Patent Application
20150299670
The carbohydrate component of lignocellulose represents a potential feedstock for renewable fuels and chemicals (1-3), an alternative to food crops and petroleum. However, the cost-effective use of lignocellulosic sugars in fermentation remains challenging (4, 5). Unlike starch, lignocellulose has been designed by nature to resist deconstruction (2, 6). Crystalline fibers of cellulose are encased in a covalently linked mesh of lignin and hemicellulose. Steam pretreatment with dilute mineral acids is an efficient approach to depolymerize hemicellulose (20-40% of biomass dry weight) into sugars (hemicellulose hydrolysate, primarily xylose) and to increase the access of cellulase enzymes (2, 3, 6). However, side reaction products (furfural, 5-hydroxymethylfurfural, formate, acetate, and soluble lignin products) are formed during pretreatment that hinder fermentation (7, 8). Furfural (dehydration product of pentose sugars) is widely regarded as one of the most important inhibitors (6-8). The concentration of furfural is correlated with the toxicity of dilute acid hydrolysates (9). Although overliming to pH 10 with Ca(OH)2 can be used to reduce the level of furfural and toxicity, inclusion of this step increases process complexity and costs (9, 10).
Escherichia coli and yeasts have proven to be excellent biocatalysts for metabolic engineering (11, 12). However, both are inhibited by furans (7, 8, 13-15) and both contain NADPH-dependent oxidoreductases that convert furfural and hydroxymethylfurfural (dehydration product of hexose sugars) into less toxic alcohols (15-17). It is this depletion of NADPH by oxidoreductases such as YqhD (low Km for NADPH) that has been proposed as the mechanism for growth inhibition in E. coli (
Four genetic traits have been identified that increase furfural tolerance in microorganisms, such as ethanol-producing Escherichia coli LY180 (strain W derivative). Increased expression of fucO, ucpA or pntAB, and deletion of yqhD were associated with the increase in furfural tolerance. As a proof of concept, plasmids and integrated strains were used to characterize epistatic interactions among traits and to identify the most effective combinations. Furfural resistance traits were subsequently integrated into the chromosome of LY180 to construct strain XW129 (LY180 ΔyqhD ackA::PyadC′fucO-ucpA) for ethanol production. This same combination of traits was also constructed in succinate biocatalysts (E. coli strain C derivatives) and found to increase furfural tolerance. Strains engineered for resistance to furfural were also more resistant to the mixture of inhibitors in hemicellulose hydrolysates, confirming the importance of furfural as an inhibitory component. With resistant biocatalysts, product yields (ethanol and succinate) from hemicellulose syrups were equal to control fermentations in laboratory media without inhibitors. The combinations of genetic traits disclosed in this application can be applied, generally, to other microorganisms, such as Gram negative and Gram positive bacterial cells, yeast and fungi) to increase furfural tolerance in microorganisms used to make industrially useful products.
(A) Promoter-probe plasmid pLOI4870 was used to isolate Sau3A1 fragments that serve as surrogate promoters for expression of fucO-ucpA. Two rounds of the growth-based screen were employed in AM1 medium containing furfural. (B) Isolation and identification of promoter fragment by sequencing pLOI5237 and pLOI5259. A putative promoter (boxed region) was predicted within this fragment using BPROM and Neural Network Promoter Prediction. (C) Growth of strains containing furfural-resistance plasmids expressing the fucO-ucpA cassette. Tube cultures (n=3) were grown for 48 h in AM1 medium containing 50 g/L xylose, 20 mg/L chloramphenicol and 12.5 or 15 mM furfural as previously described (2). (D) The NADH-linked furfural reductase activity in plasmid strains containing fucO-ucpA cassettes. (E) SDS-PAGE of cytoplasmic extracts from strains harboring fucO-ucpA cassettes. Arrows indicates the predicted size of FucO (MW 40.5 kDa; thick band) and UcpA (MW 27.8 kDa; not easily seen).
SEQ ID NO: 1: promoter sequence derived from E. coli.
SEQ ID NO: 2: E. coli DNA fragment containing promoter sequence (SEQ ID NO: 1).
SEQ ID NO: 3: E. coli DNA fragment containing promoter sequence (SEQ ID NO: 1).
SEQ ID NOs: 4-5: ucpA nucleic acid and amino acid sequences.
SEQ ID NOs: 6-7: fucO nucleic acid and amino acid sequences.
SEQ ID NOs: 8-9: yqhD nucleic acid and amino acid sequences.
SEQ ID NOs: 10-11: pntA nucleic acid and amino acid sequences.
SEQ ID NO: 12: adhE promoter sequence.
SEQ ID NO: 13: nucleic acid sequence for adhE::pntAB.
SEQ ID NO: 14: nucleic acid sequence for PyadC′fucO-ucpA.
SEQ ID NOs: 56-57: pntB nucleic acid and amino acid sequences.
The invention provides organisms for production of renewable fuels and other chemicals. Particularly, the invention provides bacteria, fungi and yeast that can grow and produce renewable fuels and other chemicals in the presence of increased furfural. The invention provides for an isolated or recombinant cell/microorganism (bacterial, yeast or fungal cell) having increased expression of ucpA and fucO in combination with the deletion of the gene encoding yqhD or chromosomal integration of genes encoding pntA and pntB behind the adhE promoter (adhE::pntAB) and that exhibits improved ability to produce a desired product in the presence of furfural and 5-HMF as compared to a reference cell (e.g., a reference bacterial, yeast or fungal cell). In various embodiments, the bacterial, fungal or yeast cell contains a genetic construct comprising ucpA and fucO operably linked to a promoter comprising SEQ ID NO: 1. In various other embodiments, the bacterial, fungal or yeast cell may comprise, in addition to the aforementioned genetic modifications, a nucleic acid sequence encoding fucO that is integrated into the genome of the bacterial, fungal or yeast cell and operably linked to a native promoter within the genome of the bacterial, fungal or yeast cell (for example, the promoter for alcohol/acetaldehyde dehydrogenase (adhE)). In various embodiments, the bacterial, fungal or yeast cell having increased furfural and/or 5-HMF tolerance can produce ethanol; lactic acid; succinic acid; malic acid; acetic acid; 1,3-propanediol; 2,3-propanediol; pyruvate; dicarboxylic acids; adipic acid; butanol; and amino acids, including aliphatic and aromatic amino acids.
Various publications have disclosed bacterial, fungal or yeast cells in which ethanol; lactic acid; succinic acid; malic acid; acetic acid; 1,3-propanediol; 2,3-propanediol; 1,4-butanediol; 2,3-butanediol; butanol; pyruvate; dicarboxylic acids; adipic acid; and amino acids, including aliphatic and aromatic amino acids can be produced. Many of these microorganisms have been genetically manipulated (genetically engineered) in order to produce these desired products. Exemplary publications in this regard include U.S. Published Patent Applications US-2010/0184171A1 (directed to the production of malic acid and succinic acid), 2009/0148914A1 (directed to the production of acetic acid; 1,3-propanediol; 2,3-propanediol; pyruvate; dicarboxylic acids; adipic acid; and amino acids, including aliphatic and aromatic amino acids), 2007/0037265A1 (directed to the production of chirally pure D and L lactic acid) and PCT application PCT/US2010/029728 (published as WO2010/15067 and directed to the production of succinic acid). The teachings of each of these publications, with respect to the production of bacterial cells producing a desired product, is hereby incorporated by reference in its entirety.
In another aspect of the invention, bacterial, fungal or yeast cells disclosed herein demonstrate increased growth in the presence of furfural and/or 5-HMF as compared to a reference bacterial, fungal or yeast cell. In another embodiment, the bacterial, fungal or yeast cell has increased growth in the presence of furfural and/or 5-HMF at concentrations of about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM or higher (or between about 5 mM and about 20 mM furfural and/or 5-HMF, about 15 mM to about 30 mM furfural and/or 5-HMF, preferably about 15 mM furfural and/or 5 HMF).
Bacterial cells can be selected Gram negative bacteria or Gram positive bacteria. In this aspect of the invention, the Gram-negative bacterial cell can be selected from the group consisting of Escherichia, Zymomonas, Acinetobacter, Gluconobacter, Geobacter, Shewanella, Salmonella, Enterobacter and Klebsiella. Gram-positive bacteria can be selected from the group consisting of Bacillus, Closridium, Cornebacterial, Lactobacillus, Lactococcus, Oenococcus, Streptococcus and Eubacterial cells. Various thermophilic bacterial cells, such as Thermoanaerobes (e.g., Thermoanaerobacterium saccharolyticum) can also be manipulated to increase furfural resistance and/or 5-HMF resistance as disclosed herein. Other thermophilic microorganisms include, but are not limited to, Bacillus spp., e.g., Bacillus coagulans strains, Bacillus licheniformis strains, Bacillus subtilis strains, Bacillus amyloliquifaciens strains, Bacillus megaterium strains, Bacillus macerans strains. Paenibacillus spp. strains or Geobacillus spp. such as Geobacillus stearothermophilus strains can be genetically modified. Other Bacillus strain can be obtained from culture collections such as ATCC (American Type Culture Collection) and modified as described herein.
Other embodiments provide for a yeast cell or fungal cell having increased expression of ucpA and fucO in combination with the deletion of the gene encoding yqhD or chromosomal integration of a gene encoding pntAB behind the adhE promoter (adhE::pntAB) and that exhibits improved ability to produce a desired product in the presence of furfural and 5-HMF as compared to a reference cell (e.g., a reference bacterial, yeast or fungal cell). In various embodiments, the bacterial, fungal or yeast cell contains a genetic construct comprising ucpA and fucO operably linked to a promoter comprising SEQ ID NO: 1. The yeast cell may be a Candida. Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
In other embodiments, the genetic modifications disclosed herein may be made to a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, Oomycota and all mitosporic fungi. A fungal cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycola and Oomycola (as defined by Hawksworth et al., Ainsworth and Bisby's Dictionary of the Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Miyceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press. Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.
In various embodiments within this aspect of the invention, the bacterial cells can be Escherichia coli or Klebsiella oxytoca that have, optionally, been genetically modified to produce a desired product. In these embodiments, an isolated or recombinant bacterial cell is modified as disclosed herein to provide increased tolerance to furfural.
Various other aspects of the invention provide methods of producing ethanol, lactic acid, succinic acid, malic acid, acetic acid, 1,3-propanediol, 2,3-propanediol, 1,4-butanediol, 2,3-butanediol, butanol, pyruvate, dicarboxylic acids, adipic acid or amino acids. In these aspects of the invention, known bacterial, fungal or yeast cells that produce ethanol, lactic acid, succinic acid, malic acid, acetic acid, 1,3-propanediol, 2,3-propanediol, 1,4-butanediol, 2,3-butanediol, pyruvate, dicarboxylic acids, adipic acid or amino acids are manipulated in a manner that results in an increase in furfural tolerance for the bacterial, fungal or yeast cell (as compared to a reference bacterial, fungal or yeast cell). In various embodiments, the methods comprise culturing a bacterial, fungal or yeast cell producing a desired product (e.g., ethanol, lactic acid, succinic acid, malic acid, acetic acid, 1,3-propanediol, 2,3-propanediol, 1,4-butanediol, 2,3-butanediol, pyruvate, dicarboxylic acids, adipic acid or amino acids) and having increased UcpA activity, as compared to a reference cell, under conditions that allow for the production of the desired product. The desired product (e.g., ethanol, lactic acid, succinic acid, malic acid, acetic acid, 1,3-propanediol, 2,3-propanediol, 1,4-butanediol, 2,3-butanediol, pyruvate, dicarboxylic acids, adipic acid or amino acids) can, optionally, be purified from the culture medium in which the bacterial, fungal or yeast cell was cultured. In various other embodiments, the bacterial, fungal or yeast cells can be cultured in the presence of a hemicellulose hydrolysate.
As used herein. “isolated” refers to bacterial, fungal or yeast cells partially or completely free from contamination by other bacteria. An isolated bacterial, fungal or yeast cell (bacterial, fungal or yeast cell) can exist in the presence of a small fraction of other bacteria which do not interfere with the properties and function of the isolated bacterial, fungal or yeast cell (e.g., a bacterial, fungal or yeast cell having increased furfural tolerance). An isolated bacterial, fungal or yeast cell will generally be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% pure. Preferably, an isolated bacterial, fungal or yeast cell according to the invention will be at least 98% or at least 99% pure.
A “recombinant cell” is a bacterial, fungal or yeast cell that contains a heterologous polynucleotide sequence, or that has been treated such that a native polynucleotide sequence has been mutated or deleted. A “mutant” bacterial, fungal or yeast cell is a cell that is not identical to a reference bacterial, fungal or yeast cell, as defined herein below.
A wild-type bacterial, fungal or yeast cell is the typical form of an organism or strain, for example a bacterial cell, as it occurs in nature, in the absence of mutations. Wild-type refers to the most common phenotype in the natural population. “Parental bacterial, fungal or yeast strain”, “parental bacterial strain”, “parental fungal strain” or “parental yeast strain” is the standard of reference for the genotype and phenotype of a given bacterial, fungal or yeast cell and may be referred to as a “reference strain” or “reference bacterial, fungal or yeast cell”. A “parental bacterial, fungal or yeast strain” may have been genetically manipulated or be a “wild-type” bacterial cell depending on the context in which the term is used.
The terms “increasing”, “increase”, “increased” or “increases” refers to increasing by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100% or more, a particular activity (e.g., increased UcpA activity). The terms “decreasing”, “decrease”, “decreased” or “decreases” refers to reducing by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100% or more, a particular activity (e.g., any decreased activity). An increase (or decrease) in activity includes an increase (or decrease) in the rate and/or the level of a particular activity (e.g., furfural tolerance). “Growth” means an increase, as defined herein, in the number or mass of a bacterial, fungal or yeast cell over time.
The nucleic and amino acid sequence of the ucpA gene (SEQ ID NO: 4) and polypeptide (UcpA; SEQ ID NO: 5) are known in the art (see, for example, EMBL-Bank Accession No. X99908.1 which is hereby incorporated in its entirety and are provided in the sequence listing appended hereto). Likewise, the nucleic acid and polypeptide sequences for FucO are also known in the art. The nucleic and amino acid sequence of the FucO gene (SEQ ID NO: 6) and polypeptide (SEQ ID NO: 7) are known in the art (see GenBank Accession Nos. ADT76407.1, for example and GenBank Accession No. CP002185, REGION: 3085103-3086251, VERSION CP002185.1 GI:315059226, Archer et al., BMC Genomics 12 (1), 9 (2011), each of which is hereby incorporated by reference in its entirety) and are provided in the sequence listing appended hereto.
In one aspect of the invention, bacterial cells having increased UcpA and FucO activity can also have the activity of YqhD decreased or altered, as compared to the activity of YqhD in a reference bacterial cell. Activity is decreased or altered by methods known in the art, including but not limited to modification of yqhD (e.g. by inserting, substituting or removing nucleotides in the gene sequence or complete chromosomal deletion of the gene). Thus, this aspect of the invention can also provide a bacterial cell wherein expression of UcpA and FucO is increased, as compared to a reference bacterial cell and expression of the yqhD is decreased as compared to the expression of yqhD in a reference bacterial cell. Methods for altering the activity of YqhD and inactivating yqhD are known in the art, see for example PCT/US2010/020051 (PCT publication WO 2010101665 A1) which is hereby incorporated by reference in its entirety.
The invention provides for a bacterial, fungal or yeast cell that has an increased resistance to furfural, increased expression of FucO and UcpA protein or mRNA and in combination with the deletion of the gene encoding yqhD or chromosomal integration of a gene encoding pntAB behind the adhE promoter (adhE::pntAB) and that exhibits improved ability to produce a desired product in the presence of furfural and 5-HMF as compared to a reference cell (e.g., a reference bacterial, yeast or fungal cell). In various embodiments, the bacterial, fungal or yeast cell contains a genetic construct comprising ucpA and fucO operably linked to a promoter comprising SEQ ID NO: 1 and which, as compared to a reference bacterial, fungal or yeast cell, exhibits at least one of: 1) increased growth in the presence or absence of furfural as compared to a reference bacterial, fungal or yeast cell; 2) increased growth and increased production of a desired product as compared to a reference bacterial, fungal or yeast cell; 3) increased growth and increased production of a desired product, in the presence of furfural, as compared to a reference bacterial, fungal or yeast cell; 4) increased growth in the presence of a hydrolysate as compared to a reference bacterial, fungal or yeast cell; and 5) increased production of a desired product as compared to a reference bacterial, fungal or yeast cell.
Various aspects of the invention provide for the use of a variety of hydrolysates for the production of a desired product, including but not limited to, hydrolysate derived from a biomass, a hemicellulosic biomass, a lignocellulosic biomass or a cellulosic biomass. Yet other aspects of the invention provide a bacterial, fungal or yeast cell with increased resistance to furfural, wherein the bacterial, fungal or yeast cell is capable of producing a desired product as a primary fermentation product, wherein optionally, the primary fermentation product is produced under anaerobic or microaerobic conditions.
The invention also provides for a method for producing a desired product from a biomass, a hemicellulosic biomass, a lignocellulosic biomass, a cellulosic biomass or an oligosaccharide source comprising contacting the biomass, hemicellulosic biomass, lignocellulosic biomass, cellulosic biomass or oligosaccharide with any of the isolated or recombinant bacterial, fungal or yeast cell of the invention thereby producing the desired product from a biomass, hemicellulosic biomass, lignocellulosic biomass, cellulosic biomass or an oligosaccharide source.
Further, the invention provides for a method for producing a desired product from a biomass, a hemicellulosic biomass, a lignocellulosic biomass, a cellulosic biomass or an oligosaccharide source in the presence of furfural comprising contacting the biomass, hemicellulosic biomass, lignocellulosic biomass, cellulosic biomass or oligosaccharide with the isolated or recombinant bacterial, fungal or yeast cell of the invention, thereby producing the desired product from a biomass, hemicellulosic biomass, lignocellulosic biomass, cellulosic biomass or an oligosaccharide source.
Other aspects of the invention provide for isolated polynucleotides and isolated polypeptides comprising any one of SEQ ID NOs: 1-3 and 13 or 14. In particular embodiments, any one of SEQ ID NOs: 1-3 can be operably linked to a heterologous polynucleotide sequence (i.e., a gene other than yadC) in order to facilitate expression of the heterologous sequence within a host cell. Various other embodiments include vectors comprising any one of SEQ ID NOs: 1-3 operably linked to a heterologous polynucleotide sequence or vectors comprising SEQ ID NO: 13 or 14. Host cells comprising such vectors are another aspect of the disclosed invention.
Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.
The methods of seamless chromosomal deletion, gene replacement, and integration were previously described using Red recombinase technology (27). In general, primers “up” and “down” were used to amplify target genes and adjacent regions (200-400 bp upstream and downstream to ORF). Resulting PCR products were cloned into the pCR2.1 TOPO vector. Primers with the designation “1” and “2” (“10” and “20” in some cases) were used to amplify the backbone of the plasmid by inside-out PCR, omitting the coding region of target gene. The PCR fragments were ligated to cat-sacB cassette (amplified from pLOI4162) to create the template for integration (1). After removal of cat-sacB, the self-ligated plasmid contains only the adjacent regions of target region allowing a seamless deletion (27). Plasmids and primers used in strain constructions are listed in Table 1.
Constructions of Plasmids for fucO-ucpA Expression and Chromosomal Integration
pLOI5229 (pTrc fucO-ucpA)
The DNA sequence of fucO (ribosome binding site, coding region and terminator) was previously cloned into pTrc99a (pLOI4319) (17). The whole plasmid of pLOI4319 (17) was amplified by PCR using primers pTrcFucO-UcpA left and pTrcFucO-UcpA right to open the plasmid precisely after fucO stop codon and to create the fragment containing the plasmid backbone and fucO ORF. The fragment containing intergenic sequence (AATTGAAGAAGGAATAAGGT; SEQ ID NO: 15) and ucpA ORF was assembled by PCR using E. coli genomic DNA as template and primers pTrcFucO-UcpAORFup and pTrcFucO-UcpAORFdown. Both PCR fragments contain a more than 50 bp identical sequence at each end provided by primers. The two pieces of DNA were joined by CloneEZ® PCR Cloning Kit from GenScript (Piscataway, N.J.) to produce pLOI5229. The protein level of FucO produced from pLOI5229 is equal to that from pLOI4319 (approximately 0.7 U/mg protein) (
pLOI4857 (Cloning Wild-Type ackA and its Adjacent Region into pACYC184) The fragment of E. coli ackA ORF and its adjacent region (200 bp upstream and downstream from coding region) was amplified by PCR using primers ackAup200 and ackAdown200. Using primers pACYC-up and pACYC-down, the plasmid backbone of pACYC184 excluding let ORF (1.2 kb) was also amplified. After phosphorylation, these two DNA fragments were ligated to form plasmid pLOI4857.
pLOI4859 (Replacing ackA ORF with fucO-ucpA to Create ackA::fucO-ucpA Cassette)
Primers ackA 1 and ackA 2 were used to amplify the sequence from pLOI4857 precisely excluding the ackA ORF by PCR. Primers ackApAC up and ackApAC down were used to amplify the fucO-ucpA fragment from pLOI5229. The two pieces of DNA were joined by CloneEZ® PCR Cloning Kit, designated pLOI4859.
pLOI14869 (Reducing the Size of pLOI4859)
Primers pACY PacI and pACY HindIII were used to amplify the backbone of pACYC184 omitting let and downstream sequence (1.9 kb). PacI and HindIII sites in primers were added to the two ends of the PCR fragment. Primers HindIII ackA fucO and ackA fucO PacI were used to amplify the fucO-ucpA cassette with flanking ackA′ regions using pLOI4859 as a template. These primers included PacI and HindIII sites at the ends. These two PCR products ligated to create plasmid pLOI4869.
pLOI4870 (Adding Unique BamHI Site and Ribosomal Binding Region)
The full length of plasmid pLOI4869 was amplified by inside-out PCR using primers fucO RBS and fucO BamHI. After phosphorylation and self-ligation, the resulting plasmid was designated pLOI4870. This plasmid contained a promoter-probe cassette consisting of a unique BamHI site for ligation of Sau3A1 fragments followed by an adhE ribosomal binding site, fucO ORF, an intergenic sequence and ucpA ORF (
Growth-Based Screen for Surrogate Promoters to Express the fucO-ucpA Cassette
E. coli genomic DNA was completely digested with Sau3AI and ligated into BamHI-treated pLOI4870 to create a plasmid library containing varied sequences between ackA upstream sequences (ackA′) and the ribosomal binding site of fucO (
The promoter fragment in pLOI5237 (1.6 kb) was composed of 10 independent Sau3AI fragments (
Sequences of Promoter Fragments from pLOI15237 and pLOI5259 (Subclone)
The predicted promoter region (BPROM and Neural Network Promoter Prediction) is underlined and bold. The sequence of ackA′ upstream and partial fucO ORF (downstream) are italicized and underlined.
TACTTGAGTCGTCAAATTCATATACATTATGCCATTGGCTGAAAATTACGCAAAATGGCA
TAGACTCAAGATATTTCTTCCATCATGCAAAAAAAATTTGCAGTGCATGATGTTAATCAT
AAATGTCGGTGTCATCATGCGCTACGCTCTATGGCTCCCTGACGTTTTTTTAGCCAGG
CTCAGGTAAAATGTATGGCTCCCATAAATTATTTAATACATCAGTTCCTGGT
TTGTATTACACATT
ATTAATTTCAAACATGTGGTCAGCTTACGGTACCGTAACTA
TCTGAACG . . . (fucO downstream ORF)
GTCGTCAAATTCATATACATTATGCCATTGGCTGAAAATTACGCAAAATGGCATAGACTC
AAGATATTTCTTCCATCATGCAAAAAAAATTTGCAGTGCATGATGTTAATCATAAATGTC
GGTGTCATCATGCGCTACGCTCTATGGCTCCCTGACGTTTTTTTAGCCAGG(ackA′
upstream sequenee)
ACCACGTGACTTATTAAAGATCTTTACTGCGGCTATACTCCTG
TATGGCTCCCATAAATTATTTAATACATCAGTTCCTGGTTTGTATTACACATT
ORF)
.
Previous studies have shown that deletion of yqhD and increased expression of fucO, ucpA, or pntAB from plasmids each improved growth of ethanologenic E. coli LY180 in the presence of 10 mM furfural (17-19, 22). Further constructions (see Table 1) were made to allow a comparison of all combinations of these genetic traits using pTrc99a-based plasmids for expression of target genes (fucO, ucpA, and fucO-ucpA). Three new derivatives of LY180 were constructed for use as host strains: ΔyqhD, adhE::pntAB and ΔyqhD adhE::pntAB. Integration of pntAB behind the adhE promoter in LY180 provided furfural tolerance equivalent to pTrc99a expressing pntAB (uninduced). Higher levels of pntAB expression with inducer were inhibitory in the absence or presence of furfural (18).
Ethanol production from 100 g/L xylose was complete after 48 h in control cultures lacking furfural (
SEQ ID NO. 13: nucleic acid sequence for adhE::pntAB (adhE open reading frame is replaced by pntAB open reading frame: bold and italic):
Constructing Plasmid-Free Strains for Ethanol Production (Integration of fucO-ucpA)
The use of plasmids, antibiotics, and expensive inducers allowed an investigation of gene interactions but is unlikely to provide the desired genetic stability needed for commercial strains. Chromosomal integration of fucO-ucpA behind a strong promoter such as ackA (highly expressed in mRNA arrays) (18, 20, 22) was tested as a replacement for plasmid pTrc fucO-ucpA in LY180 adhE::pntAB and LY180 ΔyqhD. However, FucO activity of the integrated strains was low (
A function-based selection was used to identify a useful promoter. A promoter probe vector was constructed for fucO-ucpA as a derivative of pACYC184 (low copy) with an appropriately engineered upstream BamH1 site (
The expression cassette from pLOI5259 (ackA′::PyadC′fucO-ucpA-ackA′) was amplified by PCR (Table 1) and integrated into the chromosomes of LY180 ΔyqhD and LY80 adhE::pntAB by precisely replacing the ackA coding region including 22 bp immediately upstream. Resulting strains were designated XW129 and XW131, respectively. Although both integrated strains produced 4-fold to 6-fold higher FucO activity than the respective parent strain (
Integration of Traits Restored Ethanol Fermentation in 15 mM Furfural.
Strain XW129(LY180 ΔyqhD ackA::PyadC′fucO-ucpA) was compared to the parent LY180 during batch fermentation in AM1 mineral salts medium (100 g/L xylose) with and without 15 mM furfural (
Furfural-Resistance Traits Also Increased Resistance to Hemicellulose Hydrolylsate.
Furfural is regarded as one of the more important inhibitors in dilute acid hydrolysates of hemicellulose (6-8). This was confirmed in part by a comparison of batch fermentations containing sugarcane bagasse hemicellulose hydrolysate (
Re-Engineering E. coli KJ122 for Conversion of Hemicellulosic Hydrolysates to succinate
Strain LY180 is derived from E. coli KO11, a sequenced strain that has acquired many mutations during laboratory selections for growth in mixed sugars, high sugars, lactate resistance, and other conditions (24-26). It is possible that some of the mutations in KO11 or the heterologous genes encoding ethanol production in this strain may be critical for engineering furfural tolerance and improving resistance to hemicellulose hydrolysate. To address this concern, we have reconstructed the optimal traits for furfural-resistance in KJ122, a succinate-producing derivative of E. coli C (27). Initially, strain KJ122 was unable to effectively ferment 100 g/L xylose (
The same genetic tools used to construct furfural tolerance in ethanol-producing biocatalysts were used to engineer XW055 (
XW055 and the furfural-resistant mutant XW136 (XW055, ΔyqhD ackA::PyadC′fucO-ucpA adhE::fucO) were compared during batch fermentation using hemicellulose hydrolysate as a source of sugar (
Discussion
Importance of Furfural as an Inhibitor in Hemicellulose Hydrolysate
Microbial biocatalysts can be used to produce renewable chemicals from lignocellulosic sugars. Large scale implementation of biobased processes has the potential to replace petroleum for solvents, plastics, and fuels without disrupting food supplies or animal feed. Costs for such processes remain a challenge and can be reduced by developing biocatalysts that are tailored for specific feedstocks. Inhibitors formed during the deconstruction of lignocellulose such as furfural are part of this challenge. Our studies demonstrate that removal of furfural is essential prior to rapid growth and metabolism of sugars by E. coli biocatalysts (
Furfural, a natural product from the dehydration of pentose sugars (7, 8), serves as one of the barriers to effective fermentation of hemicellulose hydrolysates. Previous studies have shown that furfural was unique in binary combinations of inhibitors, increasing the toxicity of other compounds (soluble lignin products, formate, acetate, etc.) in hemicellulose hydrolysates (13). The starting strain for ethanol production, LY180, was more resistant to furfural than the starting strain for succinate production, XW055, (
Epistatic Interactions of Beneficial Traits for Furfural Tolerance
A general model is included to illustrate interactions among the 4 genetic traits for furfural tolerance (
Two furfural-resistant strains have been previously isolated and characterized, EMFR9 (selected for furfural tolerance; 19) and MM160 (selected for hydrolysate resistance; 17). Each contains a mutation that improves furfural tolerance by silencing YqhD using completely different mechanisms, IS10 disruption of adjacent yqhC (transcriptional activator for yqhD) and a nonsense mutation in yqhD, respectively (17, 20). Silencing genes such as yqhD can be caused by a myriad of genetic changes (29). An increase in fitness by gene silencing would be expected to emerge early in populations under growth-based selection. No mutations were found in these strains that increased expression of ucpA, pntAB, or fucO (13, 15, 18). Genetic solutions for gain of function mutants can be very limited and much less abundant (29, 30). Also, recovery of mutants with increased expression of ucpA and pntAB would be prevented by their negative interactions with yqhD silencing. Very high levels of fucO expression were needed that may require multiple mutations, dramatically limiting recovery without deliberate genetic constructions.
Succinate Fermentation from Lignocellulose Sugars
Succinic acid is currently produced from petroleum derived maleic anhydride and can serve as a starting material for synthesis of many commodity chemicals used in plastics and solvents (31). Genetically engineered strains of E. coli (32) and native succinate producers such as Actinobacillus succinogenes (33-35) and Anaerobiospirillum succiniciproducens (36) have been tested for lignocellulose conversion to succinate. However, fermentation using these strains required costly additional steps (33), nutrient supplementation (32-36), and mitigation of toxins in hydrolysates by overliming or treating with activated charcoal carbons (32, 35). Re-engineering derivatives of KJ122 using known combinations of furfural resistance traits resulted in strain XW136 that now ferments hemicellulose hydrolysates in mineral salts medium without costly detoxification steps (32 g/L succinic acid with a yield of 0.9 g/g sugars;
Materials and Methods
Strains and Growth Conditions.
Strains used are listed in Table 1. Ethanologenic E. coli LY180 (a derivative of E. coli W, ATCC 9637) and succinate-producing E. coli KJ122 (a derivative of E. coli C, ATCC 8739) were previously developed in our lab (19, 27). Strains XW092 (LY180. ΔyqhD), XW103 (LY180, adhE::pntAB), XW109(LY180. ΔyqhD adhE::pntAB), XW115 (LY180, ΔyqhD ackA::fucO-ucpA), XW116 (LY180, adhE::pntAB ackA::fucO-ucpA), XW129 (LY180, ΔyqhD ackA::PyadC′fucO-ucpA) and XW131 (LY180, adhE::pntAB ackA::PyadC′fucO-ucpA) were genetically engineered for furfural tolerance using LY180 as the parent strain. Strain KJ122 (succinate production from glucose) was serially transferred in pH-controlled fermenters (27) at 48 h intervals for approximately 40 generations to isolate a mutant with improved xylose fermentation (designated XW055). Strains XW120 (XW055, ΔyqhD ackA::PyadC′fucO-ucpA) and XW136 (XW055, ΔyqhD ackA::PyadC′fucO-ucpA adhE::fucO) were genetically engineered using XW055 as the parent strain. Cultures were grown in low salt xylose AM1 medium as previously described (37).
Genetic Methods.
Methods for seamless chromosomal deletion, gene replacement, or integration were previously described using Red recombinase technology (12, 27). Plasmids, primers, and construction details are listed in Table 1. Clone EZ® PCR Cloning Kit from GenScript (Piscataway, N.J.) was used for gene replacement on the plasmid. Constructions were made in Luria broth containing 20 g/L xylose, or 50 g/L arabinose (inducer for lambda Red recombinase; Gene Bridges GmbH, Heidelberg, Germany) or 100 g/L sucrose (for counter-selection of sacB). Antibiotics were added when required.
Identification of Promoter for fucO-ucpA Cassette.
A genome-wide promoter library with more than 10.000 clones was constructed in plasmid pLOI4870 (pACYC184 derivative) by ligating Sau3A1 fragments of E. coli genomic DNA into a unique BamHI site immediately upstream from a promoterless fucO-ucpA cassette (
NADH-Dependent Furfural Reductase Assay and SDS-PAGE.
The preparation of cell crude lysates and furfural reductase assay were as previously described (17). Soluble protein lysates (15 μg protein) were also analyzed on 12% SDS PAGE gels (Bio-Rad, Hercules, Calif.).
Furfural Tolerance in Tube Cultures.
Furfural toxicity was measured in tube cultures (13 mm by 100 mm) as previously described for ethanol strains (17, 22). For succinate strains, tubes contained 4 ml of AM1 medium with 50 g/L xylose, 50 mM KHCO3, and 100 mM MOPS as a buffer. Tubes were inoculated with starting cell density of 44 mg/L. Cell mass was measured at 550 nm after incubation for 48 h (37° C.).
Fermentation of Ethanol or Succinate.
Ethanol fermentations with xylose were carried out as previously described (17, 22), with and without furfural. For succinate production from xylose, seed pre-cultures of strains were grown in sealed culture tubes containing AM1 medium (20 g/L xylose, 50 mM KHCO3 and 100 mM MOPS). After incubation for 16 h, pre-inocula were diluted into 500-ml fermentation vessels containing 300 ml AM1 media (100 g/L xylose, 1 mM betaine and 100 mM KHCO3) at an initial density of 6.6 mg dry cell weight. After 24 h growth, these seed cultures were used to provide starting inocula for batch fermentations (AM1 medium, 100 g/L xylose and 100 mM KHCO3). Fermentations were maintained at pH 7.0 by automatic addition of base containing additional CO2 (2.4 M potassium carbonate in 1.2 M potassium hydroxide) as previously described (27). Quantitative analyses of sugars, ethanol, furfural, and succinate were as previously described (17, 27, 38).
Preparation and Fermentation of Hemicellulose Hydrolysates.
Hemicellulose hydrolysate was prepared as previously described (39, 40). Briefly, sugarcane bagasse (Florida Crystals Corporation, Okeelanta, Fla.) impregnated with phosphoric acid (0.5% of bagasse dry weight) was steam-treated for 5 min at 190° C. (39-41). Hemicellulose syrup (hydrolysate) was recovered using a screw press, discarding solids. After removal of fine particulates with a Whatman GF/D glass fiber filter, clarified hydrolysate was stored at 4° C. (pH 2.0). Hydrolysate was adjusted to pH 9.0 (5 M ammonium hydroxide) and stored for 16 h (22° C.) before use in fermentations, declining to pH 7.5. Batch fermentations (300 ml) were conducted in pH-controlled vessels containing 210 mL hemicelluloses hydrolysate supplemented with 0.5 mM sodium metabisulfite, components of AM1 medium (37), and inoculum. Potassium bicarbonate (100 mM) was included for succinate production. Final hydrolysate medium contained 36 g/L total sugar (primarily xylose), furfural 1.2 g/L, HMF 0.071 g/L, formic acid 1.1 g/L and acetic acid 3.2 g/L. Pre-cultures and seed cultures were prepared as described above. After 20 h incubation, seed cultures were used to provide a starting inoculum of 66 mg for hemicelluloses hydrolysate fermentations producing succinate or 13 mg for ethanol. Fermentations were maintained at pH 7.0 by the automatic addition of base (2.4 M potassium carbonate in 1.2 M potassium hydroxide for succinate or 2 N KOH for ethanol).
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
E. coli ackA ORF and its adjacent regions (200 bp upstream and
E. coli yqhD and its adjacent regions (PCR) cloned into the
E. coli adhE and its adjacent regions (PCR) from E. coli cloned
E. coli pntAB cloned into adhE adjacent regions in pLOI5210 to
E. coli fucO ORF cloned into pLOI5167 to replace adhE ORF by
This application claims the benefit of U.S. Provisional Application Ser. No. 61/727,360, filed Nov. 16, 2012, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.
This invention was made with government support under 2011-10006-30358 awarded by the U.S. Department of Agriculture. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US13/69987 | 11/14/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61727360 | Nov 2012 | US |
