Patent Application
20090246844
This invention relates to methods and recombinant microorganisms for the production of ethanol by a consolidated bioprocessing approach for the conversion of cellulosic material to ethanol.
Biofuels are critical to securing energy infrastructures by providing alternative fuels, which will not only limit dependence on fossil fuels, but will also reduce detrimental carbon emissions generated and released into the atmosphere. Current efforts towards the implementation of biofuels have centered on ethanol production and its use.
One problem associated with current methods for the production of biofuels is the use of food crops, such as corn and sugar, as the starting material. For example, the use of cereal grains, such as corn, for ethanol production competes directly with the food supply, and thus has the unintended consequence of driving up source material costs.
An alternative to the use of food crops is cellulosic biomass, such as agricultural and forestry residues or municipal waste, such as waste paper or cardboard. Cellulosic biomass is more abundant and would be much less expensive to use than food stuffs. Unfortunately, the production of biofuels from cellulose and lignocellulose with current technologies is very difficult because of the complex molecular structure of lignocellulose. Current methods require multiple steps either utilizing acid treatment and neutralization, and subsequent treatment with exogenously produced enzymes to hydrolyze the cellulose to sugars, or alternatively syngas technologies are used. Syngas technologies use a considerable amount of heat to turn solid biomass into a mixture of gaseous vapors, and then use a catalyst to convert these gases into liquids, primarily mixed alcohols.
Cellulose is a stable polymer with a half-life about 5-8 million years for β-glucosidic bond cleavage at 25° C. (Wolfenden and Snider, 2001). The enzyme-driven cellulose biodegradation process is orders of magnitude faster, and is vital for returning carbon in sediments to the atmosphere (Zhang et al., 2006). The widely accepted mechanism for enzymatic cellulose hydrolysis involves synergistic actions of three different cellulases: endoglucanase, exoglucanase or cellobiohydrolase and β-glucosidase (Lynd et al., 2002). Endoglucanases (1,4-β-D-glucan 4-glucanohydrolases; EC 3.2.1.4) cleave intramolecular β-1,4-glucosidic linkages randomly. Exoglucanases (1,4-β-D-glucan cellobiohydrolases; EC 3.2.1.91) cleave the accessible ends of cellulose molecules to liberate cellobiose. β-glucosidases (β-glucoside glucohydrolases; EC 3.2.1.21) hydrolyze soluble cellobiose and other cellodextrins with a degree of polymerization up to 6 to produce glucose in the aqueous phase. The hydrolysis rates decrease markedly as the degree of substrate polymerization increases (Zhang and Lynd, 2004). Currently, most commercial cellulases are produced using Trichderma and Aspergillus species. The cellulose market is expected to expand dramatically when cellulases are used to hydrolyze pretreated cellulosic materials to sugars, which can be fermented to biofuels on a large scale. Genes encoding cellulases have been cloned from various bacteria, filamentous fungi and plants (Lynd et al., 2002). Several groups have expressed multiple cellulase enzymes in attempts to recreate a fully cellulolytic, fermentative system in Saccharomyces cerevisiae (van Zyl et al., 2007). Since S. cerevisiae lacks the enzymes that hydrolyze cellulose, three types of cellulases were codisplayed on the surface of the yeast cell wall. WO 2008/064314 describes yeast strains with four cellulases codisplayed on the surface of the yeast cell wall that can grow on and bind to cellulose. A yeast strain codisplaying on the cell wall surface endoglucanase II and cellobiohydrolase II from Trichoderma reesei (T. reesei), and Aspergillus aculeatus (A. aculeatus) β-glucosidase I was able to directly produce ethanol from amorphous cellulose with a yield of approximately 2.9 gram per liter (Fujita et al., 2004). Other groups expressed secreted forms of two cellulases: endoglucanase of T. reesei and β-glucosidase of Saccharomycopsis fibuligera (S. fibuligera), in combination in S. cerevisiae (Den Haan et al., 2007). The highest ethanol titer achieved was ˜1 gram per liter.
Accordingly, there is a need for more efficient methods and microorganisms for producing ethanol from cellulosic biomass.
Recombinant microorganisms having an engineered pathway for the direct conversion of cellulosic material to ethanol are provided. Methods are provided for producing ethanol using these recombinant microorganisms. These methods integrate into a single microorganism or a stable mixed culture of microorganisms to increase production efficiency, the hydrolysis of cellulosic materials and subsequent fermentation of the resulting sugars to alcohol. More specifically, embodiments of the present invention integrate the production of alcohol, such as ethanol, with one or more of the following process steps:
In one aspect, a recombinant microbial host organism is provided, preferably genetically modified S. cerevisiae, that is capable of converting cellulose to ethanol comprising a DNA molecule encoding at least one cellulase enzyme. In a preferred embodiment, the cellulase enzymes are selected from the group consisting of: endoglucanase II, cellobiohydrolase II, and β-glucosidase I.
In another aspect, a recombinant microbial host organism is provided, preferably genetically modified S. cerevisiae, that is capable of converting cellulose and hemicellulose to ethanol comprising: (1) a DNA molecule encoding at least one polypeptide involved in the fermentation of a pentose sugar, preferably xylose; (2) a DNA molecule encoding at least one cellulase enzyme. In a preferred embodiment, the at least one polypeptide involved in the fermentation of a pentose sugar is xylose isomerase.
It is contemplated that whenever appropriate, any embodiment of the present invention can be combined with one or more other embodiments of the present invention, even though the embodiments are described under different aspects of the present invention.
The invention can be more fully understood from the following detailed description, figure, and the accompanying sequence descriptions, which form a part of this application.
Recombinant microorganisms are provided that have an engineered pathway for the direct conversion of cellulosic material to ethanol. Methods are also provided that integrate hydrolysis and fermentation into a single microorganism or a stable mixed culture of microorganisms to increase efficiency of production. More specifically, embodiments of the present invention integrate the production of alcohol, such as ethanol, with one or more of the following process steps:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present specification will control. The following definitions and abbreviations are to be used for the interpretation of the claims and the specification.
The term “ethanol biosynthetic pathway” refers to a microbial pathway to produce ethanol.
The term “carbon substrate” refers to a carbon source capable of being metabolized by host organisms of the present invention, and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, or mixtures thereof.
The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein or RNA, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as naturally found in a host organism with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in the host organism. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in that source. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign gene” or “heterologous gene” refers to a gene not normally found in the host organism, but that is introduced into the host organism. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. It is also understood, that foreign genes encompass genes whose coding sequence has been modified to enhance its expression in a particular host, for example, codons can be substituted to reflect the preferred codon usage of the host.
As used herein the term “coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structures.
The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA (e.g., rRNA). A coding sequence is located downstream of a promoter sequence. Promoters may be derived in their entirety from the promoter of a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from nucleic acid fragments. Expression also refers to translation of mRNA into a polypeptide.
As used herein, the term “transformation” refers to the insertion of an exogenous nucleic acid into a cell, irrespective of the method used for the insertion, for example, lipofection, transduction, infection or electroporation. The exogenous nucleic acid can be maintained as a non-integrated vector, for example, a plasmid, or alternatively, can be integrated into the cell's genome. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements can be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation vector” refers to a vector or linear DNA fragment containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. “Expression vector” refers to a vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
As used herein, the term “maximum theoretical yield” is defined as the maximum amount of product (e.g., ethanol) that can be generated per total amount of substrate (e.g., glucose from cellulose) as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the maximum theoretical yield of ethanol starting with 261 g/L of PASC is 4 g/L. For example, the ethanol yield of 3.65 g/L observed in Example 6 would be expressed as 91.25% of the maximum theoretical yield. Alternatively, the maximum theoretical yield can be expressed in terms of grams of product generated per gram of substrate consumed.
As used herein, the term “industrial yeast strain” refers to a yeast strain that is suitable for use for the industrial production of ethanol (e.g., for use as a biofuel or as an alcoholic beverage, such as wine or beer). Industrial yeast strains include, but are not limited to, strains used for commercial and amateur winemaking and beer brewing. An industrial yeast strain will have one or more, preferably all, of the following characteristics: intrinsic tolerance to the ethanol, high temperature tolerance, high ethanol yield, and high growth rate. For example, in some embodiments, the industrial yeast strain will tolerate ethanol concentrations of greater than about 15%, greater than about 18%, greater than about 20% or greater than about 22%. In some embodiments, the industrial yeast strain will tolerate temperatures greater than 37° C. In some embodiments, the industrial yeast strain will tolerate temperatures of at least about 34° C., at least about 35° C., at least about 36° C., or at least about 37° C. In some embodiments, the industrial yeast strain will have a growth rate, such that the doubling time of the number of yeast cells is less than about 120 minutes, for example, between about 90 minutes and about 120 minutes, or about 100 minutes, or about 90 minutes, or less than about 90 minutes. In some embodiments, the industrial yeast strain will convert cellulose to ethanol at least about 80%, about 85%, about 90%, about 95% or about 99% or greater of the maximum theoretical yield. In some embodiments, the industrial yeast strain will produce ethanol from cellulose at a yield of at least about 3 g/L, about 3.2 g/L, about 3.5 g/L, about 3.8 g/L or about 4 g/L.
Standard molecular biology techniques used herein are well known in the art and are described by Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. Techniques for manipulation of S. cerevisiae used herein are well known in the art and are described in Methods in Yeast Genetics. 2005. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., and in Guthrie C, Fink G R, (Eds.). 2002. Methods in Enzymology, Volume 351, Guide to Yeast Genetics and Molecular and Cell Biology (Part C), Elsevier Academic Press, San Diego, Calif.
Consolidated bioprocessing (CBP), as used herein, is a processing strategy for cellulosic biomass which involves consolidating the production of alcohol, such as ethanol, with one or more of the following steps into a single process:
Laccases are enzymes that catalyze the oxidation of a variety of phenolic compounds as well as diamines and aromatic amines. In fungi, laccases are involved in the degradation of lignocellulosic materials (Rodríguez-Couto and Toca-Herrera, 2006). Ligninolytic enzymes are notoriously difficult to express in non-fungal systems. However, some embodiments of the present invention use laccase genes to break down lignin and release the cellulose or hemicellulose. Other enzymes suitable for expression in yeast to breakdown lignin include: lignin peroxide and manganese-dependent peroxidase (Hammel and Cullen, 2008).
Enzymatic degradation of cellulose involves the coordinate action of at least three different types of cellulases. Such enzymes are given an Enzyme Commission (EC) designation according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (Eur. J. Biochem. 264: 607-609 and 610-650, 1999). Endo-β-(1,4)-glucanases (EC 3.2.1.4) cleave the cellulose strand randomly along its length, thus generating new chain ends. Exo-β-(1,4)-glucanases (EC 3.2.1.91) are processive enzymes and cleave cellobiosyl units (β-(1,4)-glucose dimers) from free ends of cellulose strands. Lastly, β-D-glucosidases (cellobiases: EC 3.2.1.21) hydrolyze cellobiose to glucose. All three of these general activities are required for efficient and complete hydrolysis of a polymer such as cellulose to a subunit, such as the simple sugar, glucose. Cellulose degrading yeast strains can be made, for example in S. cerevisiae, by codisplaying on the cell surface or by coexpressing secreted forms cellulolytic enzymes from the filamentous fungi T. reesei and A. aculeatus.
One of the most effective ethanol-producing yeasts, S. cerevisiae, has several advantages such as high ethanol production from hexoses and high tolerance to ethanol and other inhibitory compounds in the acid hydrolysates of lignocellulose biomass. However, because standard, wild-type, strains of this yeast cannot utilize pentoses, such as xylose, and celloligosaccharides (two to six glucose units), fermentation from a lignocellulose hydrolysate will not be completely efficient. According to certain embodiments of the present invention, a recombinant yeast strain is provided that can ferment xylose and cellooligosaccharides by integrating a gene for the intercellular expression of xylose isomerase from Piromyces sp. and a gene for displaying β-glucosidase from A. acleatus. According to some embodiments of the present invention, a recombinant yeast strain is provided that can ferment xylose and cellooligosaccharides by integrating genes for the intercellular expression of xylose reductase and xylitol dehydrogenase from Pichia stipitis (P. stipitis) and a gene for displaying β-glucosidase from A. acleatus.
Microbial hosts for ethanol production may be selected from bacteria, cyanobacteria, filamentous fungi and yeasts. The microbial hosts selected for the industrial production of ethanol, i.e., industrial strains, are preferably tolerant to ethanol and should be able to convert carbohydrates to ethanol with a high yield. Suitable microbial hosts include hosts with one or more, preferably all, of the following characteristics: intrinsic tolerance to the product, high temperature tolerance, high rate of carbon substrate utilization, efficiency in converting the carbon substrate to product, high growth rate, availability of genetic tools for gene manipulation, and the ability to generate stable chromosomal alterations.
The ability to genetically modify the host is useful for the production of a recombinant microorganism. The mode of gene transfer technology may be any method known in the art, such as by electroporation, conjugation, chemical transformation or transduction or transformation. A broad range of host conjugative plasmids and drug resistance markers are available and known to one of skill in the art.
In preferred embodiments, heterologous genes will be stably integrated into the host's genomic DNA. Stably integrating the heterologous genes will create stable transformants, which avoid the requirement for constant selection pressure. In this approach, the need for auxotrophic or antibiotic markers is eliminated. In certain preferred embodiments, multiple copies of the heterologous genes will stably integrate into the host's genomic DNA.
Additionally, the production host should be amenable to chemical mutagenesis or transposon-induced mutagenesis so that mutations to improve intrinsic ethanol tolerance may be obtained. Genome shuffling can also be used to develop and improve complex phenotypes. For example, genome shuffling can be used to increase ethanol production and tolerance in S. cerevisiae. Using yeast sexual and asexual reproduction, mutant diploid cells can be shuffled through highly efficient sporulation and adequate crossing among the resultant haploid cells, followed by selection on special selection plates. The selected strain obtained after several rounds genome shuffling not only can improve resistance to ethanol, but also can increase ethanol yield.
Suitable microbial hosts for the production of ethanol include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Preferred hosts include: Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis, Saccharomyces carlsburgenesis and Saccharomyces cerevisiae. A preferred microbial host is a Saccharomyces species, for example, Saccharomyces bayanus, Saccharomyces carlsburgenesis or Saccharomyces cerevisiae. A particularly preferred microbial host is Saccharomyces cerevisiae.
Recombinant organisms containing the genes encoding the enzymatic pathway for the conversion of cellulose substrate to ethanol are constructed using techniques well known in the art. Methods of obtaining desired genes from a bacterial genome are common and well known in the art of molecular biology. For example, if the sequence of the gene is known, suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA can be amplified using standard primer-directed amplification methods such as polymerase chain reaction (PCR; U.S. Pat. No. 4,683,202) to obtain amounts of DNA suitable for transformation using appropriate vectors. Alternatively, the gene sequence can be amplified directly using standard primer-directed amplification methods such as PCR using genomic DNA as a template. Additionally, the gene can be chemically synthesized.
Once the relevant pathway genes are identified and isolated they can be ligated to the vector DNA and transformed into suitable expression hosts by means well known in the art. Vectors and cassettes useful for the transformation of a variety of host cells are common and commercially available from companies such as EPICENTRE® (Madison, Wis.), Invitrogen Corp. (Carlsbad, Calif.), Stratagene (La Jolla, Calif.), and New England Biolabs, Inc. (Beverly, Mass.). Typically the vectors and cassettes contain sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the specific species chosen as a production host.
Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genetic elements is suitable for the present invention. Promoters useful for expression in Saccharomyces include, but are not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TP1, CUP1, FBA, GPD and GPM. A preferred promoter for expression in Saccharomyces is the GAPDH promoter from S. cerevisiae.
Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included. Termination control regions useful for expression in Saccharomyces include, but are not limited to the CYC1, FBAt, GPDt, GPMt, ERG10t, GALt1 and ADH1 terminators. A preferred terminator for use in Saccharomyces is the CYC1 terminator from S. cerevisiae.
All sequence citations, references, patents, patent applications or other documents cited are hereby incorporated by reference.
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.
Expression constructs encoding cellulases for codisplay on the yeast cell wall surface were constructed by fusing cellulase genes with the DNA encoding the secretion signal sequence of glucoamylase from Rhizopus oryzae. The secretion signal is responsible for delivery of the cellulase to the cell wall. The gene, encoding the C-terminal half of S. cerevisiae α-agglutinin was linked to the 3′-end of the cellulase. The α-agglutinin part of the recombinant protein allows for the attachment to the cell wall. Furthermore, all three cellulases were also expressed in secreted soluble forms that are not attached to the cell wall. Expression constructs for secreted forms lacked the α-agglutinin portion.
DNA sequences of cellulase genes are known, and the following genes were used: T. reesei endoglucanase II (GenBank accession number DQ178347); T. reesei cellobiohyrdolase II (GenBank accession number M55080) and A. aculeatus β-glucosidase I (GenBank accession number D64088). The cellulase DNA constructs were commercially synthesized by Blue Heron Bio using their GeneMaker® synthesis platform. Unique restriction endonuclease sites were added to the sequences to facilitate subcloning into expression vectors. Several restriction sites were removed from coding sequences via one nucleotide substitutions that did not change the amino acid sequence.
The cellulase DNA constructs were cloned into the Blue Heron pUC119 vector. The sequences of the vector inserts are shown below:
Each of the above plasmids was used to create corresponding expression plasmids for cell wall attached cellulases. The cellulase expression vectors were generated using the pUC19-based yeast-E. coli shuttle vectors YEplac112, YEplac195 and YEplac181 (containing selectable markers TRP1, URA3 and LEU2, respectively) that have the yeast 2μ plasmid DNA replication origin (Gietz and Sugino, 1988). These shuttle vectors are high copy, small size vectors that can efficiently transform S. cerevisiae. For cell wall attached CBHII, pUC119-AF101 DNA was digested with HindIII-EcoRI and the 3370 bp DNA fragment was gel purified. The purified DNA fragment was ligated into the HindIII-EcoRI digested vectors YEplac112, YEplac181 and YEplac195, to generate YEplac112-AF101-at, YEplac181-AF101-at and YEplac195-AF101-at, respectively. For cell wall attached BGLI, pUC119-AF102 DNA was digested with XbaI-BamHI and the 2520 bp DNA fragment was gel purified. The purified DNA fragment was ligated into the XbaI-BamHI digested YEplac181-AF101-at vector, to generate YEplac181-AF102-at. For cell wall attached EGII, pUC119-AF103 DNA was digested with XbaI-BamHI and the 1212 bp DNA fragment was gel purified. The purified DNA fragment was ligated into the XbaI-BamHI digested YEplac112-AF101-at vector, to generate YEplac112-AF103-at.
Expression plasmids for secreted cellulases were also generated. For secreted BGLI, pUC119-AF102 DNA was digested with XbaI-KpnI and the 2530 bp DNA fragment was gel purified. The purified DNA fragment was ligated into XbaI-KpnI digested vectors YEplac181-AF101-at and YEplac195-AF101, to generate YEplac181-AF102-sec and YEplac195-AF102-sec, respectively. For secreted EGII, pUC119-AF103 DNA was digested with XbaI-KpnI and the 1212 bp DNA fragment was gel purified. The purified DNA fragment was ligated into the XbaI-KpnI digested YEplac112-AF103-at vector, to generate YEplac112-AF103-sec. For secreted CBHII, pUC119-AF101 DNA was digested with XbaI-BamHI and the 1341 bp DNA fragment was gel purified. The purified DNA fragment was ligated into the XbaI-BamHI digested YEplac195-AF102-sec, to generate YEplac195-AF101-sec.
Derivatives of yeast strains AFY1 (MATα his3-Δ200 leu2-3, 112ura3-52 lys2-801 trp1-1) and AFY2 (MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1) (Table 1) were used. These strains can be transformed with up to five plasmids carrying different selection markers. Transformation with the expression plasmids were performed with a lithium acetate method. Co-transformation with up to 3 plasmids was performed and the Trp+Ura+Leu+ colonies containing plasmids encoding cellulases were selected.
The yeast transformation procedure used was a slightly modified version of the protocol described in Ausubel et al., (2002). Cells from an overnight culture were resuspended in 50 mL YPD (start OD600 of 0.2) and grown to an OD600 of 0.5-0.7. The cells were harvested by centrifugation (1,500 g, 5 min) and resuspended in 20 mL sterile distilled water. The cells were harvested by centrifugation and resuspended in 1.5 mL of freshly prepared sterile TE/LiOAc (prepared from 10× concentrated stocks; 10× TE -0.1 M Tris-HCl, 0.01 M EDTA, pH 7.5; 10× LiOAc-1 M LiOAc adjusted to pH 7.5 with dilute acetic acid). For each transformation, ˜5 μg of DNA was mixed with 70 μg of freshly denatured salmon sperm DNA (10 mg/mL, boiled for 20 min in a water bath, then chilled in ice/water) and 200 μL cells in TE/LiOAc were added and carefully mixed. Immediately, 1,200 μL of freshly prepared sterile 40% PEG 4,000 (prepared from stock solutions: 50% PEG 4000, 10× TE, 10× LiOAc, 8:1:1 v/v, pH 7.5) were added and carefully mixed. Cells were incubated for 30 min at 30° C. with constant agitation. Cells were incubated for 15 min at 42° C. and then collected by centrifugation (4,000 g, 1 min). Cells were resuspended in 200 μL YPD and plated onto selective plates. Plates were incubated at 30° C. until colonies appeared.
All chemicals, media components and supplements were of analytical grade standard. Phosphoric acid-swollen cellulose (PASC) was prepared as described by Den Haan et al., (2007). Briefly, Avicel® PH-101 (Fluka) (2 g) was first soaked with 6 mL of distilled water. Then, 50 mL of 86.2% phosphoric acid was added slowly to the tube and mixed well, followed by another 50 mL of phosphoric acid and mixing. The transparent solution was kept at 4° C. overnight to completely solubilize the cellulose, until no lumps remained in the reaction mixture. Next, 200 mL of ice-cold distilled water was added to the tube and mixed, followed by another 200 mL of water and mixing. The mixture was centrifuged at 3,500 rpm for 15 min and the supernatant was removed. Addition of distilled water and subsequent centrifugation were repeated. Finally, 10 mL of 2M sodium carbonate and 450 mL of water were added to the cellulose, followed by 2 or 3 washes with distilled water, until a final pH of 5-7 was obtained. Acid treatment of Whatman® Paper #1 was done as described above for Avicel®, except only 1 g of shredded paper was used.
Single colonies were inoculated into 10 mL of media with appropriate supplements and with 2% glucose as a carbon source and incubated aerobically for 24-72 hours at 30° C. Yeast cells were collected by centrifugation for 10 min at 4,000 rpm and resuspended in 100 mL of media with 2% glucose. After incubation under aerobic conditions for 24-72 hours at 30° C. cells were harvested by centrifugation and washed with distilled water twice. Cell pellets were inoculated in 10 mL of media with either 2% glucose, or 200 g/L PASC or treated Whatman® Paper and ethanol fermentations were anaerobically performed at 30° C. in 15 mL tubes with closed caps. 0.2 mL aliquots were collected at different time points and analyzed using gas chromatography for ethanol concentration.
Fermentation products, such as ethanol, were analyzed using gas chromatography (GC) (5890 Series II Agilent Technologies, Wilmington, Del.) provided with a RTX-5 capillary column (30 m×0.53 mm i.d.×1.5 μm) (Restek, Bellefonte, Pa.) and flame ionization detection. Prior to analysis, the samples were centrifuged at 14,000×rpm for 10 minutes. The samples were diluted 20-fold with a 25 ppm aqueous solution of n-propanol as an internal standard. Helium was used as a carrier gas at 5 mL/min and was split 1 to 20 before the capillary column. Following sample injection, the column was held at 40° C. for 4 minutes and then ramped to 130° C. at a rate of 30° C./min. The GC was equipped with a 7673B auto-sampler (Agilent Technologies) and data were collected through contact closures and analyzed using Peak Simple software (SRI Instruments Torrance, Calif.). Linear calibration curves were developed for ethanol covering the range of 1000 ppm to 0.8 ppm.
Several yeast strains were constructed for production of ethanol from cellulose. To ferment cellulose to ethanol, strains were constructed that codisplay three cellulases (EGII, CHBII and BGLI) on the yeast cell wall surface (Table 1). Furthermore, a second set of strains that produce secreted forms of the same cellulases was developed (Table 1). The strains with surface displayed cellulases and the strains expressing secreted cellulases are efficient hosts for the production of ethanol from either PASC or treated paper (
Xylose fermenting S. cerevisiae strains were also engineered. Wild-type strains of S. cerevisiae cannot utilize pentoses, such as xylose. However efficient fermentation of pentose sugars is advantageous to attain economically feasible processes for ethanol production from lignocellulosic biomass, as xylose could be 25%-30% of the fermentable carbon substrate depending on the feedstock. Anaerobic xylose fermentation by S. cerevisiae was first demonstrated by heterologous expression of xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis together with overexpression of the endogenous xylulokinase (XK) (Ho et al., 1998, 1999). Alcohol fermentation from xylose was also performed by a recombinant S. cerevisiae strain carrying only one heterologous xylose isomerase (XI) gene from the fungus Piromyces sp. (Kuyper et al., 2003).
The open reading frame encoding XI (GenBank accession number AJ249909) was synthesized by Blue Heron Bio. Sites for restriction endonucleases SalI and KpnI were introduced at 5′- and 3′-ends of DNA, respectively. The sites for restriction endonucleases HindIII and KpnI were removed via one nucleotide substitutions that do not change the amino acid sequences. The synthesized DNA was cloned into the Blue Heron pUC119 vector. The sequence of the vector insert is shown below:
The resulting plasmid, pUC119-AF105, was digested with SalI-KpnII and the 1326 bp DNA fragment was gel purified. The purified DNA fragment was ligated into the SalI-KpnI digested vector YEplac195-AF101-at to generate plasmid pYEplac195-AF105. This plasmid was used for the transformation of yeast cells as well as for cotransformation of cells already containing cellulase genes as described above. Alternatively, xylose fermentation by S. cerevisiae can be achieved using heterologous expression of xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis together with overexpression of the endogenous xylulokinase (XK).
In order to create stable transformants in yeast strains suitable for industrial fermentations, which avoid the requirement for constant selection pressure, the cellulases described in Example 1 were also inserted into the genomic DNA of S. cervisiae. In this approach, the need for auxotrophic markers was eliminated. Instead of using auxotrophic markers, the transformants were selected by using dominant resistant-selectable markers (typically antibiotics). Because expression vectors for industrial strains are not commercially available, an expression vector AFV1 (pYI-kanMX-18SrDNA) was created. This vector has the G418 (antibiotic) resistance gene, KanMX. To integrate multiple copies of the cellulase genes into the chromosomes of Saccharomyces cerevisiae, a fragment of 18S rDNA from S. cerevisiae was also inserted in AFV1, which results in the integration of copies of target genes into the yeast's multiple 18S sites.
The three cellulase genes were cloned into a single vector, AFV1. AFV1 was created from the integrating vector YIplac211 as follows: The KanMX gene was amplified by PCR from the pUG6 plasmid using primers 1.H9 CCTTAGCGGCGCCAGCTGATGCTTCGTACGCTGCAG (SEQ ID NO: 5) and 1.H10 CCTTAGCAGGCCTGCATAGGCCACTAGTGGATCTTATATC (SEQ ID NO: 6). The resulting PCR product was digested with NarI and StuI and inserted into the vector YIplac211 also digested with NarI and StuI. The resulting vector was named YI-KanMX. Then 18S rDNA was amplified by PCR from genomic yeast DNA using primers 1.I1 CCTTAGCGACGTCTAATGATCCTTCCGCAGG (SEQ ID NO: 7) and 1.I2 CCTTAGCGATATCTATCTGGTTGATCCTGCCAG (SEQ ID NO: 8). The resulting PCR product was digested with AatII and EcoRV and inserted into the vector YI-KanMX also digested with AatII and EcoRV. The resulting vector was named YI-KanMX-18SrDNA. The ampicillin resistance gene from YI-KanMX-18srDNA was removed by PCR amplification of the entire vector DNA using primers 2.A7 TACCAGCTTAAGTTTCACTCCTAGGCAAATAGGGGTTCCGCGCACATTTCC (SEQ ID NO: 9) and 2.A8 CATAAATGCGGCCGCTACCTAGTTTAAACAGGATCTAGGTGAAGATCCTTTTTGATA ATC (SEQ ID NO: 10). The resulting vector was named AFV 1. Primers 2.A7 and 2.A8 also introduced several unique restriction sites (AvrII, AflII, NotI and PmeI) that were used for cloning of three secreted cellulases. Insertion of the cellulase genes into AFV1 was done by procedure similar to that described into Example 1.
Two industrial diploid yeast strains, a distillery strain—SuperStart (White Labs, Boulder, Colo.) and a wine strain—K1-V1116 (Lallemand, Montreal, QC, Canada) were transformed with cellulase genes. SuperStart is intended for use in fuel ethanol and beverage alcohol fermentations. It ferments well at temperatures up to 93° F. (34° C.) and in a pH range of 3.5 to 6.0. The K1-V1116 is a vigorously fermenting, dominant strain that will overcome wild yeast, and ferments well in a must that is low in nutrients. Transformation of the yeast cells was done by electroporation using linear DNA. The AFVI vector containing three cellulases gene was linearized by digesting 10 μg plasmid DNA with 100 units of NheI enzyme, which cuts the vector only once in the 18SrDNA region. Transformation mixtures were plated on YPD agar dishes containing various concentrations of G418 (up to 10 mg/mL). Only recombinant yeast strains with integrated genes can grow on agar dishes containing G418 antibiotic.
Remarkably, both SuperStart and K1-V1116 strains were unable to form complete tetrads after sporulation and dissection. The SuperStart spores germinated very poorly, forming a few very small colonies. By contrast, segregation of viability of the K1-V1116 spores was close to a 2:2 ratio suggesting that this strain has a single copy mutation (i.e., the strain is a heterozygous diploid) in a gene essential for germination or viability. To construct a genetically tractable yeast strain useful for subsequent genetic manipulations, cells derived from viable spores were resporulated and asci were dissected. The resultant spores showed 100% viability, demonstrating that this strain can be subjected to further genetic analysis and manipulation.
Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. Other aspects, advantages, and modifications considered to be within the scope of the following claims. The claims presented are representative of the inventions disclosed herein. Other, unclaimed inventions are also contemplated. Applicants reserve the right to pursue such inventions in later claims.
Guldener U, Heck S, Fiedler T, Beinhauer J, Heenmann J. 1996. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acid Research 24:2519-2524.
This application claims priority to International Patent Application No. PCT/US2008/012186, filed Oct. 27, 2008, which claims priority to U.S. Provisional Patent Application No. 61/000,458, filed Oct. 26, 2007, each of which is incorporated by reference into this disclosure in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61000458 | Oct 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2008/012186 | Oct 2008 | US |
| Child | 12386858 | US |
