Patent Application
20140170724
This application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an ASCII text filed entitled “235-02200101_SequenceListing_ST25.txt” having a size of 12 kilobytes and created on Dec. 9, 2013. The information contained in the Sequence Listing is incorporated by reference herein.
This disclosure provides, in one aspect, a shuttle vector for transferring genetic material between Caldicellulosiruptor spp. and an amplification cell. Generally, the shuttle vector includes an origin of replication sequence from the amplification cell, an origin of replication for Caldicellulosiruptor spp., a selectable marker for the amplification cell, and a heterologous coding sequence that complements a functional deletion in the Caldicellulosiruptor spp. genome.
In some embodiments, the amplification cell can be E. coli.
In some embodiments, the selectable marker can include antibiotic resistance.
In some embodiments, the Caldicellulosiruptor spp. can be C. bescii.
In some embodiments, the heterologous coding sequence can include pyrF operably linked to a regulatory sequence.
In some embodiments, the heterologous coding sequence can include a coding region from a species different than the Caldicellulosiruptor spp. In some of these embodiments, the heterologous coding sequence can encode a polypeptide involved in plant biomass deconstruction. In some embodiments, the polypeptide encoded by the heterologous coding sequence is involved in biosynthesis of a biofuel. In some embodiments, the polypeptide encoded by the heterologous coding sequence is involved in biosynthesis of a bioproduct.
In another aspect, this disclosure provides a method that generally includes introducing the shuttle vector as summarized immediately above into a cell. In another aspect, this disclosure provides a genetically modified cell that includes such a shuttle vector.
In another aspect, this disclosure describes a genetically modified Caldicellulosiruptor spp. microbe engineered to increase biosynthesis of a bioproduct, wherein the genetically modified Caldicellulosiruptor spp. microbe exhibits an increase in biosynthesis of the bioproduct compared to a wild type control. In some embodiments, the bioproduct can include a biofuel. In some of these embodiments, the biofuel can include ethanol.
In some embodiments, the genetically modified Caldicellulosiruptor spp. microbe exhibits an increase in biosynthesis of the bioproduct compared to a wild type control when grown on lignocellulosic biomass. In some of these embodiments, the lignocellulosic biomass can include switchgrass.
In yet another aspect, this disclosure provides a method that generally includes growing a genetically modified Caldicellulosiruptor spp. microbe on a feed stock effective for the genetically modified Caldicellulosiruptor spp. microbe to biosynthesize the bioproduct.
In some embodiments, the bioproduct can include a biofuel. In some of these embodiments, the biofuel can include ethanol.
In some embodiments, the feed stock can include lignocellulosic biomass. In some of these embodiments, the lignocellulosic biomass can include switchgrass.
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
The recalcitrance of plant biomass can be a barrier to economical microbial conversion of plant biomass to products of interest such as biofuels and other bioproducts. Thermophilic microbes including members of the genus Caldicellulosiruptor can grow on non-pretreated lignocellulosic biomass and can, therefore, be an attractive option for biomass conversion. The ability to genetically manipulate members of this genus can allow one to exploit the potential of these microbes for Consolidated BioProcessing (CBP) (Lynd et al., 2005 Curr Opin Biotechnol 16:577-583). The use of Caldicellulosiruptor species for CBP can be limited, however, by a lack of molecular biology tools that permit efficient transfer of genetic material between members of the genus Caldicellulosiruptor and other microbial species.
Replicating shuttle vectors facilitate a variety of genetic manipulations including, for example, optimizing transformation protocols and homologous and heterologous expression of genes of interest. For members of the Caldicellulosiruptor genus, a replicating shuttle vector can be an important tool for extending the breadth of biomass substrates on which the microbes can grow, the metabolic pathways that may be engineered into the microbes, and the products that may be produced by the engineered metabolic pathways. Most reported shuttle vectors rely on drug resistance markers for selection, but these markers are not suitable for use in hyperthermophiles like Caldicellulosiruptor species, which grow optimally at and above 70° C. Typically, the antibiotics and/or the corresponding resistance gene products are not stable at such temperatures.
This disclosure provides a replicating shuttle vector for Caldicellulosiruptor species based on pBAS2, the smaller of two native C. bescii plasmids. pBAS2 was constructed to contain the wild type pyrF allele for uracil prototrophic selection. This plasmid is capable of stable replication and selection in both E. coli and C. bescii. Plasmid DNA was unchanged during transformation and replication in C. bescii and back transformation into E. coli. Transformation with replicating plasmid DNA is an order of magnitude more efficient than transformation with non-replicating plasmids in C. bescii (Chung et al., 2012 PLoS One 7:e43844), making this vector an important tool to screen transformability of members of this genus.
A similar approach was used to screen for transformability of other members of this genus using M.CbeI to overcome restriction as a barrier and was successful for transforming C. hydrothermalis, an attractive species for many applications. Plasmids containing a carbohydrate binding domain (CBM) and linker region from the C. bescii celA coding region were maintained with selection and were structurally stable through transformation and replication in C. bescii and E. coli. Moreover, the translation product of celA is predicted to be heavily glycosylated. Since E. coli doesn't glycosylate proteins, the ability to examine CelA produced in E. coli as well as C. bescii is an example of the use of this shuttle vector for analysis of important biological functions unique to Caldicellulosiruptor spp.
Isolation of a Spontaneous Deletion of the C. bescii pyrFA Locus for Nutritional Selection of Transformants.
Attempts to use drug resistance markers for selection of transformants in C. bescii have been unsuccessful. We therefore turned our attention to an alternative selection marker. Orotidine monophosphate (OMP) decarboxylase, encoded by pyrF in bacteria (ura3 in yeast), converts the pyrimidine analog 5-fluoroorotic acid (5-FOA) to 5-fluorouridine monophosphate, which is ultimately converted to fluorodeoxyuridine by the uracil biosynthetic pathway. Fluorodeoxyuridine is a toxic product that kills growing cells that are synthesizing uracil (Bocke et al., 1984 Mol Gen Genet 197:345-346). Mutants of pyrF are, therefore, uracil auxotrophs and resistant to 5-FOA, providing uracil prototrophy as a selection for the wild type allele and 5-FOA resistance as a counter selection for the mutant allele. We previously reported the isolation and use of a pyrF mutant for nutritional selection of transformants, but the mutation involved a deletion of most of the pyrBCF region. Complementation of the mutation therefore required all three deleted coding regions. Cloning such a large region of the chromosome for complementation is undesirable for designing a replicating plasmid vector since increased size often results in lower transformation efficiency.
Here, we instead isolated a different deletion mutant, in the pyrFA locus, that was complemented by pyrF alone. The partial deletion of pyrA is unusual in that it can be complemented with only the wild-type pyrF allele. This simple complementation by a single coding region reduces the size of the elements required to successfully construct the shuttle vector since the entire operon is not needed to complement the mutation. To obtain this new deletion strain, C. bescii cells were plated on modified DSMZ 640 media (Chung et al., 2012 PLoS One 7:e43844) containing 8 mM 5-FOA. Spontaneous resistance to 5-FOA was observed at a frequency of approximately 10−5 at 65° C. Among 30 mutants isolated, one, designated JWCB005 (Table 1), had an 878 bp deletion that spans most of the pyrF open reading frame (Cbes1377), and part of the adjacent gene, pyrA (Cbes1378) (
Construction of a Replicating Shuttle Vector Based on pBAS2.
C. bescii contains two native plasmids, pBAL (8.3 kb) and pBAS2 (3.7 kb) (Dam et al., 2011 Nucleic Acids Res 39:3240-3254; Clausen et al., 2004 Plasmid 52:131-138). Because of its relatively small size, we chose to use pBAS2 to supply replication functions for C. bescii in the shuttle vector. The pBAS2 plasmid was first reported nearly a decade ago, but the copy number was never determined. The plasmid contains sequences that show homology to a double stranded replication origin, characteristic of plasmids with rolling circle replication, however no single stranded intermediates of plasmid replication were detected (Clausen et al., 2004 Plasmid 52:131-138).
To avoid disrupting the replication functions of the pBAS2 plasmid, we linearized the plasmid DNA just upstream of the Cbes2777 ORF and inserted aac, a selection marker conferring apramycin resistance in E. coli. C. bescii pyrF, placed under transcriptional control of the promoter of Cbes2105 (30S ribosomal protein S30EA), was used for selection of uracil prototrophy in the C. bescii pyrFA deletion mutant JWCB005. The pSC101 replication origin was used for replication in E. coli. The resulting plasmid, pDCW89 (
Assessment of Plasmid Maintenance, and Relative Copy Number in C. bescii.
To assess plasmid maintenance and relative copy number, C. bescii transformants were serially sub-cultured every 16 hours for five passages in selective and nonselective liquid LOD medium (Farkas et al., 2012 J Ind Microbiol Biotechnol 40:41-49). Total DNA isolated from cells after each passage was used for Southern hybridization analysis (
Most plasmids that replicate via a rolling circle mechanism exist in high copy per chromosome (Espinosa et al., 1995 FEMS Microbiol Lett 130:111-120) and the native pBAS2 may exist in high copy as well. The relative copy number of pBAS2 was determined by qPCR with primer pairs targeting specific regions of pBAS2 and/or the chromosome. The relative copy number of pBAS2 was calculated to be seventy-five copies per chromosome based on two biologically independent analyses. The fact that the shuttle vector exists in a single copy per chromosome may be due, at least in part, to competition with the endogenous pBAS2 since they share replication and maintenance functions. The 4.6 kb band indicates the pyrF-containing fragment in wild type C. bescii (lane 12) and 8.3 kb band is non-specific hybridization with pBAL, the larger of two endogenous plasmids in C. bescii (
Plasmid maintenance was determined by assessing the presence of the plasmid after passage with and without nutritional selection over the five successive transfers. Southern analysis showed that the plasmid relative copy number remains constant with selection, but that the plasmid is quickly lost without selection (
Transformation of C. hydrothermalis with Shuttle Vector DNA Methylated with M.CbeI.
Restriction of transforming DNA is a barrier—and for C. bescii apparently an absolute barrier—to transformation of DNA from E. coli (Chung et al., 2012 PLoS One 7:e43844; Chung et al., 2011 J Ind Microbiol Biotechnol 38:1867-1877). Transformation of plasmid DNA from E. coli into C. bescii can involve in vitro methylation with an endogenous α-class N4-Cytosine methyltransferase. M.CbeI (Chung et al., 2012 PLoS One 7:e43844). To test whether modification by M.CbeI also allowed transformation of other members of this genus, a spontaneous mutation resistant to 5-FOA was isolated in C. hydrothermalis (Chung et al., 2013 J Ind Microbiol Biotechnol 40:517-521), JWCH003 (Table 1). This mutant was a tight uracil auxotroph and was used as a host for plasmid transformation. Unmethylated plasmid DNA isolated from various E. coli hosts failed to transform this mutant but DNA methylated with M.CbeI transformed at a frequency similar to that for C. bescii (typically about 500 transformants per μg of plasmid DNA). Transformants were initially confirmed by PCR amplification of aac contained exclusively on the plasmid. As shown in
Shuttle vector plasmid DNA was readily isolated from C. hydrothermalis, suggesting that the vector may exist in higher copy in C. hydrothermalis than in C. bescii. This may be due, at least in part, to the absence of a competing plasmid in C. hydrothermalis.
Cloning of a CBM and a Linker Region of the celA Coding Region into pDCW89.
To test the use of pDCW89 as a cloning vector, a 0.68 kb DNA fragment containing a carbohydrate binding domain (CBM) and linker region derived from celA (Cbes1867) was cloned into pDCW89 (
To determine the loci required for replication of shuttle vector, we constructed plasmids (pDCW154 and pDCW155,
Thus, in one aspect, this disclosure provides a novel shuttle vector than can be used to transfer one or more heterologous polynucleotides between a Caldicellulosiruptor spp. microbe and another host microbe. Typically, the host microbe may be an amplification host—i.e., a host microbial cell that maintains the shuttle vector at a high copy number and/or can be grown to high cell density with the shuttle vector so as to amplify the shuttle vector. Generally, the shuttle vector includes an origin of replication sequence from the amplification cell, an origin of replication for Caldicellulosiruptor spp., a selectable marker for the amplification cell, and a heterologous coding sequence that complements a functional deletion in the Caldicellulosiruptor spp. genome.
In some embodiments, the amplification cell can be E. coli.
In some embodiments, the selectable marker includes a marker that can select host cells transformed with the shuttle vector from host cells that lack the shuttle vector. Exemplary selectable markers include, for example, antibiotic resistance. Exemplary antibiotics against which the shuttle vector can provide resistance include and antibiotic that works against E. coli including, for example, apramycin, spectinomycin, ampicillin, kanamycin, chloramphenicol, etc.
In some embodiments, the Caldicellulosiruptor spp. can be C. bescii. In other embodiments, however, the Caldicellulosiruptor spp. can include any member of the genus Caldicellulosiruptor such as, for example, C. hydrothermalis, C. acetigenus, C. kristjanssonii, C. kronotskiensis, C. lactoaceticus, C. obsidiansis, C. owensensis, or C. saccharolyticus.
As used herein, a “functional deletion in the Caldicellulosiruptor spp. genome” refers to any decrease in expression of a coding region that may be complemented by providing a shuttle vector that includes a heterologous polynucleotide. Thus, a “functional deletion” need not require the complete deletion of the a particular coding region and/or regulatory region controlling expression of the particular coding region.
“Complement” and variations thereof refer to the curing of a biological deficiency by providing a heterologous polynucleotide effective to cure the biological deficiency. Typically, the biological deficiency can be growth rate and/or maximum cell density when grown under certain conditions. The heterologous polynucleotide may directly correspond to a functional deletion in the genome—i.e., provide a copy of a coding region lost in the genome due to a deletion in the genome.
As used herein, “heterologous polynucleotide” and “heterologous coding region” are used interchangeably and refer to a polynucleotide that encodes a polypeptide product and that has been placed under the control of a promoter and/or other regulatory sequence that does not natively regulate expression of the heterologous polynucleotide. Thus, in some cases, a heterologous polynucleotide can encode a polypeptide product that is not natively produced by a host cell. In other cases, however, a heterologous polynucleotide can encode a polypeptide that is natively expressed by the host cell but has been placed under the regulatory control of a different promoter and/or other regulatory sequence.
In some embodiments, therefore, the shuttle vector can include a Caldicellulosiruptor spp. coding region that complements a genomic functional deletion in a Caldicellulosiruptor spp. strain. One exemplary embodiment described above involves a Caldicellulosiruptor bescii strain that involves a deletion in the pyrFA region of the genome. The shuttle vector can include a wild type (or, in some cases, a genetically modified variant of) pyrF that cures the genomic pyrF deficiency.
In some embodiments, the heterologous coding sequence of the shuttle vector can include a coding sequence from a species different than the Caldicellulosiruptor spp. In one exemplary embodiment, the shuttle vector can include one or more coding regions from another thermophilic microbe such as, for example, Clostridium thermocellum. For example, a shuttle vector can include the C. thermocellum adhE coding region, which encodes the C. thermocellum bifunctional acetaldehyde/alcohol dehydrogenase (AdhE). In other embodiments, the heterologous coding sequence can encode any wild-type or mutant form of a polypeptide that has lignocellulosic activity such as, for example, CelA, related glycosyl hydrolases, transporters that extend the breadth of substrates that may be degraded by a host cell.
As will be described in more detail below, the C. thermocellum adhE can allow C. bescii to produce ethanol directly from lignocellulosic biomass without the lignocellulosic biomass having to be pretreated. Thus, in some embodiments, the heterologous coding sequence of the shuttle vector can encode a polypeptide that is involved in biosynthesis of a biofuel or another commercially or industrially relevant chemical.
As used herein, “biosynthesis” refers to any process in which at least one step in the synthesis of a chemical compound is performed by a living organism. Thus, biosynthesis can refer to a process in which a compound is synthesized by a process that includes steps performed by a living organism and also includes steps that are performed without the involvement of a living organism—e.g., a process in which a compound is produced by a living organism, collected, and then subjected to one or more additional steps that do not involve the living organism. As used herein, a “bioproduct” is any compound that is the product of “biosynthesis.”
As used herein, the term “biofuel” refers includes, but is not limited to, a bioalcohol such as, for example, ethanol, propanol, and butanol; biodiesel; bioethers; and biogas.
In another aspect, this disclosure provides methods that involve the genetic modification of one or more organisms by transferring genetic material into an organism, or between two or more organisms, using the shuttle vectors described herein. Generally, therefore, such methods can include introducing into a cell any embodiment of the shuttle vector described above. In some embodiments, the cell can be a Caldicellulosiruptor spp. such as, for example, C. bescii. In other embodiments, the cell can be, for example, E. coli.
In yet another aspect, therefore, this disclosure provides a genetically modified cell that includes any embodiment of the shuttle vector described above.
Heterologous Expression of Clostridium thermocellum Polynucleotide in C. bescii.
We introduced a heterologous nucleotide into C. bescii using a vector similar in relevant aspects to the shuttle vector described above. As a result, we successfully metabolically engineered the C. bescii to produce ethanol from plant biomass without conventional pretreatment. This was accomplished by expressing a heterologous bi-functional acetaldehyde/alcohol dehydrogenase coding region (adhE) from Clostridium thermocellum to introduce a pathway for ethanol production. The engineered strain produced 12.8 mM ethanol directly from 2% (wt/v) switchgrass—a real world plant biomass substrate—and decreased acetate production by 38% compared to wild type. Direct conversion of lignocellulosic biomass to ethanol represents a new paradigm for consolidated bioprocessing and offers the potential for carbon neutral, cost effective, sustainable fuel production.
Conventional strategies for bioethanol production from lignocellulosic feed stocks typically involve physicochemical pretreatment, enzymatic saccharification, and/or fermentation (
C. bescii is the most thermophilic cellulolytic bacterium so far described, growing optimally at ˜80° C. with the ability to use, without pretreatment, a wide range of substrates such as, for example, cellulose, hemicellulose, and lignocellulosic plant biomass (Blumer-Schuette et al., 2008 Curr Opin Biotechnol 19:210-217; Yang et al., 2009 Appl Environ Microbiol 75:4762-4769). C. bescii also efficiently ferments both C5 and C6 sugars derived from plant biomass. The shuttle vector provided herein can simplify methods for the genetic manipulation of members of the Caldicellulosiruptor genus and opens the door for metabolic engineering for the direct conversion of plant biomass to liquid fuels such as ethanol (
C. bescii uses the Embden-Meyerhof-Parnas (EMP) pathway to convert glucose to pyruvate, which supplies various pathways that ultimately produce, for example, acetate, lactate, and hydrogen (
While many mixed acid fermentation organisms use a bifunctional acetaldehyde/alcohol dehydrogenase (AdhE) to reduce acetyl-CoA into acetaldehyde and then into ethanol, bioinformatic analysis indicates that the C. bescii genome does not encode an obvious AdhE or acetaldehyde dehydrogenase (AldH) (Carere et al., 2012 BMC Microbiology 12:295). Indeed, C. bescii does not natively produce ethanol. The phylogenetically related thermophilic Firmicute Clostridium thermocellum, however, encodes an NADH-dependent AdhE (Cthe0423) that is involved in ethanol production in C. thermocellum. Based on its known thermostability, coenzyme specificity (NADH-dependent), similarity in codon usage, and favorable catalytic stoichiometry, this adhE was a promising candidate to generate an ethanol production pathway in C. bescii. We used the ldh deletion mutant strain of C. bescii to express C. thermocellum adhE and produce ethanol from lignocellulosic biomass.
First, to determine the concentration at which growth of C. bescii is inhibited by ethanol, wild-type C. bescii was grown in LOD medium with 1% cellobiose as the sole carbon source and subjected to different levels of added ethanol at 65° C. (
The C. thermocellum adhE coding region (Cthe0423) was amplified from C. thermocellum chromosomal DNA and cloned into pDCW144 (
The pDCW144 plasmid was transformed into the C. bescii ldh mutant strain JWCB018 (ΔpyrFA ldh::ISCbe4 ΔcbeI (Table 3), referred to herein as ldh−), which is a uracil auxotroph and contains a deletion of the CbeI restriction enzyme to simplify genetic manipulation (Cha et al., 2013 J Ind Microbiol Biotechnol 40:1443-1448; Chung et al., 2013 Biotechnol Biofiels 6:82). Counter-selection with 5-fluoroorotic acid (5-FOA) selected for segregation of the merodiploid as previously described (Chung et al., 2013 Biotechnol Biofuels 6:82), depicted in
Heterologous expression of the AdhE protein was detected in transformants containing the expression cassette by Western hybridization using monoclonal antibodies targeting the His-tag (
Introduction of a new fermentative pathway for ethanol production in C. bescii might have resulted in poor growth due to a redox imbalance. Therefore, we examined the growth rate and yield of the adhE-expressing strain relative to the wild type and parent strains (
To determine the functionality of AdhE in C. bescii and its effect on the redirection of flux to ethanol, the fermentation product profiles from C. bescii wild type and mutant strains were examined via high performance liquid chromatography (HPLC) during growth on a soluble substrate (cellobiose, 1%), a model microcrystalline cellulosic substrate (Avicel, 2% wt/vol), and a plant biomass substrate (switchgrass, 2% wt/vol) (
Surprisingly, with no adhE expression optimization or pathway manipulation aside from the use of an ldh mutant background, 70%-73% of the detected fermentation products (excluding hydrogen) in the ldh− adhE+ strain was ethanol during growth on cellobiose, Avicel, and switchgrass. By 39 hours, cellobiose fermentation was complete. At this point, 14.7 mM ethanol had been produced from 7.4 mM cellobiose (14.8 mM glucose equivalents) that had been removed from the culture, resulting in a molar yield of 0.99 mol ethanol/mol glucose equivalents (
Thus, this disclosure describes the metabolic engineering of a hyperthermophilic organism for the conversion of lignocellulosic biomass to a liquid fuel. Furthermore, ethanol has been produced directly from plant biomass without the use of harsh, expensive, or chemical pretreatment. Combining metabolic engineering with the native cellulolytic ability of C. bescii has the potential to transform the biofuels industry by creating a process in which the pretreatment step and/or the addition of exogenous cellulases may be eliminated.
The strains, vectors, and methods described herein can serve as platforms for further metabolic engineering to increase yield and titer to allow cellulosic biofuel production on an industrial scale. As approximately 25% of the carbon fermentation products remain to be redirected to ethanol, one strategy for improving yield can involve deleting or otherwise reducing the activity of one or more of the two hydrogenase coding regions and the acetate kinase coding region in C. bescii. For example, deletion of the NADH-dependent hydrogenase, pta and ack (enzyme 5 and enzyme 7 in
The strains, vectors, and methods described herein can serve as platforms for the production of other classes of products derived from thermophilic organisms with proper folding at high temperature and physiologically relevant post-translational modifications, such as methylation and glycosylation.
Thus, in another aspect, this disclosure provides a genetically modified Caldicellulosiruptor spp. microbe engineered to increase biosynthesis of a bioproduct. Generally, the genetically modified Caldicellulosiruptor spp. microbe exhibits an increase in biosynthesis of the bioproduct compared to a wild type control.
In some cases, a wild type control may be unable to biosynthesize a particular bioproduct. In such cases, any detectable biosynthesis of the bioproduct may be considered and increases in biosynthesis of the bioproduct compared to a wild type control. In other cases, an increase in biosynthesis of a bioproduct compared to a wild type control can be quantitatively measured and described as a percentage of the biosynthetic activity of an appropriate wild-type control. The biosynthetic activity exhibited by a genetically modified microbe can be, for example, at least 110%, at least 125%, at least 150%, at least 175%, at least 200% (two-fold), at least 250%, at least 300% (three-fold), at least 400% (four-fold), at least 500% (five-fold), at least 600% (six-fold), at least 700% (seven-fold), at least 800% (eight-fold), at least 900% (nine-fold), at least 1000% (10-fold), at least 2000% (20-fold), at least 3000% (30-fold), at least 4000% (40-fold), at least 5000% (50-fold), at least 6000% (60-fold), at least 7000% (70-fold), at least 8000% (80-fold), at least 9000% (90-fold), at least 10,000% (100-fold), or at least 100,000% (1000-fold) of the activity of an appropriate wild-type control.
In some embodiments, the bioproduct can include a biofuel such as, for example, a bioalcohol such as, for example, ethanol, propanol, and butanol; biodiesel; bioethers; and biogas.
In some embodiments, the genetically modified Caldicellulosiruptor spp. microbe can exhibit the increase in biosynthesis of the bioproduct compared to a wild type control when grown on lignocellulosic biomass. As used herein, lignocellulosic biomass refers to biomass that includes carbohydrate polymers (e.g., cellulose, and/or hemicellulose) and lignin, an aromatic polymer. Lignocellulosic biomass includes materials such as, for example, switchgrass, corn stover, straw, sugarcane bagasse, woody plants such as trees, and other terrestrial plants.
In some embodiments, at least 50% of the fermentation product of the genetically modified Caldicellulosiruptor spp. microbe can be a desired bioproduct such as, for example, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the fermentation product of the genetically modified Caldicellulosiruptor spp. microbe can be a desired bioproduct.
As used herein, the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
In the preceding description, particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
C. bescii, C. hydrothermalis, and E. coli strains are listed in Table 1. Primers are listed in Table 2.
Caldicellulosiruptor species were grown anaerobically in liquid or solid modified DSMZ516 medium (Chung et al., 2012 PLoS One 7:e43844) or in low osmolarity defined (LOD) growth medium (Farkas et al., 2012 J Ind Microbiol Biotechnol 40:41-49) with maltose as the sole carbon source as described at 75° C. for C. bescii or at 68° C. for C. hydrothermalis. For growth of auxotrophic mutants JWCB005 and JWCH003, the defined medium containing 40 μM uracil was used. E. coli strains DH5α (dam+ dcm+), BL21 (dam+ dcm+), and ET12567 (dam− dcm−) were used for plasmid DNA constructions and preparations. Standard techniques for E. coli were performed as described (Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press; 2001). E. coli cells were grown in L broth supplemented with apramycin (50 μg/ml), kanamycin (25 μg/ml), or spectinomycin (20 μg/mL), where appropriate. E. coli plasmid DNA was isolated using a miniprep kit (Qiagen, Valencia, Calif.). Chromosomal DNA from Caldicellulosiruptor species was extracted using the Quick-gDNA™ MiniPrep (Zymo Research Corp., Irvine, Calif.) according to the manufacturer's instructions. Total DNA was isolated from Caldicellulosiruptor species as described (Lipscomb et al., 2011 Appl Environ Microbiol 77:2232-2238), except that adding additional lysozyme (30 μg/ml) for 1 hour at room temperature in lysis buffer (Chung et al., 2011 J Ind Microbiol Biotechnol 38:1867-1877) and sonication were employed to enhance the cell lysis. Plasmid DNA isolation from Caldicellulosiruptor species was performed as described (Chung et al., 2011 J Ind Microbiol Biotechnol 38:1867-1877).
A spontaneous deletion within the C. bescii DSM6725 pyrFA locus (
Plasmids were generated using high fidelity pfu AD DNA polymerase (Agilent Technologies, Inc., Santa Clara, Calif.), restriction enzymes (New England Biolabs, Inc., Ipswich, Mass.), and Fast-link™ DNA Ligase (Epicentre Bioechnologies Corp., Madison, Wis.) according to the manufacturer's instructions. Plasmid pDCW89 (
Electrotransformation of JWCB005 and JWCH003 was performed as described (Chung et al., 2012 PLoS One 7:e43844). JWCB011 and JWCB014 were generated by transforming JWCB005 with M.CbeI methylated pDCW89 and/or pDCW129 as described and selecting for uracil prototrophy at 75° C. DNA transformation of C. bescii was confirmed by PCR analysis using primers DC230 and JF199 or primers DC233 and DC235, and also by back-transformation to E. coli. Transformation of the JWCH003 strain was performed similarly, but at 68° C. Transformation of pDCW89 into C. hydrothermalis was confirmed by direct plasmid DNA isolation from transformant, JWCH005. The transformation efficiencies were calculated as the number of transformant colonies per μg of DNA added and do not take into account plating efficiencies. E. coli strain DH5α cells were transformed by electroporation in a 2 mm gap cuvette at 2.5 V, and transformants were selected for apramycin resistance.
C. bescii transformants (JWCB011) were serially subcultured every 16 hours for five passages in selective (without uracil) and non-selective (supplemented with 40 M uracil) liquid media. After each passage, cells were harvested and used to isolate total DNA. For each sample, 3 μg of total DNA was digested with 10 U of EcoRV for six hours at 37° C. The restriction fragments were separated by electrophoresis in a 1.0% (wt/vol) agarose gel and transferred onto nylon membranes (Roche Diagnostics, Indianapolis, Ind.). Primers JF396 and JF397 (Table 2) were used to amplify a fragment of the pyrF coding region using JWCB005 genomic DNA as template to generate a digoxigenin (DIG)-labeled probe by random priming with DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Diagnostics, Indianapolis, Ind.). The membrane was incubated with probe at 42° C. and washed at 65° C. Band intensities were determined by using a Storm 840 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, Calif.) equipped with ImageQuaNT™ v.5.4 software (GE Healthcare, Little Chalfont, United Kingdom). Relative copy number was determined as the ratio of band intensity of the plasmid-derived band to the chromosomal pyrF fragment. Plasmid maintenance with and without selection was inferred from the change in relative copy number over the five successive cultures. To assess the structural stability of the plasmid, total DNA isolated from five independent C. bescii transformants containing pDCW89 was used to back-transform E. coli for plasmid isolation and restriction digestion analysis.
Determining the Relative Copy Number of pBAS2
Total DNA was isolated from JWCB001 and treated with RNase A (Qiagen, Valencia, Calif.). qPCR experiments were carried out with an LightCycler 480 Real-Time PCR instrument (Roche Diagnostics, Indianapolis, Ind.) with the LightCycler 480 SYBR Green I master mix (Roche Diagnostics, Indianapolis, Ind.). The relative copy number of pBAS 2 (Dam et al., 2011 Nucleic Acids Res 39:3240-3254; Clausen et al., 2004 Plasmid 52:131-138) was determined as the average of two biologically independent samples. Table 2 lists the primers used in the qPCR experiment.
Caldicellulosiruptor
C. bescii DSM6725 wild type (ura+/5-FOAS)
C. bescii ΔpyrFA (ura−/5-FOAR)
C. bescii JWCB005 transformed with pDCW89 (ura+/5-FOAS)
C. bescii JWCB005 transformed with pDCW129 (ura+/5-FOAS)
C. hydrothermalis ISCahyI insertion mutation in pyrF (ura−/5-FOAR)
a
Escherichia coli
b
b
E. coli/Caldicellulosiruptor species shuttle vector (ApramycinR)
E. coli/Caldicellulosiruptor species shuttle vector (ApramycinR)
1
German Collection of Microorganisms and Cell Culture
a Chung et al., 2013 J Ind Microbiol Biotechnol 40: 517-521.
b Chung et al., 2011 J Ind Microbiol Biotechnol 38: 1867-1877.
C. bescii strains and plasmids are listed in Table 3. Primers are listed in Table 4.
All C. bescii strains were grown anaerobically in liquid or on solid surface in low osmolarity defined (LOD) medium (Farkas et al., 2013 J Ind Microbiol Biotechnol 40:41-49), final pH 7.0, with maltose (0.5%/wt/v; Sigma-Aldrich, St. Louis, Mo.) as the carbon source unless otherwise noted. Liquid cultures were grown from a 0.5% inoculum or a single colony and incubated at 75° C. in anaerobic culture bottles degassed with five cycles of vacuum and argon. For growth of uracil auxotrophic mutants, the LOD medium was supplemented with 40 μM uracil. E. coli strain DH5α was used for plasmid DNA constructions and preparations. Standard techniques for E. coli were performed as described (Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press; 2001). E. coli cells were grown in LB broth supplemented with apramycin (50 μg/mL) and plasmid DNA was isolated using a miniprep kit (Qiagen, Valencia, Calif.). Chromosomal DNA from C. bescii strains was extracted using the Quick-gDNA™ MiniPrep (Zymo Research Corp., Irvine, Calif.) or using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, Calif.) according to the manufacturer's instructions.
C. bescii DSMZ6725 wild type/(ura+/5-FOAS)
4, 5
4
1German Collection of Microorganisms and Cell Cultures
2Cthe-adhE (Cthe0423; Bifunctional acetaldehyde-CoA/alcohol dehydrogenase derived from Clostridium thermocellum ATCC27405)
3Cthe-adhE*(EA) (Cthe0423-P704L, H734R: Bifunctional ace(aldehyde-CoA/alcohol debydrogenase derived from ethanol tolerant Clostridium thermocellum strain EA) (Brown et al., 2011 Proc Natl Acad Sci USA 108: 13752-13757).
4 Chung et al., 2013 Biotechnol Biofuels 6: 82.
5 Cha et al., 2013 J Ind Microbiol Biotechnol 40: 1443-1448.
Construction of Vectors for Knock-in of the Cthe0423 and its Derivative into C. bescii
Plasmid pDCW144 (
A 2.31 kb DNA fragment containing the targeted insertion region sequences (intergenic space between convergent genes Cbes0863-Cbes0864) in C. bescii chromosome was amplified using primers DC456 (with KpnI site) and DC457 (with EcoRI site) using C. bescii genomic DNA as a template. The 4.0 kb DNA fragments containing an apramycin resistance cassette, pyrF cassette (Chung et al., 2013 PLoS One 8:e62881), and the pSC101 replication origin, were amplified from pDCW88 (Chung et al., 2013 Biotechnol Biofuels 6:82) using primers DC081 and DC356. The DC081 and DC356 primers were engineered to contain KpnI and EcoRI sites, respectively. These two linear DNA fragments were digested with KpnI and EcoRI, and ligated to construct 6.33 kb size of pDCW139 (
Plasmid pDCW140 was constructed by inserting the 3.28 kb of DNA fragment, which contains the 134 bp of upstream sequences of Cbes2303 (S-layer protein), 3,507 bp of Cbes2303 coding sequences, and 86 bp of its downstream sequences, into the pDCW139. (
Plasmid pDCW142 was constructed by (a) adding restriction sites for cloning and C-terminal 6× Histidine-tag in front of the stop codon and (b) removing the Cbes2303 coding sequences in pDCW140. The 6.3 kb DNA fragment was amplified from pDCW139 using primers DC464 (with BamHI site) and DC466 (with SphI site, 6× Histidine-tag, and stop codon) using pDCW140 as a template. This DNA fragment was blunt-end ligated after treatment with T4 PNK (New England Biolabs Inc., Ipswich, Mass.) to construct pDCW142 (
To complete construction of pDCW144, a 2.62 kb DNA fragment containing the coding sequence of Cthe0423 was amplified by PCR using DC469 (with BamHI site) and DC470 (with SphI site) using Clostridium thermocellum ATCC 27405 genomic DNA as a template. This DNA fragment was digested with BamHI and SphI, and then cloned into pDCW142 that had been digested with BamHI and SphI (
Plasmid pDCW145 is identical to pDCW144 except for the cloning of Cthe0423 adhE* (EA) (Brown et al., 2011 Proc Nat Acad Sci USA 108, 13752-13757), which contains two point mutations in coding sequences, into pDCW142. To make this change, a 2.62 kb DNA fragment containing the coding sequence of Cthe0423* were amplified by PCR using DC469 (with BamHI site) and DC470 (with SphI site) using Clostridium thermocellum EtOH (Brown et al., 2011 Proc Nat Acad Sci USA 108, 13752-13757), genomic DNA as a template. E. coli strain DH5α cells were transformed by electroporation in a 2-mm-gap cuvette at 2.5 V and transformants were selected for apramycin resistance. The sequences of all plasmids were verified by automatic sequencing (Macrogen USA, Rockville, Md.).
Transformation, Screening, Purification, and Sequence Verification of Engineered C. bescii Mutants
To construct strain JWCB032, one microgram of pDCW144 DNA was used to electrotransform JWCB018 (ΔpyrFA ΔcbeI) as described (Chung et al., 2013 Biotechnol Biofiuels 6:82). Cells were then plated onto solid LOD medium and uracil prototrophic transformant colonies were inoculated into liquid medium for genomic DNA extraction and subsequent PCR screening of the targeted region to confirm the knock-in event of pDCW144 into the chromosome. Confirmed transformants were inoculated into nonselective liquid defined medium, with 40 μM uracil, and incubated overnight at 75° C. to allow loop-out of the plasmid DNA leaving the adhE expression cassette (
Cell-free extracts of C. bescii were prepared from 500 ml cultures grown to mid-log phase at various temperatures (60° C., 65° C., 70° C., and 75° C.), harvested by centrifugation at 6,000×g at 4° C. for 15 minutes and resuspended in Cel-Lytic B cell lysis reagent (Sigma-Aldrich, St. Louis, Mo.). Cells were lysed by a combination of 4× freeze-thawing and sonication on ice. Protein concentrations were determined using the Bio-Rad protein assay kit with bovine serum albumin (BSA) as the standard. 77 microgram protein samples were electrophoresed in a 4-15% gradient Mini-Protean TGX gels (Bio-Rad Laboratories, Inc., Hercules, Calif.) and electrotransferred to PVDF membranes (IMMOBILON-P; EMD Millipore Corp., Billerica, Mass.) using a MINI-PROTEAN 3 electrophoretic apparatus (Bio-Rad Laboratories, Inc., Hercules, Calif.). The membranes were then probed with His-tag (6×His) monoclonal antibody (1:5000 dilution; Invitrogen, Life Technologies Corp., Grand Island, N.Y.) using the ECL Western Blotting substrate Kit (Thermo Scientific, Waltham, Mass.) as specified by the manufacturer.
Analysis of growth and ethanol tolerance was conducted in stoppered 125 ml serum bottles containing 50 ml LOD medium supplemented with 10 g/l cellobiose (Sigma-Aldrich, St. Louis, Mo.) and 1 mM uracil. Duplicate bottles were inoculated with a fresh 2% (v/v) inoculum and incubated at both 65° C. and 75° C. with shaking at 150 rpm. Optical cell density was monitored using a Genova spectrophotometer (Jenway, Bibby Scientific, Burlington, N.J.), measuring absorbance at 680 nm. Batch fermentations were performed for five days, at 65° C. in the same culture conditions except using 10 g/l cellobiose, 20 g/l avicel (Fluka, Sigma-Aldrich, St. Louis, Mo.), or 10 g/l unpretreated (sieved −20/+80-mesh fraction; washed with warm water but no additional pretreatment) switchgrass as carbon sources.
Fermentation products acetate, lactate and ethanol, were analyzed on an Agilent 1200 infinity high-performance liquid chromatography (HPLC) system (Agilent technologies, Santa Clara, Calif.). Metabolites were separated on an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, Calif.) under isocratic temperature (50° C.) and a flow (0.6 ml/min) condition in 5.0 mM H2SO4 and then passed through a refractive index (RI) detector (Agilent 1200 Infinity Refractive Index Detector). Identification was performed by comparison of retention times with standards, and total peak areas were integrated and compared against peak areas and retention times of known standards for each interest.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in. e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
This application claims priority to U.S. Provisional Patent Application Ser. No. 61/739,393, filed Dec. 19, 2012, which is incorporated herein by reference.
This invention was made with government support under DE-AC05-00OR22725 awarded by the BioEnergy Science Center of the Department of Energy and 5T32GM007103-30 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61739393 | Dec 2012 | US |
