Patent Application
20250059496
The contents of the electronic sequence listing (20241106_SequenceListing_ST26_23156197US1.xml; Size: 128,189 bytes; and Date of Creation: Nov. 6, 2024) is herein incorporated by reference in its entirety.
The present invention relates to a method to produce alcohols and alcohol precursors through genetically manipulated ethanologenic bacteria that degrades cellulose and ferments the remaining sugar source.
Biofuel is increasingly becoming a necessity in order to wean off the human consumption of fossil fuels in aspects of everyday life, transport and home heating being the largest two industries of focus. As an alternative energy source to oil and coal, the main feedstock for the production of bioethanol and other bioalcohols is starch which can yield its sugar much more readily than cellulose. This is due to the difference in structure as starch contains glucose molecules connected through α-1,4 linkages and cellulose comprises of glucose molecules attached through β-1,4 linkages. The β-1,4 linkages allow for crystallization of the cellulose, leading to a more rigid structure, which is more difficult to break down using physical, chemical, and biological processes.
The limitation that comes from solely concentrating the production of bioethanol on extracting the sugars from starches is that it prevents the utilization of the larger portion of biomass which comes in the form of lignocellulosic biomass (contains lignin, cellulose, and hemicellulose) present in almost every plant on earth. A delignification reaction allows the recovery of cellulose from those lignocellulosic plants. Further degradation of the cellulose generates cellobiose and/or glucose which can be utilized for the production of ethanol using various biology-based processes.
Seen as a sustainable alternative to gasoline and with the goal of alleviating many countries' dependence on foreign oil, the bioethanol industry is still hampered by its dependence on corn or sugar cane as main sources of fuel, as they are both rich in starch. It is estimated that about 45% of all corn production in the U.S. is directed to ethanol fuel production. This is a situation which has disastrous consequences when the prices of gasoline decline significantly low making corn-based bioethanol unsustainable on a price viewpoint.
Across the world, many other large ethanol-producing countries, including China and Brazil, have shown some struggles in ethanol production from biomass as many companies are carrying large debts from the implementation of such processes in conjunction with large processing plants having to be shut down or decrease production.
In Asia, palm oil prices have recently increased to their highest levels in years, which, in turn, will hamper the ability of Indonesia and Malaysia to produce local biofuel. Oil palm trunk is a valuable and plentiful resource in those countries to generate biofuels and biochemicals. Oil palm trunk contains a large amount of starch which is more readily solubilized in water, compared to cellulose. The starch can then be heated and hydrolyzed to glucose by amylolytic enzymes without pre-treatment. However, the conventional Oil palm trunk treatment requires high capital and operational costs and is therefore prohibitive to market entry. Moreover, the treatment carries a high probability of microbial contamination during starch processing which significantly reduces purity and poses additional complications in refining procedures.
In Europe, the biofuel industry (including biodiesel and bioethanol production) depends heavily on food-based feedstocks like vegetable oils (i.e., rapeseed, palm oil, soy) for biodiesel and corn, wheat, and sugar beet for bioethanol. Simultaneously, concerns have been raised that making fuel out of crops displaces other crops and can inflate food prices. These concerns are leading to policy changes that incentivize a shift away from food-based biofuels.
The pivot from starches to cellulose for the production of glucose is preferable as it will cease to use a food source to generate glucose. However, the costs to do so are currently prohibitive. Cellulosic ethanol as it is called relies on the non-food component of a plant to be used to generate ethanol. This would allow the replacement of the current more widespread approach of making bioethanol by using corn or sugarcane. The diversity and abundance of these types of cellulose-rich plants would allow for food resources to remain largely intact and capitalize on the waste generated from these food resources (such as cornstalk and stover) to generate ethanol. Other cellulose sources such as straws, algae and even trees fall under cellulose-rich biomass sources which can be used in generating ethanol if a commercially viable process is developed.
The reason why starches are preferred to cellulose-rich sources to generate ethanol is that the extraction of glucose from cellulose is substantially more difficult and resource intensive. To better understand the conditions associated with this increased difficulty, it is worthwhile describing the structural similarities and differences between starch and cellulose.
Cellulose and starch are polymers which have the same repeat units of glucose. However, the differences between starch and cellulose can be seen in the way the repeating glucose monomers are connected to one another. In starch, the glucose monomers are oriented in the same direction. In cellulose, each successive glucose monomer is rotated 180 degrees in respect to the previous glucose monomer. This, in turn, ensures that the bonds between each monomeric glucose differs between starch and cellulose. In starch, the bonds (otherwise known as links between the monomers) are referred to as α-1,4 linkages, in cellulose these bonds are referred to as β-1,4 linkages (see
The difference between these bonds impacts the chemical characteristics of starch and cellulose. Starch can dissolve in warm water while cellulose does not. Starch can be digested by humans, cellulose cannot. In general, starch is structurally weaker than cellulose partly due to its geometrical make-up which is less crystalline than cellulose. Starch is, at its core, a method for plants to store energy due to the reversibility of the α-1,4 linkage, therefore extracting sugars from starch is much easier than to do so from cellulose as the latter's core function is to provide structural support.
As the main component of lignocellulosic biomass, cellulose is a biopolymer consisting of many glucose units connected through β-1,4-glycosidic bonds (see
The hydrolysis of cellulose is the rate limiting step in the conversion of cellulose into biofuel. The processes currently using cellulose as a starting material for bioethanol production require the conversion of cellulose into cellobiose, further processing to generate glucose, and the final generation of ethanol. The fermentation of glucose using ethanologenic organisms is what leads to the production of ethanol. While that last step in biofuel production has been mastered for some time, the rate limiting step of cellulose hydrolysis is the most crucial one which hinders a wider acceptance of biofuels. The difficulty in overcoming this conversion of cellulose into glucose lies with the fact that cellulose has a crystalline structure which renders its conversion to glucose quite difficult because of the close packing of multiple cellulose polymers. This close packing imparts cellulose its inherent stability under a variety of chemical conditions. For example, cellulose polymers are generally insoluble in water, as well as a number of organic solvents. Furthermore, cellulose is also generally insoluble when exposed to weak acids or bases.
In general, there are three main approaches to hydrolyze cellulose: mechanical or physical, chemical and enzymatic. The chemical method resorts to the use of concentrated strong acids to hydrolyze cellulose under conditions of high temperature and pressure. Many different types of acids, such as HCl and H2SO4, have been used in the past to achieve this. The use of one of these acids usually results in at least one of the following drawbacks: corrosion of the reaction vessel, difficulty of disposing of the discharged reactants, and others. The biofuel industry is generally reticent to use chemically hydrolyzed cellulose because of the presence of toxic by-products in the resulting glucose. These by-products, if introduced in the fermentation step, will negatively affect the delicate balance of the fermenting yeast.
It is known that the costs to extract biofuel from cellulose are higher than when doing so from starch. It is estimated that, on average, depending on location and availability of biomass, the cost for cellulose conversion is about 50% more that starch conversion to glucose. This means that there currently is a clear barrier to producers for using cellulose rather than corn or other starch resources to generate glucose from biomass.
It is generally understood that roughly half of the total cost of producing biofuel from cellulose stems from the price of the enzymes (cellulases and hemicellulases). The generation of enzymes for enzymatic hydrolysis of cellulose is a time-consuming process and large volumes of enzyme are required to render the process commercially viable. One possible approach is to improve the rate of the hydrolysis reaction which, in turn, would result in a decrease in the overall cost of the process.
The enzymatic approach to hydrolyzing cellulose uses enzymes to carry out the hydrolysis reaction. Enzymes, such as cellulases (comprising endoglucanases; exoglucanases; and β-glucosidases) are used for the conversion of cellulose into glucose, however each requires extensive controls in place to maximize the reaction rates the enzymatic approach is expected to provide. Conditions such as temperature, pH, salinity, concentration of substrate and product are all factors that may affect enzyme activity with even small deviations of these parameters from the enzyme's optimal conditions resulting in loss of function. Overall, with many controls needing to be taken into consideration with the enzymatic hydrolysis of cellulose along with strict enzyme-specific functional conditions, such a method can render the process cost prohibitive in some cases and/or limit their implementation.
The enzymatic hydrolysis of cellulose is, as seen from the above, limited by the structure of cellulose itself but also by the approaches taken to degrade it into a biofuel. The production of a robust, low-cost process from cellulose has not yet been achieved. In this sense, genetically modified organisms have been employed for part of the entirety of the cellulose to ethanol pathway.
U.S. Pat. No. 4,496,656A describes a process for production of cellulase according to the present invention thus comprises culturing a cellulase-producing microorganism belonging to Cellulomonas uda CB4 in a cellulose-containing medium and recovering the cellulase produced from the culture broth. According to the present invention, because the bacteria belonging to Cellulomonas uda CB4 is capable of producing a cellulase having a high activity, not found in the reports of the prior art, in a culture medium, it is possible to produce a cellulase having a high crystalline cellulose decomposing activity comparable to those produced from a mold within a short cultivation period of two days.
In the paper titled “Expression of a cellulase gene in Zymomonas mobilis” by Misawa et al. (J. Biotechnology, 1988, 7 (3), 167-177), it was reported that a cellulase (CMCase) gene of Cellulomonas uda CB4 was introduced into Zymomonas mobilis NRRL B-14023 on pZA22, a cloning vector for Zymomonas, by conjugal transfer. Z. mobilis carrying this gene synthesized cellulase immunologically identical with that of C. uda CB4, by transcriptional read-through from the promotor of the chloramphenicol resistance (chloramphenicol acetyltransferase) gene within pZA22. A strong promotor containing a translation initiation signal was obtained from Z. mobilis NRRL B-14023 chromosomal DNA. By gene fusion between this Zymomonas promotor fragment and the truncated cellulase gene, the activity of cellulase synthesized in Z. mobilis reached 0.78 units per ml culture, six-fold higher than that by transcriptional read-through from the promotor of the chloramphenicol resistance gene.
In the paper titled “Expression of an endoglucanase gene of Pseudomonas fluorescens var. cellulosa in Zymomonas mobilis” by Lejeune et al. (FEMS Microbiology Letters, 1988, 49 (3), 363-366), the authors reported that by using a broad host-range, mobilizable plasmid vector, the endoglucanase gene eglX from Pseudomonas fluorescens var. cellulosa was cloned and expressed in the bacterial ethanologen Zymomonas mobilis. The enzyme was intracellular in this new host. It was produced throughout the growth phase and was not repressed by glucose. We postulate that transcription of the eglXgene was initiated from a promoter of the vector in Z. mobilis.
In the paper titled “Heterologous Expression and Extracellular Secretion of Cellulolytic Enzymes by Zymomonas mobilis” by Linger et al. (Appl Environ Microbiol. 2010 October; 76(19): 6360-6369), the authors reported that by using a technique known as consolidated bioprocessing (CBP), the initial steps toward achieving a single organism to convert pretreated lignocellulosic biomass to ethanol in the fermentation host Zymomonas mobilis were investigated. This was achieved by expressing heterologous cellulases and subsequently examining the potential to secrete these cellulases extracellularly. Numerous strains of Z. mobilis were found to possess endogenous extracellular activities against carboxymethyl cellulose, suggesting that this microorganism may harbor a favorable environment for the production of additional cellulolytic enzymes. The heterologous expression of two cellulolytic enzymes, E1 and GH12 from Acidothermus cellulolyticus, was examined. Both proteins were successfully expressed as soluble, active enzymes in Z. mobilis although to different levels. While the E1 enzyme was less abundantly expressed, the GH12 enzyme comprised as much as 4.6% of the total cell protein. Additionally, fusing predicted secretion signals native to Z. mobilis to the N termini of E1 and GH12 was found to direct the extracellular secretion of significant levels of active E1 and GH12 enzymes. The subcellular localization of the intracellular pools of cellulases revealed that a significant portion of both the E1 and GH12 secretion constructs resided in the periplasmic space. The results obtained strongly suggest that Z. mobilis can support the expression and secretion of high levels of cellulases relevant to biofuel production, thereby serving as a foundation for developing Z. mobilis into a CBP platform organism.
In the paper titled “Evaluation of Cellulase Production by Zymomonas mobilis” by Todhanakasem and Jittjang (Bioresources, 2017, 12(1), 1165-1178), the authors report the use of Z. mobilis as a potential microbe for consolidated bioprocessing to convert lignocellulosic biomass to fermentable sugars while at the same time producing ethanol. To achieve this goal, Z. mobilis must be evaluated for the production of cellulolytic enzyme. This work reports on the potential of intracellular and extracellular crude extracts from Z. mobilis ZM4 and TISTR 551 to hydrolyze various cellulosic materials including carboxymethylcellulose (CMC), delignified rice bran, microcrystalline cellulose, and filter paper. Crude intracellular extracts from ZM4 and TISTR 551 showed high endoglucanase activity with CMC substrates at an optimal pH of 6 to 7 and temperature range of 30 to 40° C. The endoglucanase activity from the crude extracts was significantly higher than the exoglucanase activity. Of the high crystalline celluloses substrates tested, the best results were obtained for the hydrolysis of delignified rice bran by crude intracellular enzyme extracts of Z. mobilis TISTR 551.
In the paper by Vasan et al. titled “Cellulosic ethanol production by Zymomonas mobilis harboring an endoglucanase gene from Enterobacter cloacae” (Bioresource Technol. 2011, 102(3), 2585), the authors report the use of a 2.25-kb fragment conferring cellulase activity from Enterobacter cloacae (isolated from the gut of the wood feeding termite, Heterotermes indicola), cloned in Escherichia coli. The cloned fragment contained a 1083-bp ORF which could encode a protein belonging to glycosyl hydrolase family 8. The cellulase gene was introduced into Zymomonas mobilis strain Microbial Type Culture Collection centre (MTCC) on a plasmid and 0.134 filter paper activity unit (FPU)/ml units of cellulase activity was observed with the recombinant bacterium. Using carboxymethyl cellulose and 4% NaOH pretreated bagasse as substrates, the recombinant strain produced 5.5% and 4% (v/v) ethanol respectively, which was threefold higher than the amount obtained with the original E. cloacae isolate. The recombinant Z. mobilis strain could be improved further by simultaneous expression of cellulase cocktails before utilizing it for industrial level ethanol production.
In light of the above, it is clear that there is an unmet need for a process to generate alcohol and alcohol precursors from a lignocellulosic biomass material that is not reliant on enzymatic cocktails due to their cost. In that respect, the use of a single organism that contains cellulolytic as well as fermentation capabilities is much more highly attractive as it will potentially lower the costs of bioethanol production.
The embodiments of the present invention are related to novel organisms for the production of alcohols and alcohol precursors using cellulose-based materials. More particularly, the invention encompasses methods for the genetic modifications of an ethanologenic organism which allows the organism to produce alcohols and alcohol precursors in association with cellulose degradation through the introduction of genes isolated from prokaryotic and/or eukaryotic organisms. These genes associated with cellulose degradation are then integrated or introduced into another prokaryotic organism. Through the inclusion of cellulose degrading genes into an organism that natively does not contain them, this invention extends metabolic pathways beyond those present natively in the prokaryotic organism itself for the purpose of producing ethanol.
According to a first aspect of the present invention, there is provided polynucleotide sequences and their corresponding polypeptide sequences from microorganisms that allow for the expression of cellulose hydrolytic proteins in bacteria for the purpose of producing an alcohol or alcohol precursor. Gene and promoter sequences were introduced into the prokaryotic cell using techniques such as two-step allelic exchange or using expression vectors. Natural promoters and ribosomal binding sites (RBS) from the bacterial expression strain were attached upstream of the gene's start codon to induce gene expression within the bacterium. Methods to transfer these genes from cloning vectors into the organisms of interest are also briefly outlined and are modelled after the protocol seen for the knock in or knock out of genes in a different bacterium, Pseudomonas aeruginosa, using the aforementioned two-step allelic exchange, however these defined methods are not meant to limit the scope of the invention and thus CRISPR and other undefined cloning methods can also be used.
According to a preferred embodiment of the present invention, there is provided a genetically modified ethanologenic organism which comprises a β-glucosidase 1 (bgl1) polynucleotide sequence, wherein said bgl1 polynucleotide sequence has at least 70% sequence coverage to SEQ 3 or SEQ 14, and at least 70% sequence identity to SEQ 3 or SEQ 14.
According to a preferred embodiment of the present invention, there is provided a genetically modified ethanologenic organism which comprises a β-glucosidase 1 (bgl1) polynucleotide sequence, wherein said bgl1 polynucleotide sequence is selected from the group consisting of: SEQ 3; SEQ 14; SEQ 15; SEQ 16; SEQ 17; SEQ 18; SEQ 19; SEQ 20; and SEQ 21.
According to a preferred embodiment of the present invention, SEQ 3 or SEQ 14, upon transcription and translation, provides a β-glucosidase 1 (BGL1) polypeptide sequence SEQ 4. According to a preferred embodiment of the present invention, there is provided a genetically modified ethanologenic organism which comprises a BGL1 polypeptide sequence which has at least 70% sequence coverage to SEQ 4, and at least 35% sequence identity to SEQ 4.
According to a preferred embodiment of the present invention, there is provided a genetically modified ethanologenic organism which comprises a β-glucosidase 1 (BGL1) polypeptide sequence wherein said BGL1 polypeptide sequence is selected from the group consisting of: SEQ 4; SEQ 22; SEQ 23; SEQ 24; SEQ 25; SEQ 26; SEQ 27; SEQ 28; SEQ 29; SEQ 30; SEQ 31; SEQ 32; SEQ 33; SEQ 34; SEQ 35; SEQ 36; SEQ 37; SEQ 38; SEQ 39; and SEQ 40.
According to a preferred embodiment of the present invention, there is provided a genetically modified ethanologenic organism which comprises an exoglucanase (cex-like) polynucleotide sequence, wherein said cex-like polynucleotide sequence has at least 70% sequence coverage to SEQ 1 or SEQ 68, and at least 70% sequence identity to SEQ 1 or SEQ 68.
According to a preferred embodiment of the present invention, there is provided a genetically modified ethanologenic organism which comprises an exoglucanase (cex-like) polynucleotide sequence, wherein said cex-like polynucleotide sequence is selected from the group consisting of: SEQ 1, SEQ 41, SEQ 42, SEQ 43, SEQ 44, SEQ 45, SEQ 46, SEQ 47, SEQ 48, and SEQ 68.
According to a preferred embodiment of the present invention, SEQ 1 or SEQ 68, upon transcription and translation, provides an exoglucanase (CEX-like) polypeptide sequence. According to a preferred embodiment of the present invention, there is provided a genetically modified ethanologenic organism which comprises a CEX-like polypeptide sequence which has at least 70% sequence coverage to SEQ 2, and at least 35% sequence identity to SEQ 2.
According to a preferred embodiment of the present invention, there is provided a genetically modified ethanologenic organism which comprises an exoglucanase (CEX-like) polypeptide sequence, wherein said CEX-like polypeptide sequence is selected from the group consisting of: SEQ 2; SEQ 49; SEQ 50; SEQ 51; SEQ 52; SEQ 53; SEQ 54; SEQ 55; SEQ 56; SEQ 57; SEQ 58; SEQ 59; SEQ 60; SEQ 61; SEQ 62; SEQ 63; SEQ 64; SEQ 65; SEQ 66; and SEQ 67.
According to a preferred embodiment of the present invention, there is provided a genetically modified ethanologenic organism which comprises:
Preferably, said organism comprises:
According to a preferred embodiment of the present invention, there is provided a genetically modified ethanologenic organism which upon transcription and translation under the control of the native of a native or synthetic promoter and Ribosomal binding site (RBS), comprises:
Preferably, said organism comprises:
Preferably, said organism further comprises a promoter and RBS sequence which drives gene expression, wherein the genetic material for the promoter/RBS sequences comprises at least one or another of the following: SEQ 5; SEQ 6; SEQ 7; SEQ 8; SEQ 9; SEQ 10; SEQ 11; SEQ 12; and SEQ 13. Preferably, the organism is a bacterium. More preferably, the organism is selected from the group consisting of the genera Aspergillus, Mucor, Zymomonas, Escherichia, Clostridia, Bacillus, and Pseudomonas. According to a preferred embodiment of the present invention, the organism is a prokaryotic organism. Preferably, the organism belongs to the bacterial genus Zymomonas.
According to a preferred method of the present invention, the ethanologenic organism is selected from the genus of Aspergillus, Mucor, Zymomonas, Escherichia, Clostridia, Bacillus, and Pseudomonas, however this list by no means is meant to limit the scope of the invention. Preferably, the ethanologenic organism is the bacteria Zymomonas mobilis.
According to another aspect of the present invention, ethanologenic bacterial cells were genetically modified by inserting a bacterial promoter sequence into the bacterial genome, immediately followed by a cellulose hydrolytic gene(s)-related to the degradation of cellulose. In some embodiments, the native promoter and polynucleotide sequence could also include the promoter and ribosomal binding site (RBS) including various combinations from the following genes: Glyceraldehyde-3-phosphate dehydrogenase, type I (gap) (Pgap), Thiamine pyrophosphate protein TPP binding domain-containing protein (pdc) (Ppdc), the EF-Tu transcription factor (Ptuf), Phosphopyruvate hydratase (eno) (Peno), 2-Dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase (eda) (Peda), glucose-6-phosphate dehydrogenase (zwf) (Pzwf), ROK family protein (frk) (Pfrk), Carboxymethylenebutenolidase (clcD1) (PclcD1), as well as Glucosamine-fructose-6-phosphate aminotransferase (glmS) (PglmS), however this list of possible promoters is in no way meant to limit the scope of the invention. In preferred embodiments, the promoter and RBS polynucleotide sequence to regulate cellulose degradation for the production of an alcohol or alcohol precursor is the native promoter for the Glyceraldehyde-3-phosphate, dehydrogenase, type 1 (gap) gene (Pgap), obtained from Z. mobilis.
According to a preferred embodiment of the present invention, cex-like genes can be acquired from, but by no means is limited to, the following prokaryotic species: Cellulomonas uda, Cellulomonas palmilytica, Cellulomonas cellasea, Saccharothrix syringae, Promicromonospora iranensis, Glycomyces paridis, Micromonospora fulviviridis, Couchioplanes caeruleus, Streptomonospora alba, Phytoactinopolyspora halotolerans, Cellulomonas wangsupingiae, Xylanimonas cellulosilytica, Actinoplanes lutulentus, or Micromonospora saelicesensis. These listed organisms are exemplary only and are in no way meant to limit what organisms these genes can be acquired from, nor to limit the scope of the invention.
According to a preferred embodiment of the present invention, bgl1 genes can be acquired from, but by no means is limited to, the following eukaryotic species: Aspergillus niger, Aspergillus luchuensis, Penicillium vulpinum, Sanghuangporus baumii, Penicillium rolfsii, Trichoderma gamsii, Halenospora varia, Daldinia childiae, Fusarium solani, Glaciozyma antarctica, Monascus purpureus, and Trichophyton interdigitale. These listed organisms are exemplary only and are in no way meant to limit what organisms these genes can be acquired from, nor to limit the scope of the invention.
According to a preferred embodiment of the present invention, the cex-like and bgl1 genes are obtained from the organism Cellulomonas uda (cex-like) and Aspergillus niger (bgl1), respectively. In other embodiments, a native promoter sequence can be used to bolster the production of proteins and/or molecules utilized in the production of an alcohol or alcohol precursor through the hydrolysis of cellulose.
According to another embodiment of the present invention, the ethanologenic bacterial cells have been genetically modified to have improved degradation of cellulose through the addition of both prokaryotic and eukaryotic gene(s) which when transcribed and translated generate cellulose hydrolytic enzymes. In some embodiments, the transcription of these genes is driven by a promoter sequence, and can include, but is not limited to, any combination of Pgap-cex-like or Pgap-bg11 gene cassettes which have been integrated into a bacterial cell.
The invention may be more completely understood in consideration of the following description of various embodiments of the invention in connection with the accompanying FIGURE, in which:
It is known to the person skilled in the art that some species of ethanologenic bacteria contain and express endogenous endoglucanase and endogenous exoglucanase enzymes with functional cellulase activities. According to an aspect of the present invention, there is provided genetic modifications to ethanologenic bacterial strains to enhance such innate capabilities and thereby increase the efficiency of cellulose degradation by said bacteria.
According to a preferred embodiment of the present invention, there are provided genetic modifications to ethanologenic bacterial strains which allow for the degradation of cellulose with the purpose of producing an alcohol or alcohol precursor from an inexpensive cellulosic feedstock using a bacterial species modified with a cex-like polynucleotide sequence (such as SEQ 1 or SEQ 68) or, upon transcription and translation of said cex-like polynucleotide, a CEX-like polypeptide (such as SEQ 2).
According to a preferred embodiment of the present invention, there are provided genetic modifications to ethanologenic bacterial strains which allow for the degradation of cellulose with the purpose of producing an alcohol or alcohol precursor from an inexpensive cellulosic feedstock using bacterial species modified with a bgl1 polynucleotide sequence (such as SEQ 3 or SEQ 14) or, upon transcription and translation of said bgl1 polynucleotide, a BGL1 polypeptide (such as SEQ 4).
According to an aspect of the present invention, there is provided genetic modifications to ethanologenic bacterial strains which includes a combination of cex-like and bgl1 polynucleotide sequences allowing for increased degradation of cellulose with the intent to produce an alcohol or alcohol precursor from inexpensive feedstock as a source of cellulose.
Within the context of this invention all terms and technical parameters described fall within their commonly known meanings as known by individuals within the field of science that the proposed invention is associated with unless otherwise stated. Furthermore, unless otherwise indicated, all techniques utilized within the context of this invention are commonly conducted within the fields of molecular biology, cell biology, biochemistry, and microbiology.
An alcohol or alcohol precursor is any molecule that is obtained as an intermediate, by-product, or co-product of the conversion of a cellulosic material to an alcohol. It is known to the person skilled in the art that said alcohol precursor can be an oligosaccharide, di-saccharide, monosaccharide, organic acid and their corresponding salts, aldehyde, ketone, etc.
Biofuels within the context of this invention is defined as any fuel for which is produced using biological material.
Bioethanol within the context of this invention is defined as ethanol that is produced from lignocellulosic biomass.
Cellulosic ethanol is defined within the context of this invention as ethanol that is produced through the degradation of cellulose.
Cellobiose is defined within the context of this invention as the dimer of glucose formed from the degradation of cellulose.
Cellulose hydrolysis within the context of this invention is defined as the degradation of cellulose via hydrolytic processes.
Hydrolysis within the context of this invention is defined as a chemical or microbiological reaction which facilities the breaking of chemical bonds between molecules.
A monomer is defined within this invention as a single molecule that can, under specific conditions, be combined with other molecules, including itself, to generate more complex structures called polymers.
A polymer is defined within the context of this invention as a material comprised of repeating subunits of the same or different molecule.
A biopolymer is defined within the context of this invention as a polymer that is typically produced by the cells of living organisms. By way of example, cellulose is considered a biopolymer.
A polynucleotide within the context of this invention is defined as the collection of individual nucleotides in any organization or size.
A polypeptide within the context of this invention is defined as the combination of multiple peptides of any organization or size that relates to the amino acid sequence. The term polypeptide and protein within the context of this invention can be used interchangeably.
A vector within the context of this invention refers to the composition of a polynucleotide within the intended purpose of introducing nucleic acids into one or more organism types. Vectors are further defined based on their functional purpose and can be designated as expression vectors, cloning vectors, plasmids, or shuttle vectors.
The term expression within the context of this invention refers to the generation of a polypeptide sequence which is produced based on its polynucleotide sequence or gene.
The term promoter in the context of this invention is used to describe the nucleic acid sequence for the regulation and binding of polymerases for the purpose of the transcribing of a gene. This promoter can be native to an organism, or a non-endogenous promoter can be introduced into an organism to alter the regulation of gene expression.
The term gene refers to a sequence of DNA that encodes for a specific polypeptide sequence. A gene can include both sequences between coding regions (introns) and the encoding sequence itself (exon).
The term recombinant in the context of this invention refers to the modification or alteration of a sequence associated with either a polypeptide or polynucleotide sequence. Recombination can be utilized for altering expression and coding segments of a gene of interest that would produce a non-native or non-naturally occurring product.
The term homology or homologous refers to the level of similarity between two or more polypeptide or polynucleotide sequences.
The terms transfection, transformation, or introduced refer to the addition of polynucleotide sequence(s) that would normally be considered exogenous to organism. This may include the addition of a polynucleotide directly to the genome of an organism or the transfer of a plasmid and/or vector.
Within the context of this patent the terms native or natural refers to polypeptide and/or polynucleotides present within the organism prior to any modification. These native or naturally occurring polypeptides and/or polynucleotides would be present or produced by the organism without any external alterations.
The term metabolic pathway refers to the subsequential biochemical reactions involved in the formation of a biologically relevant product within an organism.
Within this invention the terms knock-in and knock-out refer to the addition or removal of DNA sequences within an organism and can also be interchangeable with the terms insertion and deletion, respectively.
The term promoter within the context of this invention refers to a sequence of DNA that is responsible for the initiation of DNA transcription and thus polypeptide formation.
A coding sequence within the context of this invention refers to a sequence of polynucleotides or DNA that facilitates the generation of a protein through transcription and translational processes.
Genetic modification or related statements herein refer to the alteration of the genetic code of an organism which includes the insertion or deletion of DNA sequences within an organism. Within the context of this invention, genetic modification could include insertion and maintenance of an expression vector into the organism, or the direct modification of the organism's genome by directly adding or deleting genes through a process like two-step allelic exchange or CRISPR.
The term ribosomal binding site (RBS) refers to the region within a polynucleotide sequence that allows for the appropriate binding of the ribosome to facilitate the translation of a polynucleotide sequence to produce the corresponding polypeptide sequence, which includes the terms protein, enzyme, and plasmid.
The term cloning vector herein refers to a polynucleotide sequence or plasmid that can be replicated within a host organism for storage or amplification purposes. A cloning vector may contain all the necessary regulatory sequences needed to facilitate the transcription and translation of a protein.
The term unmodified promoter is defined as a promoter sequence which has been unaltered or exists within the host organism itself.
The term two-step allelic exchange is referred to the process by which a gene of interest is either interested or deleted from an organism through specific selective conditions. The insertion or deletion of a specific gene of interest is done so through the utilization of distinct polynucleotide sequences which allow for the exchange of genetic material between two sources.
The term CRISPR cloning is defined as a process by which the gene of interest is inserted or removed from an organism's genome using the CRISPR-CAS9 cloning system.
The terms upstream and downstream refer to regions of polynucleotides which are found prior to or after a specific gene of interest within a plasmid and/or genome of an organism.
The term enzyme within this invention defines a polypeptide sequence, specifically in the form of a protein, that can modify a biological molecule or take part within its generation through direct or indirect interactions. The process by which an enzyme influences the modification and/or production of a biological molecule and/or product is termed enzymatic activity.
The term open reading frame (ORF) refers to the collection of nucleotides which are found in between the start and stop codons of a polypeptide encoding DNA sequence.
The term codon(s) refers to 3 adjacent nucleotides in a polynucleotide sequence that are used by the cell to “decode” the polynucleotide sequence when the polynucleotide sequence is translated to make the polypeptide sequence and are responsible defining the order of protein residues in a polypeptide sequence based on this code. Based on a 3-letter code, and 4 different nucleotide bases, these codons include 64 different combinations that are able to be used by the cell, which with some redundancy codes for 22 possible protein residues, as well as 1 start and 3 stop codons.
A start and stop codon refer to a nucleotide codon sequence comprised of three specific nucleotides in succession of each other which allows for the identification of the initiation (start) and termination (stop) for the transcription of a polypeptide sequence.
The term encoded refers to the polypeptide sequence that is obtained from a polynucleotide sequence after the polynucleotide sequence had been transcribed and translated.
The combined term transcribed and translated (or the combined process of transcription and translation) refers to the process by which a polynucleotide sequence is used to produce a polypeptide sequence.
Metabolic engineering herein refers to the alteration of an organism's metabolic pathway potential. This can include both the deactivation and/or altering of pre-existing metabolic pathways of an organism or the inclusion of additional metabolic processes.
Sequence alignment herein refers to a bioinformatic technique by which two polynucleotide sequences or two polypeptide sequences are arranged or aligned in such a way as to identify regions of similarity between a reference sequence (the sequence that is known) and the quarry sequence (the sequence to be compared to the reference sequence). Those skilled in the art know that alignment algorithms such as, but by no means limited to, the BLAST, ALIGN, or CLUSTAL algorithms can be used to obtain this information for polynucleotide or polypeptide sequences, respectively.
Percentage sequence identity or percentage identity herein refers to the similarity between 2 sequences that have been processed through a sequence alignment, to provide insight into how similar aligned sequences are at either the nucleotide or peptide level for polynucleotide or polypeptide sequences, respectively. The percentage identity is used to determine the similarity of a query sequence to a reference sequence.
Percentage sequence coverage or percentage coverage refers to the number of aligned nucleotides or peptides in a query sequence relative to the length of the reference sequence. The percentage coverage provides an indication of how much of the reference polynucleotide or polypeptide sequence is covered by the query sequence, allowing for instance the lengths of the found genes or proteins to be compared. The BLASTN algorithm was used herein as one method to determine the percentage identity and percentage coverage between one or even multiple different polynucleotide sequences with respect to an inputted reference sequence, allowing for the determination of the percentage identity and percentage coverage of one or many query sequences to said reference sequence. One of ordinary skill in the art will recognize that search results from a BLASTN search will be influenced by the search parameters used in the search. Therefore, for all BLASTN searches done with respect to this invention to identify other sequences which have been catalogued in the NCBI polynucleotide databases relative to a reference include the following parameters:
The BLASTP algorithm was used herein as one method to determine the percentage identity and percentage coverage between one or even multiple different polypeptide sequences with respect to an inputted reference sequence, allowing for the determination of the percentage identity and percentage coverage of one or many query sequences to said reference sequence. One of ordinary skill in the art will recognize that search results from a BLASTP search will be influenced by the search parameters used in the search. Therefore, for all BLASTP searches done with respect to this invention to identify other sequences which have been catalogued in the NCBI polypeptide databases relative to a reference include the following parameters:
The phrases substantially similar or substantially identical in the context of at least 2 nucleic acid sequences or at least 2 polypeptide sequences typically means that a polynucleotide, polypeptide, or region or domain of a polypeptide has a percentage coverage of at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or even 99.5%, and at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% percentage identity to the reference sequence. Some polynucleotide or polypeptide sequences that fall in this category are sequences that share genetic or protein homology to the reference sequence.
The terms genetic and protein homology, or homologous sequences refer to polynucleotide sequences or translated polypeptide sequences that have a similar or identical function in the cell. For example, 2 different proteins share a similar or identical function even though they were isolated from 2 different organisms. Polynucleotide sequences with homology are generally understood to have similar or identical biochemical functionality.
The term endoglucanase refers to a protein with a certain enzymatic activity to degrade cellulose by attacking random internal 1,4-β-linkages between the glucose units in cellulose.
The term exoglucanase refers to a protein with a certain enzymatic activity to degrade cellulose by releasing cellobiose or glucose from the nonreducing end of the cellulose polymer.
The term glucosidase refers to a protein with enzymatic activity to degrade cellobiose to glucose.
The term ethanologenic organism refers within this invention to an organism that can produce ethanol as part of its native metabolic processes.
Example polypeptide sequences for enzymes involved in cellulose degradation that can be integrated into prokaryotic organisms are provided in the sequence listing. The expression and production of these sequences within the cell are partially driven by the genetic polynucleotide promoter and ribosomal binding site sequences as provided in the sequence listings: SEQ 5 (Pgap), SEQ 6 (Ppdc), SEQ 7 (Ptuf), SEQ 8 (Penc), SEQ 9 (Peda), SEQ 10 (Pzwf), SEQ 11 (Pfrk), SEQ 12 (PclcD1), and SEQ 13 (Pglms). The invention is not limited to the use of these polynucleotide sequences and their respective polypeptide sequences. Those of ordinary skill in the art know that organisms of a wide variety of species commonly express and utilize homologous proteins, which contain insertions, substitutions and deletions in the polypeptide sequences of the polynucleotide sequences listed above, and effectively provide a similar function. For example, the protein sequences for CEX-like from Cellulomonas uda or Couchioplanes caeruleus or Promicromonospora iranensis; or BGL1 from Aspergillus niger or Penicillium vulpinum or Halenospora varia may differ to different degrees from the polypeptide sequences seen between these organisms yet maintain similar or identical functions of the protein within the organism with respect to enzymatic function. Protein sequences comprising such variations are included within the scope of the present invention and are considered substantially or sufficiently similar to the referenced polynucleotide sequences above and upon transcription and translation their respective polypeptide sequences. Although it is not intended that the present invention is limited by any theory by which it achieves its advantageous result, it is believed and supported by biochemical knowledge that the identity between polypeptide sequences that is necessary to maintain proper functionality is related to maintaining the tertiary structure (3D) of the polypeptide. This maintenance of the tertiary structure is associated with the specific interactive/catalytic portions of the protein sequence and will therefore have the desired activity, and it is contemplated that a protein including these interactive sequences in the proper special context will have this activity.
The person of ordinary skill in the art knows that many different amino acids contain properties between each other and can serve similar functions in the final polypeptide sequence. Thus, when one amino acid is changed with another amino acid from this group, such as a non-polar amino acid, an uncharged polar amino acid, a charged polar acidic amino acid, or a charged polar basic amino acid, some polypeptide functionality is generally maintained. For example, it is known that the uncharged polar amino acid serine may be substituted for the uncharged polar amino acid threonine in a polypeptide without substantially altering the protein structure and functionality. Whether a given substitution will affect the functionality of the enzyme may be determined without undue experimentation using synthetic techniques and screening assays known to one of ordinary skill in the art.
The person of ordinary skill in the art will recognize that changes in the protein sequence, resulting from individual single or multi-nucleotide substitutions, deletions, or additions to a polynucleotide will lead to changes in the resulting translated polypeptide sequence. Small mutations, such as the change of an amino acid from one to another, or the addition or elimination of single amino acids, or a small to moderate percentage of amino acids from the encoded polypeptide sequence can be considered “sufficiently similar” when the alteration results in the substitutions of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues in a polypeptide chain, selected from a group of integers from 1-50, can be so altered. Thus, for example, 1, 2, 3, 5, 10, 12, 20, 32, 41, or even 50 alterations can be made. Conservatively modified variants typically provide similar biological activity to the unmodified variants and typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, modification of CEX-like and BGL1 to yield functional proteins generally have a sequence identity of at least 40%, 50%, 60%, 70%, 80%, or 90%, preferably a sequence identity of greater than 50%, of the native protein to allow processing of its native substrate. Tables of conserved substitution provide lists of functionally similar amino acids. Amino acids in polypeptide chains that are similar to one another include the following groups: (1) Serine (S), Threonine (T); (2) Aspartic acid (D), Glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) Alanine (A), Leucine (L), and Isoleucine (I).
The person of ordinary skill in the art will recognize that many different organisms will have functionally similar polynucleotide and polypeptide sequences (or homology between the sequences), however there may be differences between these sequences when compared to a reference sequence. As examples, suitable polynucleotides and their corresponding polypeptide sequences for cellulose degradation can be seen below. Note that the following sequences by no means are meant to limit the scope of the invention. In fact, any substantially similar polynucleotide sequences or substantially similar produced polypeptide sequences for the CEX-like and BGL1 genes/proteins with similar function or similarity to these genes/proteins in the cellulose degradation pathway can also be used for producing alcohol and alcohol precursors including cellulosic ethanol production.
According to a preferred embodiment of the present invention, the β3-glucosidase 1 (bgl1) polynucleotide sequence SEQ 3 or SEQ 14 is one β-glucosidase utilized for producing alcohol and alcohol precursors including cellulosic ethanol production. In this embodiment, bgl1 is under the transcriptional control of a native promoter and a ribosomal binding site. However, in other embodiments of the invention, bgl1 polynucleotide sequences that are homologous and/or substantially similar to SEQ 3 or SEQ 14 may also be used in the present invention to produce cellulosic ethanol. The bgl1 polynucleotide sequences for β-glucosidase 1 in these embodiments will have at least 70% sequence coverage, or more preferably greater than 75%, 85%, 90%, 95%, 97% or most preferentially greater than 99% sequence coverage to SEQ 3 or SEQ 14, and sequence identities of at least 70%, or more preferentially greater than 75%, 80%, 90%, or 95% sequence identity, and most preferentially greater than 99% sequence identity to SEQ 3 or SEQ 14. The bgl1 polynucleotide sequences may include, but by no means are limited to, the following sequences: SEQ 3; SEQ 14; SEQ 15; SEQ 16; SEQ 17; SEQ 18; SEQ 19; SEQ 20; and SEQ 21.
According to a preferred embodiment of the present invention, the β3-glucosidase 1 (BGL1) polypeptide SEQ 4 is utilized to produce producing alcohol and alcohol precursors including cellulosic ethanol by the cell, whereby SEQ 4 is produced from the transcription and translation of the β3-glucosidase 1 (bgl1) polynucleotide SEQ 3 or SEQ 14. However, in other embodiments of the invention, BGL1 polypeptide sequences that are homologous and substantially similar to SEQ 4 may also be used in the present invention to produce cellulosic ethanol. BGL1 polypeptide sequences in these embodiments will have at least 70% sequence coverage to SEQ 4, or more preferentially greater than 75%, 80%, 85%, 90%, 95%, 97%, 98%, or most preferentially greater than 99% sequence coverage to SEQ 4, and a sequence identity of at least 35% to SEQ 4, or more preferably greater than 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity, or most preferentially greater than 99% sequence identity to SEQ 4. These polypeptide sequences may include, but are by no means limited to, the following sequences: SEQ 4; SEQ 22; SEQ 23; SEQ 24; SEQ 25; SEQ 26; SEQ 27; SEQ 28; SEQ 29; SEQ 30; SEQ 31; SEQ 32; SEQ 33; SEQ 34; SEQ 35; SEQ 36; SEQ 37; SEQ 38; SEQ 39; and SEQ 40.
According to a preferred embodiment of the present invention, the 1,4-beta-cellobiohdryolase (cex-like) polynucleotide sequence SEQ 1 or SEQ 68 is one exoglucanase utilized for producing alcohols and alcohol precursors including cellulosic ethanol production. In this embodiment, cex-like is under the transcriptional control of a native promoter and a ribosomal binding site. However, in other embodiments of the invention, cex-like polynucleotide sequences that are homologous and/or substantially similar to SEQ 1 or SEQ 68 may also be used in the present invention to produce cellulosic ethanol. The cex-like polynucleotide sequences for 1,4-beta-cellobiohydrolase in these embodiments will have at least 70% sequence coverage, or more preferably greater than 75%, 80%, 85%, 90%, 95%, 98% or most preferentially greater than 99% sequence coverage to SEQ 1 or SEQ 68, and sequence identities of at least 70%, or more preferentially greater than 75%, 80%, 90%, or 95% sequence identity, and most preferentially greater than 99% sequence identity to SEQ 1 or SEQ 68. These cex-like polynucleotide sequences may include, but by no means are limited to, the following sequences: SEQ 1; SEQ 41; SEQ 42; SEQ 43; SEQ 44; SEQ 45; SEQ 46; SEQ 47; SEQ 48; and SEQ 68.
According to a preferred embodiment of the present invention, the exoglucanase (CEX-like) polypeptide SEQ 2 is utilized to produce producing alcohols and/or alcohol precursors including cellulosic ethanol by the cell, whereby SEQ 2 is produced from the transcription and translation of the exoglucanase (cex-like) polynucleotide SEQ 1 or SEQ 68. However, in other embodiments of the invention, CEX-like polypeptide sequences that are homologous and substantially similar to SEQ 2 may also be used in the present invention to produce cellulosic ethanol. The CEX-like polypeptide sequences for exoglucanase in these embodiments will have at least 70% sequence coverage to SEQ 2, or more preferentially greater than 75%, 80%, 85%, 90%, 95%, 97%, 98%, or most preferentially greater than 99% sequence coverage to SEQ 2, and a sequence identity of at least 35% to SEQ 2, or more preferably greater than 40%, 45%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% sequence identity, or most preferentially greater than 99% sequence identity to SEQ 2. These CEX-like polypeptide sequences may include, but are by no means limited to, the following sequences: SEQ 2; SEQ 49; SEQ 50; SEQ 51; SEQ 52; SEQ 53; SEQ 54; SEQ 55; SEQ 56; SEQ 57; SEQ 58; SEQ 59; SEQ 60; SEQ 61; SEQ 62; SEQ 63; SEQ 64; SEQ 65; SEQ 66; and SEQ 67.
Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
mobilis)
mobilis)
mobilis)
mobilis)
mobilis)
mobilis)
mobilis)
mobilis)
Zymomonas mobilis codon optimized polynucleotide
carbonacea)
iranensis)
halotolerans)
Zymomonas mobilis codon optimized polynucleotide
| Number | Date | Country | Kind |
|---|---|---|---|
| 3208555 | Aug 2023 | CA | national |
