Patent Application
20240287549
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII file, created on Jun. 23, 2022, is named M065670505WO00-SEQ-KVC.TXT and is 9,174 bytes in size.
Provided herein are methods and compositions related to efficiently producing biofuels from toxic hydrolyzed biomass feedstocks with the use of modified yeast cells.
The increased use of renewable transportation fuels such as bioethanol is one of the most widely accepted strategies to combat global climate change. However, the toxicity of ethanol and other alcohols to the industrial production organism, Saccharomyces cerevisiae, is a primary factor limiting greater output. The high cell density (“pitch”) and very high sugar (“gravity”) conditions of large-scale fermentation produce preternaturally high concentrations of ethanol that lead to significant losses in cell viability and productivity. Ethanol tolerance is a complex phenotype with an elusive biological basis; genetic analysis has shown that no single modification is capable of eliciting greater resistance.
Meaningful displacement of greenhouse gas emissions from continued oil consumption requires a renewable feedstock transformable into products fungible with petrofuels and petrochemicals and, importantly, is deployable on a similar scale. Despite the declining cost of carbon-free electricity and rise of emission-free vehicles, studies estimate that this segment will comprise at most 31% of the global fleet by 2040 due to non-road modes of carriage and long average ownership in the established internal combustion fleet (1). As the transportation sector remains the largest generator of carbon dioxide, the sheer number of legacy vehicles necessitates that liquid biofuels play a dominant role in any future energy mix in order to minimize net emissions (2). Lignocellulosic biomass, the largest renewable terrestrial resource, provides a realistic intermediate-term route to sustainable fuel and non-fuel commodities at significant scale when paired with suitable fermentation infrastructure (3). In addition to quantities on the magnitude of fossil carbon, lignocellulose addresses issues such as food-fuel competition and arable land use that beset present-generation feedstocks like corn (4). Fermented fuel products, notably ethanol, can be blended directly into the gasoline supply at 15-85% or chemically dehydrated to ethylene and upgraded into jet fuel (5). However, the severe pretreatments needed to deconstruct the highly recalcitrant plant fibers into fermentable sugars typically result in feedstocks toxic to microorganisms (6-8). Partly saddled by such technical challenges, the U.S. cellulosic ethanol industry has dwindled sharply (a single pre-production plant run by POET-DSM remains) and pretreatment research has refocused on conversions that yield clarified, biocatalyst-friendly substrates (9, 10). Even then, the greater complexities required by these processes have generally increased costs (estimates as high as 30¢/gallon ethanol) as well as eroded scalability and competitiveness (11). Engineering elevated microbial tolerance to the inhibitors released in simpler, but more aggressive, hydrolytic methods would, therefore, address one of the major obstacles impeding greater utilization of cellulosic feedstocks (12-15).
Ethanol toxicity in yeast S. cerevisiae limits the production of biofuels globally, yet its biological underpinnings remain enigmatic. Surprisingly, the present disclosure shows that yeast engineered for enhanced aldehyde reduction together with elevated extracellular potassium and pH are sufficient to enable ethanol production from inhibitor-laden feedstocks produced from biomass. By specifically targeting the universal hydrolysate inhibitors, yeast strains are enhanced to tolerate a broad diversity of highly toxic feedstocks and consistently achieve industrial-scale titers of ethanol.
One aspect of the present disclosure provides a genetically modified yeast cell (modified cell) comprising a first exogenous gene operably linked to a promoter, wherein the first exogenous gene encodes an enzyme having methylglyoxal reductase (GRE2) activity. In some embodiments, the enzyme having GRE2 activity is derived from Saccharomyces cerevisiae. In some embodiments, the enzyme having GRE2 activity comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the enzyme having GRE2 activity comprises a sequence set forth in SEQ ID NO: 1. In some embodiments, the enzyme having GRE2 activity comprises at least one substitution mutation at a position corresponding to position P48, 1290, and/or D133, relative to SEQ ID NO: 1. In some embodiments, the substitution mutation at the position corresponding to position P48 of SEQ ID NO: 1 is a mutation to serine. In some embodiments, the substitution mutation at the position corresponding to position 1290 of SEQ ID NO: 1 is a mutation to valine. In some embodiments, the substitution mutation at the position corresponding to position D133 of SEQ ID NO: 1 is a silent mutation that retains aspartate. In some embodiments, the promoter is selected from the group consisting of pTDH3, pTEF3, and pPDC1.
In some embodiments, the yeast cell is of the genus Saccharomyces. In some embodiments, the yeast cell is of the species Saccharomyces cerevisiae. In some embodiments, the yeast cell is modified to ferment xylose to ethanol in the absence of glucose.
In some embodiments, the modified cell further comprises a second exogenous gene, wherein the second exogenous gene encodes an enzyme having D-lactate dehydrogenase (D-LDH) activity. In some embodiments, the enzyme having D-LDH activity is derived from Leuconostoc mesenteroides. In some embodiments, the enzyme having D-LDH activity comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the enzyme having D-LDH activity comprises a sequence set forth in SEQ ID NO: 2.
Another aspect of the present disclosure provides a method of producing biofuel from toxic biomass or feedstocks produced from biomass comprising contacting the modified cell described herein with a medium comprising a potassium salt and a pH modulator. In some embodiments, the biofuel is ethanol. In some embodiments, the potassium salt is selected from potassium phosphate monobasic (KH2PO4), potassium bicarbonate (KHCO3), potassium phosphate dibasic (K2HPO4), potassium chloride (KCl), potassium hydroxide (KOH), and potassium sulfate (K2SO4). In some embodiments, the potassium salt is K2HPO4. In some embodiments, the concentration of potassium salt in the medium is between about 15 mM to about 200 mM. In some embodiments, the concentration of potassium salt in the medium is about 50 mM. In some embodiments, the pH modulator is selected from potassium hydroxide (KOH), potassium phosphate dibasic (K2HPO4), and calcium carbonate (CaCO3). In some embodiments, the pH modulator is CaCO3. In some embodiments, the CaCO3 is in an amount sufficient to maintain, in culture medium, a pH of at least 3.5.
The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.
The accompanying drawings are not intended to be drawn to scale. In the drawings. each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
Lignocellulosic biomass remains unharnessed for the production of renewable fuels and chemicals due to challenges in deconstruction and the toxicity its hydrolysates pose to fermentation microorganisms. The present disclosure demonstrates in Saccharomyces cerevisiae that engineered aldehyde reduction combined with elevated extracellular potassium and pH are sufficient to enable near-parity production between inhibitor-laden and inhibitor-free feedstocks. By specifically targeting the universal hydrolysate inhibitors, a single strain is enhanced to tolerate a broad diversity of highly toxic genuine feedstocks and consistently achieve industrial-scale titers (cellulosic ethanol >100 g/L). Furthermore, a functionally orthogonal, lightweight design enables seamless transferability to existing metabolically engineered chassis strains: full, multi-feedstock tolerance is endowed on a xylose-consuming strain as well as one producing the biodegradable plastics precursor lactic acid. The demonstration of “drop-in” hydrolysate competence enables the potential of cost-effective, at-scale biomass utilization for cellulosic fuel and non-fuel products alike.
The present disclosure demonstrates that a targeted combination of genetic and feedstock modifications is sufficient to enhance a single strain to tolerate a wide variety of highly toxified biomass hydrolysates and deliver cellulosic ethanol with performance comparable to current clean sugar ethanol. This rationally-designed approach is, additionally, highly modular: with introduction of a single gene and no further engineering, feedstock-agnostic hydrolysate tolerance is conferred on or transferred to previously engineered metabolic chassis strains (including one synthesizing a biodegradable plastic) to enable novel cellulosic products beyond ethanol.
The results provided in the present disclosure describe a functionally independent, lightweight platform that both endows yeast with general lignocellulosic hydrolysate tolerance and integrates harmoniously with preexisting metabolically engineered chassis strains. Through systematic characterization of the three dominant and universal toxicities released from biomass pretreatment, the present disclosure demonstrates that tolerance to each inhibitor can be realized through standard acid neutralization (for acetic acid) or conversion of the aldehydes to alcohols which are subsequently ameliorated by elevated K+ and pH treatment (for furfural and HMF). The general practice of hydrolysate tolerance can, therefore, be reduced to two specific and readily modifiable parameters: in a genetic background enhanced by GRE2evol for the accelerated reduction of furfural and HMF, a large diversity of feedstocks-regardless of plant source and/or pretreatment process—can be accommodated via tailored adjustment of K+ and pH. That such a diversity of substrates can be accommodated indicates that the complex spectrum of hydrolytic byproducts other than furfural, HMF, and acetic acid (for example, the various acidic and phenolic inhibitors shown in Table 3) may be qualitatively immaterial. These benefits, collectively, renew and boost the value proposition of cellulosic fermentation. Wide feedstock compatibility can reduce the dependence on specific crop types or pretreatments and, consequently, ameliorate the supply variability (e.g., from seasonality, storage stability) and cost uncertainties surrounding biomass (13). Similarly, heightened tolerance, in addition to harnessing toxic sugar streams or transport-friendlier concentrates, enables production conditions of minimized contaminant growth that would otherwise require the standard-practice, but public health-concerning, use of antibiotics (55). Finally, the targeted specificity of this detoxification approach underlies the high decoupling with native metabolism and straightforward integration with such engineered pathways as those for xylose consumption and lactic acid synthesis. This underscores the notion of a “drop-in” tolerance phenotype extensible to even more non-native capabilities and high-volume biofuels and biochemicals.
Thus, provided herein are furfural and hydroxymethyl-furfural (HMF) tolerant yeast cells that are engineered to express a reductase with elevated detoxifying activity toward furfural and HMF. An “engineered” yeast cell refers to a yeast cell that is modified to contain a recombinant or synthetic nucleic acid. As used herein, a “furfural and HMF tolerant yeast cell” refers to an engineered yeast cell with increased viability relative to an unmodified cell (e.g., wild-type “WT” cell) when cultured in the presence of furfural and HMF. It should be understood that, in some instances, the furfural and HMF tolerance (e.g., viability) of a yeast cell may depend on a combination of factors such as, for example, the furfural and HMF concentration and the fermentable sugar concentration in which the yeast cell is cultured.
In some embodiments, the fermentable sugar concentration of the cell culture medium is about 50 g/L to about 400 g/L (e.g., of culture medium). For example, in some embodiments, the fermentable sugar concentration of the cell culture medium is about 50 g/L, about 100 g/L, about 150 g/L, about 200 g/L, about 250 g/L, about 300 g/L, about 350 g/L or about 400 g/L. In some embodiments, the fermentable sugar concentration is more than 400 g/L.
Any yeast capable of fermentation may be used herein. Examples of yeast strains for use in accordance with the present disclosure include, without limitation, the following: Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., Yarrowia spp. and industrial polyploid yeast strains. In some embodiments, the yeast strain is a Saccharomyces cerevisiae (S. cerevisiae) strain. In some embodiments, the yeast strain is an industrial yeast strain (S. cerevisiae strain) used in bioethanol production. An “industrial” yeast strain, as used here, refers to a yeast strain used in the commercial production of alcohol (e.g., ethanol). In some embodiments, an industrial yeast strain is a polyploid strain that has been selected over time for alcohol (e.g., ethanol) productivity and tolerance to alcohol, temperature and/or sugar. For example, in some embodiments, the yeast strain is a sake yeast strain (e.g., strains of Saccharomyces cerevisiae such as NCYC 479/Kyokai no. 7), PE-2 or JAY270 (Argueso JL et al. Genome Res. 19(12), 2258-70 (2009), incorporated by reference herein) or Ethanol Red® (Lesaffre Yeast Corporation). Other examples of industrial yeast strains include NCYC 73, NCYC 177, NCYC 431, NCYC 478, NCYC 975 and NCYC 1236.
The furfural and HMF tolerant yeast cells provided herein may be engineered to comprise a modified potassium transport gene encoding a polypeptide (e.g., protein) that increases cellular influx of potassium relative to an unmodified yeast cell and a modified proton transport gene encoding a polypeptide that increases the cellular efflux of protons relative to an unmodified yeast cell. “Cellular influx” of potassium refers to a process by which potassium ions are transported across a cell membrane into the intracellular compartments of a cell. “Cellular efflux” of protons refers to a process by which protons are transported across a cell membrane out of a cell into extracellular space.
An “unmodified yeast cell,” as used herein, refers to a yeast cell that is not engineered such as, for example, a wild-type (WT) yeast cell.
The furfural and HMF tolerant yeast cells provided herein may be engineered to comprise a modified NADH- or NADPH-dependent reductase gene encoding a polypeptide (e.g., protein) that decreases the toxicity of furfural and HMF.
A “NADH- or NADPH-dependent reductase gene,” as used herein, refers to a gene encoding a polypeptide that functions in the process of irreversibly reducing cytotoxic aldehydes using NADH or NADPH as an electron donor. For example, GRE2 encodes an NADPH-dependent methylglyoxal reductase that can reduce and detoxify furfural and HMF. In some embodiments, the modified cell is engineered to express a wild-type or mutant GRE2 gene.
In some embodiments, the GRE2 enzyme is a GRE2 derived from Saccharomyces cerevisiae. The Saccharomyces cerevisiae GRE2 corresponds to UniprotKB Accession No. Q12068 and is provided by SEQ ID NO: 1:
A “lactate dehydrogenase gene,” as used herein, refers to a gene encoding a polypeptide that functions to convert lactate to pyruvate and back. It also facilitate the reversible reaction of NAD+ to NADH and back. A dehydrogenase is an enzyme that transfers a hydride from one molecule to another.
In some embodiments, the lactate dehydrogenase enzyme is a D-LDH derived from Leuconostoc mesenteroides. The Leuconostoc mesenteroides D-LDH corresponds to UniprotKB Accession No. Q2ABS1 and is provided by SEQ ID NO: 2:
In some embodiments, the modified gene (e.g., GRE2 or D-LDH) is operably linked to a promoter. A “promoter,” as used herein, refers to a transcription regulatory sequence (nucleic acid sequence) that initiates transcription of an RNA molecule from the DNA downstream of it. In some embodiments, the promoter operably linked to GRE2 or D-LDH is a TDH3 promoter (pTDH3), TEF3 promoter (pTEF3), or a PDC1 promoter (pPDC1).
A “modified” gene, as used herein, refers to a gene that is mutated, overexpressed or misexpressed. In some embodiments, the mutation is a deletion mutation, or a deletion. A “deletion mutation” refers to a region of a chromosome that is missing (i.e., loss of genetic material), which affects the function of a gene, or gene product (e.g., polypeptide encoded by the gene). Any number of nucleotides can be deleted. In some embodiments, a deletion mutation may render a gene, or gene product, non-functional. The symbol “Δ” denotes a deletion mutation. For example, engineered gre2Δ/gre2Δ yeast have deletion mutations in homologous alleles of GRE2. Methods of introducing genetic mutations in yeast are well-known, any of which may be used in accordance with the present disclosure (Sherman, F. in Encyclopedia of Molecular Biology and Molecular Medicine (Meyers, R. A.) 6, 302-325 (Wiley-Blackwell, 1998); Orr-Weaver, T. L., et al. Proc Natl Acad Sci USA 78, 6354-6358 (1981); Sikorski, R. S. & Hieter, P. Genetics 122, 19-27 (1989); and Wach, A., et al. Yeast 10, 1793-1808 (1994), each of which is incorporated by reference herein). A modified gene, or gene product, is herein considered to be “overexpressed” if the expression levels of the gene, or gene product, are increased relative to the expression levels of an unmodified (e.g., wild-type) gene, or gene product. A modified gene, or gene product, is herein considered to be “misexpressed” if the gene, or gene product, is expressed at a cellular location where or at a developmental time when it is not normally expressed. Methods of overexpression and misexpression in yeast are well-known, any of which may be used in accordance with the present disclosure (Mumberg, D., et al. Gene 156, 119-122 (1995); and Mumberg, D., et al. Nucleic Acids Res 22, 5767-5768 (1994), each of which is incorporated by reference herein).
Ethanol resistance is increased substantially and concomitantly with ethanol production under the high sugar (e.g., 300 g/L) and high cell density (e.g., OD600˜20-30) conditions that are typical of large-scale industrial fermentation. As used herein, “industrial fermentation” refers to the use of fermentation by yeast to produce useful products such as biofuel (e.g., ethanol, or bioethanol). A fermentation process (e.g., conversion of sugar to alcohol) is herein considered to be “large-scale” if the process includes culturing fermenting yeast cells (e.g., engineered yeast cells) in a volume of at least 5 liters (L) (e.g., of culture medium). In some embodiments, a large-scale industrial fermentation process may include culturing fermenting yeast cells in a volume of at least 10 L, at least 15 L, at least 20 L, at least 25 L, at least 50 L, at least 100 L, at least 500 L, at least 1,000 L, at least 5,000 L or at least 10,000 L. In some embodiments, a large-scale industrial fermentation process may include culturing fermenting yeast cells in a volume of at least 100,000 L, at least 500,000 L, or at least 1,000,000 L. The yeast cells may be cultured in, for example, shake flask cultures or bioreactors.
Industrial fermentation processes may also include culturing yeast in the presence of a high concentration of fermentable feedstock or fermentable sugar. “Fermentable feedstock” herein refers to feedstock that can be converted (e.g., by yeast) to sugar and then to alcohol or other engineered end-products. Non-limiting examples of a fermentable feedstock include lignocellulosic biomass (e.g., (corn stover, sugarcane bagasse, straw), composed of carbohydrate polymers (e.g., cellulose, hemicellulose) and an aromatic polymer (e.g., lignin) A “fermentable sugar” herein refers to a sugar that can be converted (e.g., by yeast) to alcohol or other engineered end-products. Examples of fermentable sugars for use in accordance with the present disclosure include, without limitation, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, tagatose, arabinose, lyxose, ribose, xylose, ribulose and xylulose. Sources of fermentable sugars include, without limitation, feedstock such as corn, wheat, sorghum, potato, sugarbeet, sugarcane, potato-processing residues, sugarbeet, cane molasses and apple pomace. Fermentable sugars can be produced directly or derived from polysaccharides such as cellulose and starch. In some embodiment, the fermentable sugar is from (e.g., derived from) a lignocellulosic substance. Thus, in some embodiments, the fermentable sugar is a hexose such as glucose. In some embodiments, the fermentable sugar is from xylan hemicellulose. Xylose can be recovered by acid or enzymatic hydrolysis. Thus, in some embodiments, the fermentable sugar is a pentose such as xylose. Enzymatic hydrolysis using mixtures of enzymes, such as cellulase and hemicellulases, may be used herein to minimize the destruction of sugars associated with higher severity pretreatments (e.g., concentrated acid hydrolysis) of lignocellulosic material. These enzymes, when combined with effective pretreatment of lignocellulosics, provide high yields of glucose, xylose, and other fermentable sugars with minimal sugar losses.
High concentrations of fermentable sugars include concentrations that are about 100 g/L to about 400 g/L. Thus, in some embodiments, the yeast (e.g., engineered yeast) is cultured in medium having a fermentable sugar concentration of at least 100 g/L. In some embodiments, the yeast is cultured in medium having a fermentable sugar concentration of about 100 g/L to about 400 g/L. For example, in some embodiments, the yeast is cultured in medium having a fermentable sugar concentration of 100 g/L, 150 g/L, 200 g/L, 250 g/L, 300 g/L. 350 g/L or 400 g/L.
Industrial fermentation processes may also include culturing yeast at a high cell density. Thus, in some embodiments, the yeast (e.g., engineered yeast) is cultured at a cell density of about 1×106 to about 1×109 viable cells/ml. For example, in some embodiments, the yeast is cultured at a cell density of about 1×106, about 2×106, about 3×106, about 4×106, about 5×106, about 6×106, about 7×106, about 8×106, about 9×106, about 1×107, about 2×107, about 3×107, about 4×107, about 5×107, about 6×107, about 7×107, about 8×107, about 9×107, about 1×108, about 2×108, about 3×108, about 4×108, about 5×108, about 6×108, about 7×108, about 8×108, about 9×108 or about 1×109 viable cells/ml.
In some embodiments, the yeast (e.g., engineered yeast) is cultured at an optical cell density, measured at a wavelength of 600 nm, of about 1 to about 150 (i.e., OD600 is about 1 to about 150). For example, in some embodiments, the OD600 of a cell culture containing fermenting yeast cells is about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150. In some embodiments, the OD600 of a cell culture containing fermenting yeast cells is about 20 to about 30.
In accordance with the present disclosure, the yeast (e.g., engineered yeast) may be cultured in standard laboratory synthetic complete medium with nutrient drop-out for selection when appropriate (Sherman, F. Meth Enzymol 350, 3-41 (2002), incorporated by reference herein). For example, yeast synthetic complete (YSC) medium may contain a nitrogen base without amino acids and ammonium sulfate (e.g., BD-Difco Yeast Nitrogen Base catalog #233520) with or without nutrients. In some embodiments, the culture medium is adjusted for K+, H+ and/or Na+ concentration.
The present disclosure also provides methods of ethanol production that comprise culturing yeast cells in culture medium that comprises fermentable feedstock and a potassium salt selected from potassium phosphate monobasic (KH2PO4 or K-Pi), potassium phosphate dibasic (K2HPO4), potassium bicarbonate (KHCO3), and potassium sulfate (K2SO4).
The potassium salt may be present in the culture medium in an amount sufficient to produce at least 100 g/L, or at least 150 g/L ethanol. In some embodiments, the potassium salt is in an amount sufficient to produce about 100 g/L to about 300 g/L of ethanol. For example, in some embodiments, the potassium salt is in an amount sufficient to produce about 100 g/L, about 150 g/L, about 200 g/L, about 250 g/L or about 300 g/L.
In some embodiments, the culture medium further comprises potassium hydroxide (KOH), which is present in an amount sufficient to maintain, in the culture medium, a pH of at least 3. Thus, in some embodiments, KOH may be used to adjust the pH of culture medium comprising a potassium salt such as, for example, KCl. In some embodiments, KOH is used to adjust the pH of the culture medium to about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5 or about 8. In some embodiments, the pH of culture medium (e.g., containing KCl) is adjusted or maintained at a pH within a range of 3 to 8 or about 3 to about 8 (e.g., a pH of 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8).
In some embodiments, the culture medium further comprises calcium carbonate (CaCO3), which is present in an amount sufficient to maintain, in the culture medium, a pH of at least 3. Thus, in some embodiments, CaCO3 may be used to adjust the pH of culture medium comprising a potassium salt such as, for example, KHCO3. In some embodiments, CaCO3 is used to adjust the pH of the culture medium to about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5 or about 8. In some embodiments, the pH of culture medium (e.g., containing KHCO3) is adjusted or maintained at a pH within a range of 3 to 8 or about 3 to about 8 (e.g., a pH of 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8).
The concentration of potassium salt in the culture medium may be about 15 mM to about 200 mM. For example, in some embodiments, the concentration of potassium salt in the culture medium is about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 150 mM, or about 200 mM. In some embodiments, the concentration of potassium salt in the culture medium is about 25 to about 50 mM, about 35 to about 65 mM, or about 50 mM to about 75 mM.
In order that the invention described in the present disclosure may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the systems and methods provided in the present disclosure and are not to be construed in any way as limiting in their scope.
Prior research conducted by the inventors of the present disclosure has demonstrated that in yeast Saccharomyces cerevisiae, responsible for the current global output of biofuel ethanol, increases in media potassium (K+) and pH were sufficient to strengthen membrane potential and enhance production universally across laboratory and industrial strains (16). Therefore, the impact of these extracellular adjustments on the fermentation of toxic lignocellulosic feedstocks was investigated. Unrefined hydrolysates of all cellulosic biomass, particularly those pretreated under acidic conditions, contain a spectrum of inhibitory byproducts but are dominated by the furan aldehydes furfural and 5-hydroxymethyl-furfural/HMF (from dehydration of pentoses and hexoses, respectively), and acetic acid (from deacetylation of hemicellulose) (17-23).
To systematically characterize the impact of these constituent toxicities, ethanol production was assessed in yeast synthetic complete medium (YSC) with the 3 inhibitors added individually at equimolar concentration. As a completely chemically-defined formulation with trace vitamins, minerals, and amino acids, YSC lacks the undefined extracts contained in “rich” media that could supply unknown components to boost or hinder tolerance. A diploid prototroph of laboratory strain S288C was used to preclude nutrient liabilities, and fermentations conducted for 48 h at 30° C. under high pitch (cell density of OD600=20) and high gravity (250 g/L glucose) conditions to mimic industrial production (24). Compared to an ethanol titer of 64±0.2 g/L in unmodified YSC, the presence of 100 mM acetic acid (Ac), furfural (FF), or HMF (HMF) reduced production respectively to 17±0.1, 4±0.3, and 34±0.3 g/L, establishing furfural as the most deleterious component (
Elevated K+ and pH also conferred improvements to furfural and HMF, but to titers substantially below that of equivalently-elevated YSC. Based on prior work that delineated a mechanism of multi-alcohol tolerance, these improvements, however small, were unlikely to have arisen from tolerance elicited to the 4±0.3 or 34±0.3 g/L of ethanol produced under inhibition (16). Rather, given reports that S. cerevisiae naturally possesses various dehydrogenases sufficiently promiscuous to reduce furfural and HMF, it was surmised that these extracellular adjustments were conferring tolerance to their furan alcohol products (27-30). Indeed, 2-furanmethanol (FF—OH) and furan-2,5-dimethanol (HMF-OH) were detected in significant amounts (p≤3.35×105), along with the disappearance of furfural and HMF, after just 24 h of fermentation in an unmodified strain (
Based on literature describing reductases with detoxifying activity toward furfural and HMF, yeast strains over-expressing ADH6, ADH7, or GRE2 from S. cerevisiae, or ADH4 from Scheffersomyces stipitis were constructed (29-33). Fermentation benchmarking over two repressive conditions combining the trio of inhibitors revealed that S. cerevisiae GRE2 evoked the greatest improvement among the candidates. When compared to the wildtype (WT), these improvements amounted to as much as 32% (
To characterize productivity under combined furfural and HMF stress, as well as the impact from modulating reductase activity, the relationship between tolerance and ethanol production for the WT, the GRE2 over-expression strain, and one deleted for GRE2 was quantified. Prior work has demonstrated that the viable fraction in an actively fermenting yeast population declined rapidly with accumulating ethanol due to the toxicity of the ethanol itself. However, such mortality could be rescued by K+ and pH adjustments in a dose-dependent manner. Furthermore, time integrations of these viable population fractions (“integrated viable cell density”) from progressively higher adjustments, and correlation with ethanol titers, established the relationship between tolerance and production. Importantly, a readout of the time-averaged specific productivity exclusive to the differentially decaying viable fractions was revealed in the correlation slope (16). Therefore, titers and viabilities were measured from a series of fermentations containing furfural, HMF, and incrementally higher adjustments of K+ and/or pH (
Given the efficacy exhibited by the GRE2 over-expression strain, various adaptation approaches were considered to further improve furfural and HMF hydrolysate tolerance. While whole-strain laboratory adaptive evolution is well practiced for augmenting fitness, selective advantages from genome-wide drift have been shown to incur costs in robustness (34, 35). To minimize the risk of pleiotropic deficits undermining feedstock range and strain performance, the detoxification capabilities of GRE2 specifically were honed via directed evolution. Therefore, a yeast library consisting of plasmid-borne PCR-mutagenized GRE2 variants was cultured under combined furfural, HMF, and acetic acid stress, and challenged to increasing toxicity loads over approximately 1 month (36) (
The allele exhibiting the greatest gain was a triple mutant containing a proline to serine substitution at amino acid 48, isoleucine to valine at amino acid 290, and a silent aspartate mutation at amino acid 133 (GRE2P48S+1290V+D133D; hereafter as GRE2evol). Across several toxicity combinations mimicking a range of pretreatment severities, GRE2evol consistently conferred improvements over unevolved GRE2 in ethanol production (
As rates of detoxification are directly proportional to cell biomass (
Expression of GRE2evol was, moreover, capable of conferring near-parity ethanol production between inhibitor-free and fully toxified conditions. To first establish a reference upper bound for inhibitor-free productivity and titer, the WT was fermented in traditional (i.e., non-toxic) high glucose synthetic medium supplemented with potassium bicarbonate (KHCO3) and calcium carbonate (CaCO3)-selected for their widespread industrial and agricultural availability—to provide elevated K+ and pH buffering. Under these optimal conditions, ethanol reached 109±1 g/L in under 32 h (
The targeted detoxification provided by the combination of GRE2evol expression and feedstock K+/pH adjustments was applicable beyond laboratory media to a wide range of genuine lignocellulosic feedstocks. Influenced partly by their history of toxicity and limited utility, the hydrolysates currently available are produced largely at research scale and focused on maximally detoxified cellulosic sugars (6-8, 10, 38, 39). A collection of 7 samples were procured representing a diversity of plant sources (corn stover, sugar cane bagasse, wheat straw, giant miscanthus, and switchgrass) and various pretreatment methods, and inhibitors were confirmed to be present in the ranges of 0.1-21 mM furfural, 0.1-6 mM HMF, and 0-178 mM acetic acid (Table 1). These quantities were indeed sufficiently mild such that, aside from the standard supplementation with urea (to provide nitrogen) and adjustment to pH 5, the WT was capable of fermenting all glucose to completion under otherwise unmodified conditions (
Triticum
aestivum L.
Miscanthus ×
giganteus
P. virgatum
Since inhibitor and glucose loads inherent in the majority of samples fell below that of the formulated benchmark, all samples were toxified to impinge on engineered tolerance limits (Table 2). Furfural was raised to an average 45 mM, HMF to 35 mM, and acetic acid to 100 mM to more closely align with published inhibitory combinations, and glucose to an average 220 g/L for increased osmotic, and ensuing ethanol, stress (17-23). Importantly, supplementation with pure chemical forms of these components was necessary as pre-hydrolyzed solids (and hydrolysis methods) were unavailable to us to boost toxicity using raw cellulosic material. Under these conditions, the WT became repressed even with adjustments to pH 5 (
giganteus hydrolysate; courtesy of Stefan Bauer (LBNL).
Since genuine hydrolysates are highly undefined mixtures with unknown levels of salts, uninformed additions of K+ and particular buffer counter-cations could potentially exceed osmotic shock thresholds in yeast (43-45). As it had been previously established that K+ has fermentation-beneficial effects to at least 110 mM and Ca2+ is fermentation-neutral, it was deemed important to maintain total K+ within this limit and modulate pH via Ca2+-based buffers or bases (16). Thus, cation concentrations in each sample were determined by mass spectrometry and used to determine the specific mixes of KHCO3, CaCO3, and calcium hydroxide to provide as supplementation (Table 1 and Table 2). The atypically high K+ concentration in wheat straw, in fact, provided an opportunity to validate these constraints: when 50 mM KHCO3 and 100 mM CaCO3 were used in lieu of 150 mM CaCO3, the oversupply of K+ resulted in decreases to performance despite the same pH buffering capacity (
These tolerance capabilities were fully transferrable to the fermentation of inhibitor-laden xylose, the pentose comprising a significant portion of lignocellulosic sugars that unmodified S. cerevisiae cannot consume (6, 17). The inventors of the present disclosure had previously engineered a strain (XYL+) that efficiently fermented xylose to ethanol, but preferentially utilized glucose if present (46). Therefore, a YSC-based hydrolysate was formulated to favor xylose metabolism, yet mimic genuine cellulosic proportions, by combining xylose with starch whose glucan polymers were slowly digested to glucose via incrementally-dosed amylases. In the absence of inhibitors, the XYL+ chassis completely consumed available xylose and glucose in this medium and produced 62±0.9 g/L of ethanol (
Moreover, the GRE2evol-enhanced XYL+ strain maintained its tolerance capabilities on multiple toxified genuine hydrolysates. Due to catabolite repression, xylose went unconsumed by the XYL+ chassis strain in minimally modified hydrolysates of sugarcane bagasse and high acid corn stover (
The recapitulation of phenotype and substrate robustness on a pre-existing metabolic chassis suggested that GRE2evol expression and K+/pH feedstock adjustment could constitute a functionally orthogonal tolerance platform integrable with other engineered end products. Given that the alcohol-focused countermeasures may have conferred a bias toward ethanol production, an effort was made to endow hydrolysate tolerance on a non-alcohol product, namely, a glucose-consuming strain synthesizing the biodegradable plastics precursor lactic acid (D-LA), a chemical commodity projected to reach US$9 billion by 2025 (47). Given the evolutionary predilection of S. cerevisiae for alcoholic fermentation, elimination of ethanol has been a major goal in all efforts to reengineer yeast for non-ethanol products. Here, the inventors of the present disclosure opted to minimize, rather than eliminate, glycolytic flux toward ethanol in order to maintain subsistence ATP generation for cell growth and active lactate export (48). As lactate and ethanol share a common precursor in pyruvate, pyruvate decarboxylase activity and losses to ethanol were curtailed by creating a pdc1Δ/pdc1Δ pdc5Δ/pdc5Δ:: pTEF1m4-PDC5 strain where a sole chromosomal pyruvate decarboxylase gene was transcribed using a handicapped variant of the TEF1 promoter (49-51). Reductive conversion of pyruvate to lactate was fulfilled via expression of D-lactate dehydrogenase from L. mesenteroides (52).
In synthetic medium supplemented with KHCO3 and sufficient CaCO3 to buffer lactic acid accumulation, and likewise in DMR corn stover minimally supplemented with CaCO3, the D-LA+ chassis generated inhibitor-free reference titers of 66±0.3 and 65±0.4 g/L, respectively (
All plasmids were assembled using the Gibson method from segments generated via PCR. Amplification of plasmid backbone, yeast promoter, protein coding, and transcription termination fragments (see below) was carried out using the Phusion High-Fidelity DNA polymerase (New England Biolabs #M0530, neb.com) in 50 μL reactions following the manufacturer's directions. Primers were designed with 25-30 bp 5′ overhangs to serve as assembly junctions and annealing temperatures of primer pairs optimized using the vendor-provided calculator (tmcalculator.neb.com). The suggested extension times of 30 s/kb were often inadequate empirically and extended to 60 s/kb for problematic amplicons (fragments >2-3 kb tended to require the higher extension rate). For templates containing a bacterial origin of replication (e.g., plasmid backbone segment), PCR products were further digested with 20 U of DpnI (New England Biolabs #R0176) added directly to the reaction sample post-thermocycling (i.e., no additional restriction enzyme buffer), incubated for 90 min at 37° C., and heat inactivated for 20 min at 80° C. All fragments were purified and concentrated (up to 3 pooled PCR reactions per column) using the QIAquick PCR Purification Kit (QIAGEN #28106, qiagen.com) and DNA concentrations quantified with a NanoDrop Microvolume UV-Vis spectrophotometer. Gibson reactions were prepared from these eluates using 50-100 ng of vector and molar ratios of 1 part vector to 4-8 parts of each insert in the lowest volume possible (i.e., no additional H2O to meet the instructed minimum of 10 μL). Assembly enzymes were supplied through a cocktail (New England Biolabs #E2611) and reactions incubated for 30 min at 50° C. followed by an additional 1 h to overnight at room temperature. Chemically competent E. coli (New England Biolabs #C2992) were transformed and cultured per manufacturer instructions, and ampicillin-resistant isolates screened by PCR using vector- and insert-specific primers. Plasmids derived from positively scoring transformants were extracted using the QIAprep Spin Miniprep Kit (QIAGEN #27106) and validated by Sanger sequencing (QuintaraBio, quintarabio.com).
Minimal backbone segments-containing solely the bacterial replication origin, ampicillin marker, yeast replication origin, and yeast selection marker-were sourced from the p415 and p426 expression series developed by Mumberg et al. (56). For strong transcription in yeast coupled to glycolytic activity, either the TDH3 (“GPD”) or TEF1 promoters were retained from the Mumberg vectors or the −703 to −1 fragment of the PDC1 promoter was cloned from FY4/5 genomic DNA. Protein coding sequences for ADH6, ADH7, and GRE2 were likewise amplified directly from FY4/5 genomic DNA. For ADH4 from S. stipitis, Bio Basic (biobasic.com) was contracted to synthesize a S. cerevisiae codon-optimized sequence from the publicly available protein translation (GenBank accession no. XM_001387085). Similarly, IdhA from L. mesenteroides subsp. mesenteroides was produced by Eurofins Genomics (eurofinsgenomics.com) from the amino acid sequence available from UniProt (gene entry LEUM_1756). For transcription termination, either the CYC1 element from Mumberg or from FY4/5 genomic DNA were retained and the 166 bp immediately following the stop codon of ADH1 or the 295 bp following ACTI was cloned.
To clone the GRE2 mutants emerging from toxicity selection, the plasmid-based coding sequences were distinguished from chromosomal GRE2 via an initial PCR using primers binding to library plasmid elements. Specifically, the collection of mutagenized genes, including their non-mutagenized promoters and terminators, were originally subcloned via Gateway recombination and positioned between attB1 and attB2 sequences (36). Thus, from total DNA isolated from the final inhibitor-tolerant culture (
S. cerevisiae ADH6
S. cerevisiae ADH7
S. cerevisiae GRE2
S. stipitis ADH4
S. cerevisiae
S. cerevisiae
Leuconostoc mesenteroides
Recombinant strains were created following the lithium acetate chemical transformation method of Gietz et al. (57). For single plasmid introduction, 50 ng of p(RS)415-based DNA was used with 1 OD600 unit of cells and selection carried out on solid YSC-Leu dropout medium. For strains generated from introduction of two simultaneous plasmids, 150-300 ng each of p426TEF- and p(RS)415-based DNA was used with 3-4 OD600 units, and selection done on YSC-Ura-Leu solid medium. For chromosomal integrations, 800 ng-1 μg of linear DNA was used with 7-10 OD600 units. Low transformation efficiencies (e.g., from variability in strain, locus, DNA secondary structure) were typically resolved by increasing the DNA to cell ratio, amount of salmon sperm carrier DNA, or heat shock incubation time (up to 40-50 min at 42° C.).
In addition to laboratory standard BY4743, the gre24:: kanMX4/gre2A:: kanMX4 diploid used for LAMy629 preexisted this study and was obtained from the Saccharomyces Genome Deletion Project collection (sequence.stanford.edu/group/yeast_deletion_project/).
To create the diploid xylose-consuming chassis (XYL+), a xylose-enabled MATa leu2-3 haploid (internal strain F258) available from the development efforts of Zhou et al., 2012 was transformed with plasmid pJH727 (GAL:: HO LEU2; gift from Jim Haber of Brandeis University) to generate a MATa equivalent (46, 58). Induction of the HO gene in Leu+ transformants was conducted in liquid medium containing 20 g/L galactose for 6 h at 30° C. (complete mating type switching protocol, including pre-induction, is available from the Haber Lab website: bio.brandeis.edu/haberlab/jehsite/protocol.html). Individual colonies, recovered from growth in glucose medium additionally containing leucine to discard pJH727, were screened for MATα haploids by α- and a-factor sensitive mating type tester strains (59). A validated MATa leu2-3 haploid was subsequently mated with F258 to create the homozygous XYL+ leu-chassis strain LAMy435 that preceded LAMy419 and 665.
To create the S288C leu-predecessor of LAMy660, 661, and 663, the defective his3Δ1/and ura3Δ0 alleles in BY4743 were corrected sequentially by targeted chromosomal replacements. In brief, a PCR product encompassing the full length coding sequence of HIS3 was amplified from FY4/5 genomic DNA, introduced into BY4743, and transformants selected for histidine prototrophy. To repair ura3Δ0, which spans a segment larger than the open reading frame of URA3, a PCR product including 320 bp of the URA3 promoter and 194 bp beyond the stop codon was amplified from FY4/5 genomic DNA, introduced into the His+ intermediate, and transformants selected on minimal yeast nitrogen base (YNB) medium supplemented solely with leucine to yield chassis strain LAMy651.
To create the diploid ethanol-handicapped chassis for lactic acid production, a fully ethanol-deficient pdc-haploid was first generated by creating a markerless deletion of PDC5 in a MATa pdc1Δ:: kanMX4 strain sourced from the Saccharomyces Genome Deletion Project collection (PDC6, while intact, is functionally inert). Briefly, plasmid PCRSPR,PDC1±5 expressing a Candida albicans/S. cerevisiae codon-optimized version of Cas9, and a guide RNA targeting the PAM-proximal sequence TGCTAAGAACCCAGTTATCT (SEQ ID NO: 3) common to PDC1 and PDC5, was co-electroporated with the double-stranded linear repair template:
into the MATa pdc1Δ:: kanMX4 haploid according to the protocol of Vyas et al. (plasmid and template are gifts of Boon Uranukul) (60). Transformants were selected on solid YP (yeast bacto-peptone) medium containing 3% ethanol, 3% glycerol, and 100 μg/mL nourscothricin (YPEG+NAT), and isolates comparatively grown on YPEG+NAT and YPD (YP with 2% glucose) solid media to identify the pdc-phenotype (development strain LAMy399).
Separately, a haploid containing PDC5 transcribed by the low strength TEF1m4 promoter mutant was generated in a MATα pdc1Δ:: kanMX4 strain sourced from the Saccharomyces Genome Deletion Project collection. Here, the in-locus markerless edit was likewise accomplished through a similar CRISPR protocol with plasmid LAMb66 (featuring uracil selection in yeast and improved Cas9 expression) encoding a guide RNA targeting sequence TTCTCGATCAATATACTGTA (SEQ ID NO: 5) in the PDC5 promoter and the double-stranded repair template:
where lowercase letters designate the TEF1m4 promoter mutations (50). Transformants were selected on YSC-Ura solid medium and small colonies suggesting handicapped glucose growth validated further by PCR using primer pairs identifying the TEF1m4 promoted-PDC5 fusion. This haploid was mated to LAMy399 to produce the PDC5 heterozygote, and the diploid subsequently made His+ via the chromosomal integration described above to yield the ethanol-handicapped chassis strain LAMy670 preceding LAMy690 and 692.
All final strains were re-validated by PCR using plasmid- or modification-specific primers and the relevant regions Sanger sequenced as appropriate before fermentation experiments. Table 5 shows a complete list of strains used in this study.
pPDC1-GRE2-tCYC1
pPDC1-GRE2(P48S + I290V + D133D)-
tCYC1
GRE2(P48S + I290V + D133D)-tCYC1
LmLDHA-tADH1 + pRS415
LmLDHA-tADH1 + p415-
pGPD-GRE2(P48S + I290V + D133D)-
tCYC1
To provide a consistent but modifiable medium to accommodate the entire collection of strains, all baseline culturing was performed in yeast synthetic complete medium (YSC) composed of 1.5 g/L Yeast Nitrogen Base without Amino Acids & Ammonium Sulfate (BD-Difco #233520, bd.com), 5 g/L ammonium sulfate, 0.2 mM inositol, 0.1 g/L of each of the 20 amino acids, and 0.1 g/L each of adenine and uracil (all from Sigma-Aldrich, sigmaaldrich.com). Strains containing a p(RS)415 plasmid were maintained in medium lacking leucine and those with a p426 plasmid lacking uracil. Unless indicated otherwise, individual strains were expanded and acclimated to high cell density and high sugar conditions in singlicate YSC-based cultures and divided into triplicate biological samples upon inoculation into fermentation (
For the constituent toxicity studies, prototrophic strain FY4/5 was expanded in a starter culture of YSC containing 150 g/L glucose and grown overnight to approximately OD600=10. To mimic industrial high cell density conditions, 100 OD600 units of cells per fermentation were harvested, washed with an equal volume of room temperature distilled and deionized water, and cell pellets resuspended in 4 mL media for a production cell density of approximately OD600=25 (9.9 g DCW/L). Fermentation media consisted of 250 g/L glucose in YSC and were supplemented with (left to right in
Inhibitor supplementation for the additionally screened conditions in
To screen the panel of over-expressed reductases on fermentation (
To prepare samples for mass spectrometric quantification of furfural, HMF, FF—OH, and HMF-OH (
To assess the impact of GRE2 and increasing K+ and pH conditions on cell viability under combined furan aldehyde stress (
To screen for a fermentation advantage conferred by over-expression of GRE2evol vs. GRE2 (
For assaying a fitness advantage conferred by GRE2evol vs. GRE2 over-expression (
For the fermentation time courses of
To assess performance in genuine biomass feedstocks (
For demonstrating extensibility of hydrolysate tolerance to xylose-consuming strains (
For demonstrating extensibility of hydrolysate tolerance to lactic acid-producing strains (
The PCR-mutagenized GRE2 yeast library was revived from the “functional variomics” collection (gift of Xuewen Pan of Baylor University) in YSC-Ura containing 30 g/L glucose and supplemental 10 mM KH2PO4 (36). To maintain the >2×105 diversity, 20 μL of thawed cells were expanded to saturation, diluted, and cultured overnight in fresh medium to approximately OD600=2.5. Cell biomass totaling 3.5 OD600 units was harvested and resuspended in 4 mL of YSC-Ura containing 30 g/L glucose, 32/25/52 mM furfural/HMF/acetic acid, supplemental 10 mM KH2PO4, and adjusted to pH 6 with NH4OH. Following the time course of
Several attempts were required to converge on the conditions used in the successful iteration depicted in
The coding sequence for the GRE2P48S+1290V+D133D allele is as follows:
Quantification of ethanol, lactic acid, glucose, xylose, glycerol, furfural, HMF, and acetic acid was performed on cell-free, 0.45 μm-filtered samples using an Agilent 1200 Infinity Series HPLC configured with G1362A Refractive Index Detector and Aminex HPX-87H carbohydrate analysis column (Bio-Rad #125-0140, bio-rad.com). Analytes were separated isocratically in 5 mM sulfuric acid at 65° C. using a flow rate of 0.6 m/min. Under these conditions, retention times were approximately as follows: glucose 9.2 min, xylose 9.9 min, lactic acid 13.1 min, glycerol 13.8 min, acetic acid 15.3 min, ethanol 22.3 min, HMF 30.2 min, and furfural 45.0 min. Chromatogram peaks auto-integrated by the Agilent OpenLab CDS ChemStation software were converted to concentrations through interpolation off standard curves calibrated over the ranges of 0-100 g/L glucose, 0-50 g/L xylose, 0-100 g/L lactic acid, 0-8 g/L glycerol, 0-60 g/L acetic acid, 0-150 g/L ethanol, 0-8 g/L HMF, and 0-8 g/L furfural defined from chemically-pure dilution series. To compensate for the minor overlap between the peaks for glucose and xylose, standards were employed incorporating the two sugars at a ratio of 3:1 g/L glucose:xylose to reflect typical proportions. Likewise, lactic acid and glycerol standards incorporated 10:1 g/L lactic acid:glycerol.
Given the likelihood of calcium salts precipitating from the low pH in the running solvent and obstructing instrument fluidic lines, all samples derived from calcium-containing fermentations were acidified with 1% sulfuric acid (vol/vol), rotated for ≥1 h at 4° C., precipitates removed via centrifugation, and supernatants 0.45 μm-filtered before HPLC analysis.
For targeted quantification of furfural, FF—OH, HMF, and HMF-OH, cell-free samples collected from fermentation were extracted 1:1 with 100% UHPLC-grade methanol containing seventeen 13C labeled amino acids (Cambridge Isotope Laboratories, isotope.com) added as internal standards, and stored at −80° C. for further processing. Amino acid-methanol extraction buffer, downstream method development, and analysis were provided by the Metabolite Profiling Core Facility at the Whitehead Institute (metabolomics.wi.mit.edu).
In brief, additional dilutions to 1:20 and 1:80 (final) in 50% methanol were required to reach the linear range of the spectrometer. Samples of 1 μL were injected into a Dionex UltiMate 3000 ultra-high performance liquid chromatography (UPLC) unit equipped with an Ascentis Express C18 (2.1×150 mm, 2.7 μm particle) column (Sigma-Aldrich #53825-U) maintained at 35°° C. Analytes were reverse phase separated at a flow rate of 0.25 mL/min using buffers A (0.1% formic acid in LCMS grade water) and B (0.1% formic acid in LCMS grade acetonitrile) under the following gradient conditions: 0-2 min (5% B); 2-20 min (5-75% B. linear gradient); 20.1-24 min (95% B); 24.1-28 min (5% B). Mass analysis was performed on a Thermo Scientific QExactive Orbitrap operating with a spray voltage of 3.0 kV, capillary temperature 275° C., HESI probe temperature 350° C., sheath gas flow 40 units, auxiliary gas flow 15 units, and sweep gas flow 1 unit. For targeted isolation of furfural, HMF, FF—OH, and HMF-OH, positive ionization mode was used with resolution set to 70,000, automatic gain control to 1×105 with maximum injection time of 250 ms, and isolation window to 4.0 m/z. Fragmentation patterns from MS/MS were matched with reference spectra available in the METLIN online database (metlin.scripps.edu). Quantification was performed using the Thermo Scientific XcaliburTM Software calibrated against chemically-pure dilution series of 3 μM-3 mM furfural, 30 μM-3 mM FF—OH, 0.1 μM-3 mM HMF, and 30 μM-3 mM HMF-OH.
For measurement of salt concentrations in hydrolysates, 10 mL of each sample was centrifuged (3,500×g, 5 min) to remove large particulates, and the supernatants submitted to Environmental Testing & Research Laboratories (ctrlabs.com) for quantification of K+ and Ca2+(available as components of their water testing suite). Acid-digested samples were assayed in three replicate reads by inductively coupled plasma mass spectrometry.
Yeast population viabilities measured via methylene blue staining and subsequent procedures to calculate correlation with ethanol titers described previously (16).
Calculation of standard deviation (SD), propagation of error, hypothesis testing (two-sample, two-tailed t-test, α=0.05), and p-value determination were performed using MATLAB (The Math Works, mathworks.com) on independent biological triplicates following standard procedures.
Kothandaraman, Techno-economic comparison of process technologies for biochemical ethanol production from corn stover. Fuel. 89, S20-S28 (2010).
19.
Y. H. Jung. I. J. Kim, H. K. Kim, K. H. Kim, Dilute acid pretreatment of lignocellulose for whole slurry ethanol fermentation. Bioresour Technol. 132, 109-114 (2013).
contacting the modified cell of any one of Embodiments 1-16 with a medium comprising a potassium salt and a pH modulator.
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 here. Such equivalents are intended to be encompassed by the following claims.
All references, including patent documents, are incorporated by reference in their entirety.
This application claims the benefit under 35 U.S.C. § 119(c) of U.S. Provisional Application No. 63/214,304, filed Jun. 24, 2021, entitled “METHODS AND COMPOSITIONS FOR EFFICIENT PRODUCTION OF BIOFUELS AND BIOPLASTICS FROM TOXIC FEEDSTOCKS,” the entire disclosure of which is hereby incorporated by reference in its entirety.
This invention was made with Government support under Grant No. DE-EE0007531 awarded by the Department of Energy. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/034720 | 6/23/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63214304 | Jun 2021 | US |
