Numbers: EC 1.2.1.10 and 1.1.1.2. In some embodiments, the bifunctional acetaldehyde-alcohol dehydrogenase corresponds to a polypeptide selected from the group consisting of SEQ ID NOs: 44, 90, and 98
In some embodiments, transaldolase is up-regulated. In some embodiments, the up-regulated transaldolase is from an enzyme that corresponds to EC 2.2.1.2. In some embodiments, the transaldolase is from S. cerevisiae and corresponds to a polypeptide encoded by SEQ ID NO: 36. In some embodiments, transketolase is up-regulated. In some embodiments, the up-regulated transketolase is from an enzyme that corresponds to EC 2.2.1.1. In some embodiments, the transketolase is from S. cerevisiae and corresponds to a polypeptide encoded by SEQ ID NO: 38. In some embodiments, ribose-5-P isomerase is up-regulated. In some embodiments, the up-regulated ribose-5-P isomerase is from an enzyme that corresponds to EC 5.3.1.6. In some embodiments, the ribose-5-P isomerase is from S. cerevisiae and corresponds to a polypeptide encoded by SEQ ID NO: 40. In some embodiments, ribulose-5-P epimerase is up-regulated. In some embodiments, the up-regulated ribulose-5-P epimerase is from an enzyme that corresponds to EC 5.1.3.1. In some embodiments, the ribulose-5-P 3-epimerase is from S. cerevisiae and corresponds to a polypeptide encoded by SEQ ID NO: 42. In some embodiments, pyruvate decarboxylase is up-regulated. In some embodiments, the up-regulated pyruvate decarboxylase is from an enzyme that corresponds to EC 4.1.1.1. In some embodiments, alcohol dehydrogenase is up-regulated. In some embodiments, the up-regulated alcohol dehydrogenase is from an enzyme that corresponds to EC 1.1.1.1 and 1.1.1.2. In some embodiments, pyruvate formate lyase is up-regulated. In some embodiments, the up-regulated pyruvate formate lyase is from an enzyme that corresponds to EC 2.3.1.54. In some embodiments, the pyruvate formate lyase corresponds to a polypeptide encoded by SEQ ID NO: 48. In some embodiments, pyruvate formate lyase activating enzyme is up-regulated. In some embodiments, the up-regulated pyruvate formate lyase activating enzyme is from an enzyme that corresponds to EC 1.97.1.4. In some embodiments, the pyruvate formate lyase activating enzyme corresponds to a polypeptide encoded by SEQ ID NO: 46.
In some embodiments, the host cells of the invention contain genetic constructs that lead to the expression or down-regulation of one or more genes encoding a polypeptide at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to one or more of the polypeptides encoded by SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32 or the polynucleotide encoded by SEQ ID NO: 33. In some embodiments, the host cells of the invention contain genetic constructs that leads to the expression or down-regulation of a polypeptide encoding the activity associated with EC Nos.: 1.2.1.3, 1.2.1.4, 1.2.1.10, 1.1.1.8 and 3.1.3.21.
In some embodiments, GPD is down-regulated. In some embodiments, the down-regulated GPD is from an enzyme that corresponds to EC 1.1.1.8. In some embodiments gpd1 and gpd2 from S. cerevisiae are down-regulated. In some embodiments, the glycerol-3-phosphate dehydrogenase is selected from the group consisting of glycerol-3-phosphate dehydrogenase 1, glycerol-3-phosphate dehydrogenase 2, and combinations thereof. In some embodiments, the glycerol-3-phosphate dehydrogenase 1 is from S. cerevisiae and corresponds to a polypeptide encoded by SEQ ID NO: 28. In some embodiments, the glycerol-3-phosphate dehydrogenase 2 is from S. cerevisiae and corresponds to a polypeptide encoded by SEQ ID NO: 30. In some embodiments, formate dehydrogenase is down-regulated. In some embodiments, the down-regulated formate dehydrogenase corresponds to an EC number selected from the group consisting of: EC 1.2.1.43 and EC 1.2.1.2. In some embodiments, formate dehydrogenase from S. cerevisiae is down-regulated. In some embodiments, the formate dehydrogenase from S. cerevisiae corresponds to a polypeptide corresponding to SEQ ID NO: 32 or a polynucleotide corresponding to SEQ ID NO: 33. In some embodiments, glycerol-3-phosphate phosphatase is down-regulated. In some embodiments, the down-regulated glycerol-3-phosphate phosphatase corresponds to EC 3.1.3.21.
In some embodiments, aldehyde dehydrogenase is down-regulated. In some embodiments, aldehyde dehydrogenase is up-regulated. In some embodiments, the aldehyde dehydrogenase is an enzyme that corresponds to EC 1.2.1.10, EC 1.2.1.3, or EC 1.2.1.4. In some embodiments, the aldehyde dehydrogenase is acetaldehyde dehydrogenase. In some embodiments, the acetaldehyde dehydrogenase enzyme is selected from Ald2, Ald3, Ald4, Ald5, and Ald6 in S. cerevisiae. In some embodiments, the acetaldehyde dehydrogenase from S. cerevisiae corresponds to a polypeptide sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26.
In some embodiments, phosphoketolase, phosphotransacetylase and bifunctional acetaldehyde-alcohol dehydrogenase are up-regulated, and at least one glycerol-3-phosphate dehydrogenase enzyme is down-regulated.
In some embodiments, phosphotransacetylase, bifunctional acetaldehyde-alcohol dehydrogenase, and at least one aldehyde dehydrogenase are up-regulated. In some embodiments, phosphotransacetylase, bifunctional acetaldehyde-alcohol dehydrogenase, at least one aldehyde dehydrogenase are up-regulated, and at least one glycerol-3-phosphate dehydrogenase enzyme is down-regulated. In some embodiments, phosphotransacetylase, acetate kinase, bifunctional acetaldehyde-alcohol dehydrogenase, and at least one aldehyde dehydrogenase are up-regulated. In some embodiments, phosphotransacetylase, acetate kinase and bifunctional acetaldehyde-alcohol dehydrogenase are up-regulated, and at least one glycerol-3-phosphate dehydrogenase enzyme is down-regulated. In some embodiments, at least one aldehyde dehydrogenase enzyme is also down-regulated. In some embodiments, the aldehyde dehydrogenase is acetaldehyde dehydrogenase.
In some embodiments, phosphoketolase, phosphotransacetylase, bifunctional acetaldehyde-alcohol dehydrogenase, pyruvate formate lyase, and pyruvate formate lyase activating enzyme are up-regulated, and at least one glycerol-3-phosphate dehydrogenase enzyme is down-regulated.
In some embodiments, phosphoketolase, acetate kinase and bifunctional acetaldehyde-alcohol dehydrogenase are up-regulated, and at least one glycerol-3-phosphate dehydrogenase enzyme is down-regulated.
In some embodiments, phosphoketolase, acetate kinase, bifunctional acetaldehyde-alcohol dehydrogenase, pyruvate formate lyase, and pyruvate formate lyase activating enzyme are up-regulated, and at least one glycerol-3-phosphate dehydrogenase enzyme is down-regulated.
In some embodiments, gpd1, gpd2, fdh1, and/or fdh2 are down-regulated. In some embodiments, gpd1, gpd2, fdh1 and fdh2 are down-regulated, and PHK, bifunctional acetaldehyde-alcohol dehydrogenase (AdhE), and PTA are up-regulated. In some embodiments, gpd1, gpd2, fdh1 and fdh2 are down-regulated, and B. adolescentis PHK, AdhE, and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and fdh2 are down-regulated, and L. plantarum PHK1, AdhE, and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and fdh2 are down-regulated, and L. plantarum PHK2, AdhE, B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and fdh2 are down-regulated, and A. niger PHK, AdhE, and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and fdh2 are down-regulated, and L. casei PHK1, AdhE, and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and fdh2 are down-regulated, and L. casei PHK2, AdhE, and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, PHK, B. adolescentis AdhE, and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, the up-regulated enzymes are integrated. In some embodiments, the up-regulated enzymes are expressed using an expression vector. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, and B. adolescentis PHK, B adolescentis bifunctional acetaldehyde-alcohol dehydrogenase, B adolescentis PTA, B. adolescentis pyruvate formate lyase, and B adolescentis pyruvate formate lyase activating enzyme are up-regulated. In some embodiments, fcy is down-regulated.
In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, and PHK, bifunctional acetaldehyde-alcohol dehydrogenase (AdhE), ACK and PTA are up-regulated. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, and B. adolescentis PHK, AdhE, B. adolescentis ACK, and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, and L. plantarum PHK1, AdhE, B. adolescentis ACK and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, and L. plantarum PHK2, AdhE, B. adolescentis ACK, and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, and A. niger PHK, AdhE, B. adolescentis ACK and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, and L. casei PHK1, AdhE, B. adolescentis ACK and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, and L. casei PHK2, AdhE, B. adolescentis ACK, and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, PHK, B. adolescentis AdhE, B. adolescentis PTA and B. adolescentis ACK are up-regulated in S. cerevisiae cells. In some embodiments, the up-regulated enzymes are integrated. In some embodiments, the up-regulated enzymes are expressed using an expression vector. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, and B. adolescentis PHK, B adolescentis bifunctional acetaldehyde-alcohol dehydrogenase, B. adolescentis PTA, B. adolescentis ACK B. adolescentis pyruvate formate lyase, and B adolescentis pyruvate formate lyase activating enzyme are up-regulated.
In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, and bifunctional acetaldehyde-alcohol dehydrogenase (AdhE), ACK and PTA are up-regulated. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, and AdhE, B. adolescentis ACK, and B. adolescentis PTA are up-regulated in S. cerevisiae cells. In some embodiments, the up-regulated enzymes are integrated. In some embodiments, the up-regulated enzymes are expressed using an expression vector. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, B. adolescentis AdhE, B. adolescentis PTA and B. adolescentis ACK are up-regulated in S. cerevisiae cells. In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, and B adolescentis bifunctional acetaldehyde-alcohol dehydrogenase, B. adolescentis PTA, B. adolescentis ACK B. adolescentis pyruvate formate lyase, and B adolescentis pyruvate formate lyase activating enzyme are up-regulated. In some embodiments, S. cerevisiae ALD2, ALD3, ALD4, or combinations thereof are down-regulated and S. cerevisiae ALD6, ALD5, or combinations thereof are up-regulated.
In some embodiments, gpd1, gpd2, fdh1 and/or fdh2 are down-regulated, B. adolescentis AdhE, B. adolescentis PTA and B. adolescentis ACK are up-regulated in S. cerevisiae cells.
For a microorganism to produce ethanol most economically, it is desired to produce a high yield. In one embodiment, the only product produced is ethanol. Extra products lead to a reduction in product yield and an increase in capital and operating costs, particularly if the extra products have little or no value. Extra products also require additional capital and operating costs to separate these products from ethanol.
Ethanol production can be measured using any method known in the art. For example, the quantity of ethanol in fermentation samples can be assessed using HPLC analysis. Additionally, many ethanol assay kits are commercially available, for example, alcohol oxidase enzyme based assays. Methods of determining ethanol production are within the scope of those skilled in the art from the teachings herein.
In some embodiments of the invention where redirected carbon flux generates increased ethanol production, the ethanol output can be improved by growth-coupled selection. For example, continuous culture or serial dilution cultures can be performed to select for cells that grow faster and/or produce ethanol (or any desired product) more efficiently on a desired feedstock.
One embodiment of the present invention relates to a method of producing ethanol using a microorganism described herein wherein said microorganism is cultured in the presence of a carbon containing feedstock for sufficient time to produce ethanol and, optionally, extracting the ethanol.
Ethanol may be extracted by methods known in the art. (See, e.g., U.S. Appl. Pub. No. 2011/0171709, which is incorporated herein by reference.)
Another embodiment of the present invention relates to a method of producing ethanol using a co-culture composed of at least two microorganisms in which at least one of the organisms is an organism described herein, and at least one of the organisms is a genetically distinct microorganism. In some embodiments, the genetically distinct microorganism is a yeast or bacterium. In some embodiments the genetically distinct microorganism is any organism from the genus Issatchenkia, Pichia, Clavispora, Candida, Hansenula, Kluyveromyces, Saccharomyces, Trichoderma, Thermoascus, Escherichia, Clostridium, Caldicellulosiruptor, Thermoanaerobacter and Thermoanaerobacterium.
In some embodiments, the recombinant microorganism produces about 2 to about 3% higher ethanol titer than a wildtype, non-recombinant organism; at least about 1 to at least about 2% higher ethanol titer than a wildtype, non-recombinant organism; at least about 1 to at least about 5% higher ethanol titer than a wildtype, non-recombinant organism; at least about 1 to at least about 7% higher ethanol titer than a wildtype, non-recombinant organism; at least about 1 to at least about 10% higher ethanol titer than a wildtype, non-recombinant organism; at least about 1 to at least about 15% higher ethanol titer than a wildtype, non-recombinant organism; at least about 1 to at least about 20% higher ethanol titer than a wildtype, non-recombinant organism; at least about 1 to at least about 30% higher ethanol titer than a wildtype, non-recombinant organism; at least about 1 to at least about 50% higher ethanol titer than a wildtype, non-recombinant organism; at least about 1 to at least about 75% higher ethanol titer than a wildtype, non-recombinant organism; at least about 1 to at least about 100% higher ethanol titer than a wildtype, non-recombinant organism.
In some embodiments, the recombinant microorganism produces at least about 0.5 g/L ethanol to at least about 2 g/L ethanol, at least about 0.5 g/L ethanol to at least about 3 g/L ethanol, at least about 0.5 g/L ethanol to at least about 5 g/L ethanol, at least about 0.5 g/L ethanol to at least about 7 g/L ethanol, at least about 0.5 g/L ethanol to at least about 10 g/L ethanol, at least about 0.5 g/L ethanol to at least about 15 g/L ethanol, at least about 0.5 g/L ethanol to at least about 20 g/L ethanol, at least about 0.5 g/L ethanol to at least about 30 g/L ethanol, at least about 0.5 g/L ethanol to at least about 40 g/L ethanol, at least about 0.5 g/L ethanol to at least about 50 g/L ethanol, at least about 0.5 g/L ethanol to at least about 75 g/L ethanol, or at least about 0.5 g/L ethanol to at least about 99 g/L ethanol per 24 hour incubation on a carbon-containing feed stock.
In some embodiments, the recombinant microorganism produces ethanol at least about 55% to at least about 75% of theoretical yield, at least about 50% to at least about 80% of theoretical yield, at least about 45% to at least about 85% of theoretical yield, at least about 40% to at least about 90% of theoretical yield, at least about 35% to at least about 95% of theoretical yield, at least about 30% to at least about 99% of theoretical yield, or at least about 25% to at least about 99% of theoretical yield.
In some embodiments, methods of producing ethanol can comprise contacting a biomass feedstock with a host cell or co-culture of the invention and additionally contacting the biomass feedstock with externally produced saccharolytic enzymes. In some embodiments, the host cells are genetically engineered (e.g., transduced, transformed, or transfected) with the polynucleotides encoding saccharolytic enzymes.
A “saccharolytic enzyme” can be any enzyme involved in carbohydrate digestion, metabolism and/or hydrolysis, including amylases, cellulases, hemicellulases, cellulolytic, and amylolytic accessory enzymes, inulinases, levanases, and pentose sugar utilizing enzymes. Exemplary externally produced saccharolytic enzymes are commercially available and are known to those of skill in the art and include glucoamylases.
In some embodiments of the invention where redirected carbon flux generates increased ethanol production, the glycerol output can be decreased by growth-coupled selection. For example, continuous culture or serial dilution cultures can be performed to select for cells that produce less glycerol on a desired feedstock. Glycerol can be measured, for example, by HPLC analysis of metabolite concentrations.
In some embodiments, the recombinant microorganism produces at least about 20% to at least about 30% less glycerol than a wildtype, non-recombinant organism; at least about 15% to at least about 30% less glycerol than a wildtype, non-recombinant organism; at least about 10% to at least about 40% less glycerol than a wildtype, non-recombinant organism; at least about 10% to at least about 50% less glycerol than a wildtype, non-recombinant organism; at least about 10% to at least about 60% less glycerol than a wildtype, non-recombinant organism; at least about 10% to at least about 70% less glycerol than a wildtype, non-recombinant organism; at least about 10% to at least about 80% less glycerol than a wildtype, non-recombinant organism; at least about 10% to at least about 90% less glycerol than a wildtype, non-recombinant organism; at least about 10% to at least about 99% less glycerol than a wildtype, non-recombinant organism; at least about 10% to at least about 100% less glycerol than a wildtype, non-recombinant organism; at least about 5% to at least about 100% less glycerol than a wildtype, non-recombinant organism; at least about 1% to at least about 100% less glycerol than a wildtype, non-recombinant organism.
In some embodiments, the recombinant microorganism produces glycerol at least about 50% to at least about 99% of theoretical yield, at least about 45% to at least about 80% of theoretical yield, at least about 40% to at least about 85% of theoretical yield, at least about 30% to at least about 80% of theoretical yield, at least about 25% to at least about 50% of theoretical yield, at least about 20% to at least about 50% of theoretical yield, at least about 15% to at least about 50% of theoretical yield, at least about 10% to at least about 50% of theoretical yield, at least about 5% to at least about 50% of theoretical yield, at least about 4% to at least about 50% of theoretical yield, at least about 3% to at least about 50% of theoretical yield, at least about 2% to at least about 50% of theoretical yield, at least about 1% to at least about 50% of theoretical yield, at least about 1% to at least about 25% of theoretical yield, at least about 1% to at least about 20% of theoretical yield or at least about 1% to at least about 10% of theoretical yield. In some embodiments, the recombinant microorganism produces glycerol at less about 1% of theoretical yield. In some embodiments, the recombinant microorganism produces no glycerol.
In some embodiments, the recombinant microorganism has a growth rate at least about ½ to at least about equal to the growth rate of a wildtype, non-recombinant organism, at least about ¼ to at least about equal to the growth rate of a wildtype, non-recombinant organism, at least about ⅛ to at least about equal to the growth rate of a wildtype, non-recombinant organism, at least about 1/10 to at least about equal to the growth rate of a wildtype, non-recombinant organism, at least about 1/25 to at least about equal to the growth rate of a wildtype, non-recombinant organism, at least about 1/50 to at least about equal to the growth rate of a wildtype, non-recombinant organism or at least about 1/100th to at least about equal to the growth rate of a wildtype, non-recombinant organism.
In some embodiments, glycerol production is reduced as compared to a wildtype-non-recombinant organism and the organism produces ethanol at a rate of at least about 8-11 mM glycerol per gram dry cell weight (DCW) during anaerobic growth.
Phosphoketolase (PHK) enzymes from Bifidobacterium adolescentis, Lactobacillus plantarum, or Aspergillus niger were integrated at the S. cerevisiae FCY1 site in combination with the Bifidobacterium adolescentis phosphotransacetylase (PTA) gene. The genetic modification techniques utilized to develop Saccharomyces cerevisiae strain relied upon directed integration to insert genetic material at specific and known sites within the yeast chromosome. The directed integration approach creates transgenic strains with integration events that are stable and easy to characterize.
The MX cassettes are the most commonly used engineering tool when an insertion or deletion of a genetic element is desired at a given chromosomal loci (Wach A., et al.,. Yeast. 10(13):1793-1808 (1994)). A recyclable MX cassette contains one or more markers which enable both dominant and negative selection (Goldstein, A. L., et al., Yeast. 15: 507-511 (1999); Hartzog, P. E. et al., Yeast. 22: 789-798 (2005)). The dominant marker enables selection for the modification and the counter selectable marker enables subsequent removal of the marker system via Cre-Lox mediated recombination (Güldener, U., et al., Nucleic Acids Research. 24(13): 2519-2524 (1996)) or recombination between duplicated homologous regions flanking the cassette. Because the markers are removed, they can be reused during subsequent engineering steps and ensures no undesirable foreign genetic material remains in the strain.
To create stable homozygous integrations two new HSV-thymidine kinase (TDK) based MX cassettes were developed. Expression of thymidine kinase in S. cerevisiae results in sensitivity to the compound fluoro-deoxyuracil (FUDR). The cellular toxicity of FUDR is dependent on the presence of two enzymes involved in pyrimidine metabolism: thymidine kinase (Tdk) and thymidilate synthetase (ThyA). Tdk converts FUDR to fluoro-dUMP (F-dUMP) which is a covalent inhibitor of ThyA and the basis for counter selection in a variety of eukaryotic organisms (Czako, M., and Marton, L., Plant Physiol. 104:1067-1071 (1994); Gardiner, D. M., and Howlett, B. J., Curr Genet. 45:249-255 (2004); Khang, C. H., et al., Fungal Genet Biol. 42:483-492 (2005); Szybalski, W., Bioessays. 14:495-500 (1992)).
The HSV-TDK expression cassette was independently fused to two commonly used dominant selectable markers which confer resistance to the drugs G418 (Kan) or nourseothricin (Nat) (Goldstein, A. L., et al., Yeast. 15: 507-511 (1999)). Transformation of both double expression cassettes, referred to as KT-MX and NT-MX, enabled positive selection for integration into both chromosomes as illustrated in
After each engineering step taken in the construction, all markers were subsequently deleted and/or replaced with a desired expression cassette (Mascoma Assembly) resulting in a strain free of antibiotic markers (
The integration scheme for each strain construction is shown in
S. cerevisiae strains.
B. adolescentis PTA
B. adolescentis PTA
plantarum
adolescentis
B. adolescentis PTA
Niger PHK and
B. adolescentis
The anaerobic growth of the strains expressing PHK and PTA from Example 1 was compared to the anaerobic growth of strains that did not express PHK and PTA. M2390,which has a complete anaerobic glycerol-production pathway, was used as a wildtype control strain. M3293, which contains deletions of both GPD1, GPD2, FDH1 and FDH2 and expression of the B. adolescentis adhE, did not have the ability to grow anaerobically unless acetate was supplied. A functional heterologous phosphoketolase/phosphotransacetylase pathway complemented the anaerobic growth defect of M3293.
Cells from all strains were grown overnight in YPD and inoculated to a final concentration of 0.1 g/l DCW in 96 well plates containing 100 μl YPD. The inoculated plates were allowed to equilibrate in the anaerobic chamber for 20 minutes prior to sealing the plates. As seen in
In another embodiment, the ability of strains containing a deletion of both GPD1 and GPD2 expressing PHK and PTA from Example 1 to ferment corn mash was compared to the ability of strains containing deletions of both GPD1 and GPD2 but that did not express PHK and PTA to ferment corn mash (M3293). The fermentation was started by inoculating yeast to an initial concentration of 0.1 g/l DCW in 4 mls of 30% solids corn mash. The corn mash fermentation began with oxygen in both the medium and the headspace. Limited growth of M3293 was expected because the fermentation does not start anaerobically and there was acetate present. (During the corn mash fermentation, there was 0.3-0.5 g/l acetate present. The presence of the phosphoketolase/phosphotransacetylase pathway enabled growth after the supply of oxygen and acetate was depleted. All three phosphoketolase-containing strains reached a significantly higher titer than the M3293 control strain (
All strains described in Table 2 were inoculated into defined medium without amino acids. Purged anaerobic bottles were prepared with medium as described below. The fermentations were started by inoculating these bottles with yeast strains to an initial concentration of 0.1 g/l DCW cells. After inoculation, bottles were placed in a shaking incubator at held at 35 C. Samples were withdrawn and metabolite concentrations were measured by HPLC analysis.
The phosphoketolase containing strains M4408 and M4410 had improved growth relative to M3293 (
S. cerevisiae phosphoketolase expression vectors were created using the PMU1689 vector. L. plantarum PHK1, L. plantarum PHK2, A. niger PHK, L. casei PHK, B. adolescentis PHK or N. crassa PHK were cloned into PMU1689 To create, phosphoketolase expression vectors, PCR products were amplified from genomic DNA templates using primers and templates listed in Table 3 and recombined with linearized pMU1689 such that the ORF of each gene is immediately downstream of the ADH promoter and immediately upstream of the PDC1 terminator. Schematics of the PHK expression vectors can be seen in
S. cerevisiae phosphotransacetylase expression vectors were created using the pMU1771 expression vector (
B. adolescentis
B.
adolescentis
L. plantarum(1)
L.
plantarum
L. plantarum(2)
L.
plantarum
N. crassa
N. crassa
A. niger
A. niger
L. casei
L. casei
To evaluate components of the phosphoketolase pathway, the strains listed in Table 4 were inoculated into vials containing 4 ml of YP medium (10 g/l yeast extract, 20 g/l peptone) supplemented with 225 g/L maltodexdrin. Maltodextrin hydrolysis was initiated upon addition of 0.6 AGU/gTS glucoamylase at the start of the fermentation. Vials were incubated at 32° C. for 68 hrs and sampled for HPLC analysis.
M2390 is a conventional wild type yeast. M3293 contains deletion of both glycerol-3-phosphate dehydrogenase genes (GPD1 and GPD2), both formate dehydrogenase genes (FDH1 and FDH2), and expression of the B. adolescentis bifunctional alcohol/aldehyde dehydrogenase. This strain grows poorly under anaerobic conditions and is only able to produce a titer of 42 g/l ethanol whereas M2390 was able to reach 113 g/l ethanol (
The addition of components of the phosphoketolase pathway to M3293 allowed for improved ethanol production. Expression of both B. adolescentis and L. casei PHK in combination with the B. adolescentis PTA gene resulted in production of 60.7 and 72.3 g/l respectively (
A description of the cassettes used to create the strains is seen below and in
All documents cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued or foreign patents, or any other documents, are each entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited documents.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This is a continuation of U.S. Pat. No. 15,828,729 filed Dec. 1, 2017, which is a continuation of U.S. Ser. No. 14/442,408 filed May 13, 2015, now U.S. Pat. No. 9,988,650, which is a § 371 of PCT/US13/70964 filed Nov. 20, 2013, which claims priority to U.S. provisional applications Nos. 61/728,450 filed Nov. 20, 2012 and 61,792,731 filed Mar. 15, 2013, each application of which is hereby incorporated by reference in its entirety. The contents of the sequence listing attached herewith (entitled “115235-266_seq_listing.txt”; of size 349 kb, created on Sep. 24, 2019) are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61728450 | Nov 2012 | US | |
| 61792731 | Mar 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15828729 | Dec 2017 | US |
| Child | 16581664 | US | |
| Parent | 14442408 | May 2015 | US |
| Child | 15828729 | US |
