# Patent

## Title
Recombinant Caldicellulosiruptor bescii  and methods of use

## Abstract

This disclosure describes recombinant Caldicellulosiruptor bescii microbes designed to produce greater amounts of acetate, H2, and/or ethanol than a comparable wild type control. this disclosure also describes methods that generally include growing such recombinant microbes under conditions effective for the recombinant microbes to produce acetate, H2, and/or ethanol.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to U.S. Provisional Patent Application No. 61/684,430, filed Aug. 17, 2012, which is incorporated herein by reference.


GOVERNMENT FUNDING
This invention was made with government support under DE-AC05-000R-22725 awarded by the DOE/BioEnergy Science Center (BESC). The government has certain rights in the invention.


SEQUENCE LISTING
This application contains a Sequence Listing submitted electronically via EFS-Web to the United States Patent and Trademark Office as an ASCII text filed entitled “235-02120101_SequenceListing_ST25.txt” having a size of 15 kilobytes and created on Jan. 3, 2014. The information contained in the Sequence Listing is incorporated by reference herein.
SUMMARY
This disclosure describes, in one aspect, a recombinant Caldicellulosiruptor bescii that produces a greater amount of acetate than a comparable wild type control.
In another aspect, this disclosure describes a recombinant Caldicellulosiruptor bescii that produces a greater amount of H2 than a comparable wild type control.
In another aspect, this disclosure describes a recombinant Caldicellulosiruptor bescii that produces a greater amount of ethanol than a comparable wild type control.
In some embodiments of each aspect, the recombinant Caldicellulosiruptor bescii produces lactate in an amount less than a comparable wild type control. In some of these embodiments, the recombinant Caldicellulosiruptor bescii can include a deletion of at least a portion of a lactate dehydrogenase coding region. In certain embodiments, the recombinant Caldicellulosiruptor bescii can include a deletion of at least a portion of Cbes_1918.
In another aspect, this disclosure describes a method that generally includes growing a recombinant Caldicellulosiruptor bescii designed to produce acetate in a greater amount than a comparable wild type control, and doing so under conditions effective for the recombinant Caldicellulosiruptor bescii to produce acetate. In some embodiments, the method can further collecting at least a portion of the acetate.
In another aspect, this disclosure describes a method that generally includes growing a recombinant Caldicellulosiruptor bescii designed to produce H2 in a greater amount than a comparable wild type control, and doing so under conditions effective for the recombinant Caldicellulosiruptor bescii to produce H2. In some embodiments, the method can further collecting at least a portion of the H2.
In another aspect, this disclosure describes a method that generally includes growing a recombinant Caldicellulosiruptor bescii designed to produce ethanol in a greater amount than a comparable wild type control, and doing so under conditions effective for the recombinant Caldicellulosiruptor bescii to produce an alcohol (e.g., ethanol). In some embodiments, the method can further collecting at least a portion of the alcohol.
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.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a simplified version of predicted metabolic pathways for fermentation, glycolysis and electron transfer in Caldicellulosiruptor bescii. (1) L-Lactate dehydrogenase; (2) Pyruvate-ferredoxin oxidoreductase; (3) Phosphotransacetylase; (4) Acetate kinase; (5) Bifurcating (reduced ferredoxin:NADH-dependent) hydrogenase. The thick grey arrows represent potential heterologous pathways that do not exist in Caldicellulosiruptor species but could be used for renewable fuel and chemical production.
FIG. 2. (A) A modified metabolic pathway for pyruvate metabolism in Caldicellulosiruptor bescii. This strategy includes knocking-out the coding region encoding lactate dehydrogenase to produce more acetate and providing a coding region encoding an aldehyde dehydrogenase to convert acetate to acetaldehyde to produce ethanol. (B) An alternative modified metabolic pathway for pyruvate metabolism in C. bescii. This strategy includes double knock-out of the coding region encoding lactate dehydrogenase and the coding region encoding acetate kinase, and expressing a coding region encoding acetaldehyde dehydrogenase to covert acetyl-coA to acetaldehyde which is then converted to ethanol by native alcohol dehydrogenase.
FIG. 3 shows a strategy for the deletion of the ldh coding region in Caldicellulosiruptor bescii. Chromosomal DNA of a ΔpyrF C. bescii, which is a uracil auxotroph and resistant to 5-FOA, is transformed with methylated plasmid DNA. A knock-out vector was constructed with a WT pyrF coding region and a deletion cassette including the 5′ and 3′ flanking regions of the ldh coding region. Transformants were first selected for uracil prototrophy and then selected for 5-FOA resistance.
This requires a double crossover and results in markerless deletion of the ldh coding region.
FIG. 4 shows the deletion of the ldh coding region in Caldicellulosiruptor bescii. (A) A deletion cassette for the ldh coding region was constructed in a non-replicating plasmid that contained a wild type copy of the pyrF coding region, resulting in plasmid pDCW121. The cassette contained ldh 5′ and 3′ flanking DNA fragments. The plasmid was transformed into JWCB005, and uracil prototrophs were selected (resulting from plasmid insertion). Counter-selection with 5-FOA selected for strains that underwent a second recombination event, resulting in deletion of the marker and ldh to produce strain JWCB017. (B) Agarose gel showing PCR products amplified from the ldh locus in the wild type (lane 2), JWCB005 (ΔpyrFA parent strain, lane 3) and JWCB017 (ΔpyrFA, lane 4). Lane 1: DNA MW standards; Lane 5: no template PCR control. Expected bands: wild type ldh locus—3 kb; ldh deletion—2.0 kb.
FIG. 5 shows fermentation products by Caldicellulosiruptor bescii mutant strains. (A) Lactic acid and (B) acetic acid production were measured during growth on 0.5% maltose by the parent strain (JWCB005 ΔpyrFA; filled circles) and JWCB017 (ΔpyrFA Δldh; open circles). (C) Production of lactic and acetic acids by wild type and JWCB017 were further measured by NMR analysis after 48 hours incubation. (D) End products of C. bescii wild-type and mutant strains were measured by HPLC on cellobiose after 30 hours incubation, and (E) switchgrass after 120 hours incubation. Acetate, Black; Lactate, Light gray; Hydrogen, Dark gray.
FIG. 6 shows growth (O.D.680 nm) of wild-type and mutant strains on (A) 0.5% of maltose and (B) 0.5% cellobiose as the carbon source; filled circles, Wild-type; open circles, ΔpyrFA (JWCB005); filled triangles, ΔpyrFA Δldh (JWCB017). Error bars based on three biologically independent experiments.
FIG. 7 shows restriction endonuclease digests of chromosomal DNA isolated from Caldicellulosiruptor species. The nine restriction enzymes employed in this analysis are indicated on the top of the gel. (A) C. bescii chromosomal DNA. (B) C. saccharolyticus chromosomal DNA. M: 1 kb DNA ladder (New England Biolabs; Ipswich, Mass.).
FIG. 8 shows a strategy for construction of a cbeI (Cbes2438) and PCR analysis of the cbeI deletion in JWCB005. (A) A diagram of the cbeI genome region is shown with the cbeI knock-out plasmid having  ̃0.5 kb regions from each up- and downstream of cbeI for homologous recombination and also containing the pyrF cassette (Chung et al., PLoS one, 2013, 8:e62881 (Example 3)) for selection of transformants. Homologous recombination can occur at the upstream or downstream cbeI flanking regions, integrating the plasmid into the genome and generating a strain that is uracil prototroph. Counter-selection with 5-fluoroorotic acid (5-FOA) selects for loss of the plasmid and deletion of the cbeI coding region. Bent arrows depict primers used for verification of the cbeI deletion. (B) Gel depicting PCR products amplified from the cbeI genome region in JWCB018 (ΔpyrFA/ΔcbeI) compared to the parental strain JWCB005 (ΔpyrFA), amplified by primers (DC277 and DC239). Lane 1: JWCB005; lane 2: JWCB018; M: 1 kb DNA ladder (New England Biolabs; Ipswich, Mass.).
FIG. 9 shows a diagram of the cbeI (Cbes2438) knock-out vector. The gray colored boxes indicate sequences originating from C. bescii. Restriction sites and primers are indicated. aac, apramycin resistant cassette; pSC 101, low copy replication origin in E. coli; repA and par, plasmid-encoded coding regions required for pSC101 replication and partition.
FIG. 10 shows electrotransformation of unmethylated pDCW89 into JWCB018. (A) JWCB005 (ΔpyrFA) and JWCB018 (ΔpyrFAΔcbeI) were transformed with un-methylated pDCW89 DNA and plated onto defined medium either with or without uracil. Controls with no DNA transformation are also presented. (B) Restriction analysis of pDCW89 plasmid DNA before and after transformation of C. bescii and back-transformation to E. coli. Lane 1, pDCW89 plasmid DNA isolated from E. coli DH5α, digested with either EcoRI (5.8 kb and 1.9 kb cleavage products), or with HindIII (4.4 kb and 3.3 kb cleavage products). Lanes 2, 3 and 4 plasmid DNA isolated from three biologically independent E. coli DH5α back-transformants using total DNA isolated from C. bescii transformants, digested with either EcoRI or HindIII. M: 1 KB DNA ladder (New England Biolabs; Ipswich, Mass.).
FIG. 11 shows PCR analysis for the presence of pDCW89 after transformation into JWCB005. pDCW89 DNA isolated from C. hydrothermalis transformants was used to transform C. bescii (ΔpyrFA) and the presence of the plasmid was confirmed by PCR amplification of sequences contained only on the plasmid (A) the 0.9 kb PCR product of aac (Apramycin Resistance cassette), (B) the 1.3 kb PCR product of pyrF cassette and (C) the 1.6 kb PCR product of pSCJ101 ori region. Lane 1: total DNA isolated from JWCB005; lane 2: total DNA isolated from C. bescii transformant; lane 3: pDCW 89 isolated from E. coli. M: 1 kb DNA ladder (New England Biolabs; Ipswich, Mass.).
FIG. 12 shows a chromosomal map and PCR analysis of the Uridine Monophosphate (UMP) biosynthetic coding region cluster in C. bescii DSM 6725 and the spontaneous deletion in pyrFA (JWCB005) locus. (A) A diagram of the pyr operon region with the 878 bp deletion in the pyrFA ORFs. The line below the diagram indicates the length of the deletion. Bent arrows depict primers used for verification of the structure of the chromosome in the JWCB005 (ΔpyrFA) strain. pyrF and pyrE loci indicated as black color filled arrow and black dashed filled arrow, respectively. (B) Gel depicting PCR products of the pyrFA region in wild type (3.44 kb) compared to the ΔpyrFA (2.52 kb) strain amplified by primers (JH020 and FJ298). (C) Gel depicting the 2.66 kb PCR products of pyrE region in wild type and the ΔpyrFA strain by primers (DC326 and DC331). M: 1 KB DNA ladder (New England Biolabs; Ipswich, Mass.).
FIG. 13 shows a plasmid map of shuttle vector (pDCW89) and verification of its presence in C. bescii transformants. (A) A linear DNA fragment containing the pyrF expression cassette as well as the entire sequence of pBAS2, generated by PCR amplification using primers DC283 and DC284, was ligated to a DNA fragment containing E. coli replication and selection functions to generate the final shuttle vector. The cross-hatched box corresponds to the pBAS2 plasmid sequences. ORFs from C. bescii are indicated as empty arrows and those from E. coli as black arrows. The apramycin resistant cassette (AprR); PSC101 low copy replication origin in E. coli; repA, a plasmid-encoded required for PSC101 replication; par, partition locus are indicated. The proposed replication origin (115 bp) of pBAS2 is indicated. The primers and restriction sites (AatII and EcoRI) used for the verification are indicated. A detailed description of the construction of pDCW89 is described in FIG. 14 and in Example 3. (B) Gel showing the 1.6 kb PCR products containing the pSC101 ori sequences only presence in pDCW89 using primers DC230 and JF199, total DNA from JWCB005 (Lane 1), a C. bescii transformant with pDCW89 (Lane 2), and pDCW89 isolated from E. coli (Lane 3) as template. (C) Restriction analysis of plasmid DNA before and after transformation of C. bescii and back-transformation to E. coli. Lanes 1 and 4, pDCW89 plasmid DNA isolated from E. coli DH5α, and digested with AatII (Lane 1, 4.4 kb and 3.3 kb cleavage products), and EcoRI (Lane 4, 1.9 kb and 5.8 kb cleavage products); lane 2, 3, 5, 6, plasmid DNA isolated from two biologically independent E. coli DH5α back-transformed from C. bescii transformants, and digested with AatII (Lane 2 & 3), and EcoRI (Lane 5 & 6). M: 1 KB DNA ladder (New England Biolabs; Ipswich, Mass.).
FIG. 14 shows construction of shuttle vector pDCW89. The cross-hatched box corresponds to pBAS2 plasmid sequences. ORFs from C. bescii are indicated as empty arrows and those from E. coli as black arrows. The apramycin resistant cassette (AprR); PSC101 low copy replication origin in E. coli; repA, a plasmid-encoded coding region required for PSC101 replication; par, partition locus; pyrF cassette are indicated. The proposed replication origin (115 bp) of pBAS2 is indicated. All primers and two restriction sites (KpnI and XhoI) used in this construction are also indicated.
FIG. 15 shows plasmid constructions to determine the minimal sequence requirement for replication in C. bescii. DNA sequences derived from C. bescii are indicated as empty arrows and boxes. All features in these plasmid DNAs are described in the brief description of FIG. 14. The proposed replication origin (115 bp) of pBAS2 is indicated. All primers and two restriction sites (KpnI and PvuII) used in this construction are also indicated. (A) Diagram of pDCW154. (B) Diagram of pDCW155.
FIG. 16 shows determination of copy number and maintenance of pDCW89 in C. bescii. (A) Diagram of the pyrF chromosomal region. EcoRV sites (“E”) are indicated, as are the locations of primers used to generate the pyrF hybridization probe. (B) Southern blot of the pDCW89 transformant (JWCB011). Lanes 1 to 5, DNA isolated from 5 successive passages in non-selective medium; lanes 6 to 10, 5 successive passages in selective medium; lane 11, JWCB005; lane 12, C. bescii wild type; Lane 13, pDCW89 isolated from E. coli. 
FIG. 17 shows a comparison of DNA modification status between shuttle vector DNA isolated from E. coli (Lane 1) and C. hydrothermalis transformants (Lane 2) by Restriction analysis. (A) Undigested, (B) Digested with HindIII (4.3 and 3.4 kb cleavage products); (C) Digested with EcoRI (4.6 and 1.9 kb cleavage products); (D) Digested with CbeI (11 cleavage products are expected). M: 1 KB DNA ladder (New England Biolabs; Ipswich, Mass.).
FIG. 18 shows a plasmid map of pDCW129 and verification of its ability to structurally stable maintenance of inserted DNA fragment through transformation and replication in C. bescii. (A) Diagram of pDCW129. A linear DNA fragment containing the CBM3 and linker region derived from celA (Cbes1867) was inserted into pDCW89 shuttle vector. The cross-hatched box corresponds to a 0.68 kb of inserted DNA fragment. All features in pDCW129 are indicated in the legend for FIG. 13A. The primers and restriction site (EcoRV) used for the construction and verification are indicated. (B) Gel showing the 2.2 kb DNA fragment containing the pyrF cassette and inserted DNA fragment, amplified by using primers DC233 and DC235. Lane 1, total DNA isolated from JWCB005; lane 2, total DNA isolated from C. bescii transformant with pDCW129; lane 3, pCW129 isolated from E. coli. (C) EcoRV restriction digestion analysis of plasmid DNA before and after transformation of C. bescii and back-transformation to E. coli. Lane 1, pDCW129 plasmid DNA isolated from E. coli DH5α; lane 2, 3 and 4, plasmid DNA isolated from three biologically independent E. coli DH5α back-transformed from C. bescii transformants. M: 1 KB DNA ladder (New England Biolabs; Ipswich, Mass.).
FIG. 19 shows the construction of knock-in vector pDCW144. Plasmid pDCW144 was constructed in four cloning steps. ORFs from C. bescii and Clostridium thermocellum are indicated as empty arrows. ORFs from E. coli indicated as black arrows. The apramycin resistant cassette (AprR); pSC101, low copy replication origin in E. coli; repA, a plasmid-encoded coding region required for pSCJ101 replication; par, partition locus; pyrF cassette; 5′ and 3′ franking sequences of the targeted insertion site in C. bescii chromosome; regulatory and rho independent terminator sequences of Cbes2303 (marked as a cross-hatched box); C-terminal 6× Histidine-tag in front of stop codon are indicated. All primers and two restriction sites (BamHI and SphI) used in this construction are also indicated.
FIG. 20 shows targeted insertion and expression of the Clostridium thermocellum ATCC27405 adhE coding region (Cthe0423) in C. bescii. (A) A diagram of the targeted knock-in chromosome region is shown with the knock-in plasmid (pDCW144, Fig. S1 for detail description), which contains PS-layer Cthe-adhE expression cassette and pyrF cassette (Chung et al. (2013) PLoS ONE 8: e62881 (Example 3)) for selection of transformants. pDCW144 is also containing  ̃1.0 kb regions from each up- and downstream of targeted region for homologous recombination. Homologous recombination can occur at the upstream or downstream targeted chromosome regions, integrating the plasmid into the genome and generating a strain that is a uracil prototroph. Counter-selection with 5-fluoroorotic acid (5-FOA) selects for loss of the plasmid sequences but not the adhE expression cassette. Bent arrows depict primers used for verification of the knock-in of expression cassette (2.6 kb). Apr is the apramycin resistance cassette. (B) Gel depicting PCR products amplified from the targeted chromosome region in JWCB032 and JWCB033 compared to the parental strain JWCB018, amplified by primers (DC477 and DC478). Lane 1: JWCB018; lane 2: JWCB032; lane 3: JWCB033; M: 1 kb DNA ladder (New England Biolabs; Ipswich, Mass.). (C) Western blot analysis of C. bescii strains used in this study. The 77 μg of total cell protein lysate isolated from the mid-log phase cultures grown at various temperatures either 60° C., 65° C., and 70° C. were electrophoresed and probed with His-tag antibody as described in Material and Methods Lane 1: JWCB018; lane 2: JWCB032; lane 3: JWCB033; M: MagicMarkTM XP Western Protein Standard (Invitrogen; Grand Island, N.Y.; Grand Island, N.Y.).
FIG. 21 shows analysis of fermentation products on cellobiose (1%, wt/v) at 65° C. Ethanol (A), acetate (B), and lactate (C). Closed circle, wild type; open square, JWCB018; closed diamond, JWCB032; open triangle, JWCB033. Error bars represent the standard deviation and each point is the average of two biological independent samples.
FIG. 22 shows analysis of fermentation products on AVICEL (2%, wt/v) at 65° C. Ethanol (A), acetate (B), and lactate (C). Closed circle, wild type; open square, JWCB018; closed diamond, JWCB032; open triangle, JWCB033. Error bars represent the standard deviation and each point is the average of two biological independent samples.
FIG. 23 shows analysis of fermentation products on unpretreated switchgrass (2%, wt/v) at 65° C. Ethanol (A), acetate (B), and lactate (C). Closed circle, wild type; open square, JWCB018; closed diamond, JWCB032; open triangle, JWCB033. Error bars represent the standard deviation and each point is the average of two biological independent samples.
FIG. 24 shows a growth property analysis of wild type and mutant strains. Growth of various C. bescii strains on 1.0% of cellobiose as the carbon source at 65° C. (A) and 75° C. (B) was monitored by measuring culture turbidity (log10OD680nm). Error bars represent the standard deviation and each point is the average of two biological independent samples. Closed circles, JWCB001; open triangle JWCB018; open square, JWCB032; closed diamond, JWCB033.
FIG. 25 shows analysis of ethanol tolerance of the C. bescii wild-type DSM 6725. Growth of C. bescii ori 1.0% of cellobiose as the carbon source supplemented with different amounts of ethanol at 65° C. (A) and 75° C. (B) was monitored by measuring culture turbidity (log10OD680nm). Error bars represent the standard deviation and each point is the average of two biological independent samples. Closed circles, no ethanol; open circle, 200 mM; closed square, 300 mM; open square, 400 mM; closed triangle, 450 mM; open triangle, 500 mM; closed diamond, 600 mM; open diamond, 700 mM. Error bars based on two biologically independent experiments.



DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Caldicellulosiruptor spp. are the most thermophilic and cellulolytic bacteria known and have the ability to degrade unpretreated biomass (4). We recently developed methods for genetic manipulation of members of this genus (U.S. patent application Ser. No. 13/439,069, filed Apr. 4, 2012) and the ability to metabolically engineer these microbes offers the possibility of direct conversion of biomass to biofuels and bioproducts.
Current methods for the use of lignocellulosic biomass as a substrate for microbial conversion to products of interest rely on pretreatment of the biomass with an acids, a base, and/or an organic solvent, often at high temperature and accompanied by treatment with one or more hydrolytic enzymes that partially digest the plant cell walls. Enzymatic pretreatment is particularly expensive and often prohibitive for the production of low value commodity products from biomass.
Thermophilic microorganisms offer special advantages for biomass conversion, in part, because they offer the potential to decrease hydrolysis times by several-fold with the same cellulase loading or to decrease cellulase loading by several-fold at constant hydrolysis times. Organisms that can use complex biomass as substrate reduce the need for pretreatment and enzymatic hydrolysis and, therefore, the cost of the process. Caldicellulosiruptor species have the ability to use unpretreated biomass including both low-lignin napier and Bermuda grasses as well as high-lignin switchgrass and a hardwood, popular, for growth. Members of this genus are among the most thermophilic of all known organisms capable of using unpretreated cellulosic biomass.
The sequences of eight Caldicellulosiruptor genomes have been published and reveal enzymes likely to be important in lignocellulose utilization. In addition, microarray analysis of cells grown on various substrates implicates specific coding regions and coding region clusters in biomass degradation.
Strategies for Engineering Pyruvate Metabolism in C. bescii for Biofuel Production.
FIG. 1 shows a simplified version of native pyruvate metabolic pathways in C. bescii based on predictions from genomic analysis of genome sequence. C. bescii has been shown to produce lactate, acetate and hydrogen as metabolic products from pyruvate. None of the members of this genus (Caldicellulosiruptor) contains a coding region that encodes an acetaldehyde dehydrogenase, an enzyme that can convert acetyl CoA to acetaldehyde, which can subsequently be converted to ethanol by an alcohol dehydrogenase. While there are reports in the literature of some species making trace amounts of ethanol under some conditons, it may be that under special conditions the alcohol dehydrogenase becomes bifunctional allowing trace amounts of ethanol to be produced in the absence of an acetaldehyde dehydrogenase.
We used two strategies for engineering C. bescii to produce ethanol and hydrogen. Both strategies use coding regions from C. thermocellum because this organism is the best known thermophilic ethanol producer. Coding regions from other bacterial thermophilic strains, like Thermoanaerobacter spp., Geobacillus spp., etc. also can be cloned and introduced into C. becii based on the same strategies.
Our first strategy involves deleting the coding region that encodes lactate dehydrogenase (FIG. 2A). Based on genomic analysis, there is only one coding region predicted to provide this function in C. bescii, Cbes_1918. Thus, in such a mutant, pyruvate should be directed away from the production of lactate and toward the production of Acetyl-CoA and/or hydrogen (H2).
To construct the deletions, we have developed a genetic system that relies on nutritional selection and electrocompetent cells for transformation by electroporation. Nutritional selection is used because antibiotics and drug resistance markers from mesophiles often do not work at the high temperatures used for the growth of these organisms (optimal growth temperature: 75° C.). A random mutation of the pyrF locus was selected on 5-FOA (pyrF converts 5-FOA to a toxic product that kills the cells). Deleting pyrF results in a strain that is a uracil auxotroph resistant to 5-FOA, allowing prototrophic selection and counter selection of the wild type pyrF. Restriction is apparently an absolute barrier to transformation with DNA from E. coli, but it can be overcome by methylation with a cognate methylase. The method is efficient enough to allow marker replacement of chromosomal coding regions using non-replicating plasmids.
The C. bescii host containing a pyrF deletion was used for deletion of the ldh coding region by doing the 5′ and 3′ flaking regions of ldh and joining them together (FIG. 3). Since the plasmid is non-replicating, prototropy requires integration of plasmid into C. bescii chromosome at the ldh locus. Subsequent plating of transformant on 5-FOA selects a second crossover event that eliminates the wild type pyrF allele and plasmid resulting in a deletion of the ldh coding region.
The ΔpyrF Δldh double mutant produces lactate in amounts much less that the wild type control, and makes more actate and hydrogen than the wild type control, in a 2:1 ratio (FIG. 5).
At least a portion of the acetate and/or H2 may be collected using routine, well-known techniques.
The ΔpyrF Δldh double mutant may be further modified by introducing an aldehyde dehydrogenase—e.g., Cthe_2238 from Clostridium thermocellum—that converts acetate to acetaldehyde, as shown in FIG. 2A. The acetaldehyde may be subsequently converted to ethanol. Ethanol so produced may be collected using routine, well-known techniques.
The second strategy involves further modifying the ΔpyrF Δldh double mutant by deleting the coding regions for lactate dehydrogenase phosphate transacetylase (pta) and acetate kinase (ak), respectively (FIG. 2B). The pta and ak coding regions are contiguous on the C. bescii chromosome, so this further modification may be accomplished using a single marker replacement to delete both coding regions. In some embodiments, the ΔpyrF Δldh Apta Aak may be further modified by introducing a coding region that encodes an enzyme that converts acetyl-CoA to ethanol such as, for example, Cthe_0423. Ethanol so produced may be collected using routine, well-known techniques.
Deletion of Lactate Dehydrogenase (ldh) from the C. bescii Chromosome.
We describe herein a method for DNA transformation and marker replacement in Caldicellulosiruptor bescii based on uracil prototrophic selection (Example 2 and Example 3). C. bescii strain JWCB005 (ΔpyrFA, ura−/5-FGAR, described in more detail below) contains a deletion of the pyrFA locus making the strain a uracil auxotroph resistant to 5-fluoroorotic acid (5-FOA), allowing the use of pyrF as both a selectable and counter-selectable marker (FIG. 4A). A deletion of the L-lactate dehydrogenase coding region (Cbes1918) was constructed by fusing the 5′ and 3′ flanking regions of the ldh coding region and cloning the fused product into a non-replicating plasmid vector, resulting in plasmid pDCW121. This vector also contains the wild type pyrF allele under the transcriptional control of a ribosomal protein promoter (Cbes2105, 30S ribosomal protein S30EA), allowing both positive (uracil prototrophy) and negative (5-FOA sensitivity) selection. Plasmid pDCW121 was transformed into C. bescii JWCB005 selecting uracil prototrophy resulting from plasmid recombination into the targeted region, followed by counter-selecting 5-FOA resistance (resulting from plasmid excision). The resulting strain, JWCB017, contained a deletion of the ldh wild type coding region in the chromosome. To confirm the ldh deletion in JWCB017, the region of the ldh locus was amplified by PCR using primers outside of the plasmid regions of homology used to construct the deletion (FIG. 4B). The wild type and the ΔpyrFA strain (JWCB005) gave the same 3.0 kb bands, while PCR from JWCB017 resulted in the smaller 2.0 kb band. The PCR product was also sequenced to verify that the deletion in the chromosome was the same as that constructed on the plasmid.
Deletion of ldh Eliminates Lactate Production and Increases Acetate and H2 Production.
Cbes1918 is the only predicted lactate dehydrogenase coding region encoded in the C. bescii genome. To confirm that this coding region is solely responsible for the production of lactate in C. bescii, wild type, JWCB005 and JWCB017 were grown on 0.5% maltose, and fermentation products were analyzed by high-performance liquid chromatography (HPLC) (FIG. 5A and FIG. 5B) and nuclear magnetic resonance (NMR) analysis (FIG. 5C). No lactate was detected in the mutant by either method, as compared to approximately 5.0 mM lactate from the wild-type and parental strains.
To compare the production of lactate, acetate and hydrogen, C. bescii wild-type and mutant strains were grown in LOD medium with soluble cellodextrans (cellobiose) or plant biomass (switchgrass) as carbon source. When grown on 0.5% cellobiose for 30 hours, JWCB017 showed 29% and 21% more acetate production and 37% and 34% more hydrogen production than wild type and parent strains, respectively (FIG. 5D). Cells grown for 120 hours on LOD medium supplemented with 0.5% switchgrass as the sole carbon source showed a similar profile to that on cellobiose, with the Δldh strain producing 38% and 40% more acetate and 55% and 70% more hydrogen than wild-type and parent strains (FIG. 5E).
Growth Yield Increases Upon Deletion of ldh.
Growth of JWCB017 was compared to the wild type and parental strains in defined media supplemented with either 0.5% maltose or 0.5% cellobiose. While growth of the ΔpyrFA parent strain on both maltose (FIG. 6A) and cellobiose (FIG. 6B) was indistinguishable from the wild type, the JWCB017 mutant strain reached a 34-53% higher final optical density than the wild type and parent. Interestingly, while the growth rate was comparable, the exponential growth phase of JWCB017 was extended resulting in higher cell densities.
These results demonstrate the genetic manipulation of Caldicellulosiruptor to delete the coding region encoding lactate dehydrogenase. While the wild type strain produced roughly equimolar amounts of acetate and lactate, the JWCB017 mutant strain no longer produced lactate, instead rerouting carbon and electron flux to acetate and H2, respectively. The hydrogen yield observed for JWCB001 ( ̃1.8 mol/mol of glucose) and JWCB005 ( ̃1.7 mol/mol of glucose) was somewhat lower than the hydrogen yield reported values for C. saccharolyticus grown in culture media with added yeast extract, which improves yields ( ̃2.5 mol/mol of glucose; Kadar et al., Appl Microbiol Biotechnol 2007, 74:1358-1367). JWCB017 ( ̃3.4 mol/mol of glucose), however, provided a higher hydrogen yield than that reported for C. saccharolyticus. Yield and titer of acetate and H2 were increased in the C. bescii ldh deletion strain using either model soluble substrates or real-world plant biomass.
Members of the genus Caldicellulosiuptor offer special advantages for biomass conversion to products of interest in that they are hyperthermophiles with optimal growth temperatures between 78° C.-80° C. and they are capable of using biomass without conventional pretreatment.
Interestingly, deletion of ldh resulted in a higher cell yield and longer exponential growth phase relative to the wild type. The increase in cell density may involve an increase in acetate production, which may increase ATP production per glucose via acetate kinase providing more energy for biosynthesis and growth. The fact that C. bescii JWCB017 grows to a higher density without an obvious effect on growth rate suggests that engineered strains may be able to compete well with the wild type strain and thrive in an industrial setting.
More advanced metabolic engineering strategies allowing genetic manipulation of C. bescii may increase the utility of C. bescii for industrial applications. In addition to the construction of deletions, this can enable insereting one or more heterologous coding regions into the C. bescii chromosome (so called genetic knock-ins), simplifying the process of heterologous coding region expression by eliminating the need for plasmid maintenance and increasing the number of coding regions that can be stably expressed. Thus, we have created a new platform for rational strain design in C. bescii. Genetic design of C. bescii may be exploited for applications such as, for example, lignocellulosic bioconversion involving, for example, increasing the titer of H2, expressing heterologous pathways for production of, for example, liquid fuels and/or other chemicals, increasing robustness, and/or improving upon the native ability of Caldicellulosiruptor species to deconstruct and convert biomass without conventional pretreatment.
As used herein, the term “heterologous” refers to a biomolecule or bioprocess that is not natively present in a host cell. Thus, for example, a “heterologous polynucleotide” refers to a polynucleotide that does not native exist in a host cell, a “heterologous coding region” refers to a polynucleotide that encodes a polypeptide that is not natively produced by a host cell, and a “heterologous pathway” refers to a biosynthetic pathway that includes at least one biosynthetic step that is not natively performed by the host cell.
One obstacle to genetic manipulation of C. bescii involves restriction by CbeI endonuclease. Restriction by CbeI was shown to be an absolute barrier to DNA transformation (Chung et al., Journal of industrial microbiology & biotechnology 2011, 38:1867-1877), but could be overcome by in vitro methylation of DNA by a cognate methyltransferase, M.CbeI (Chung et al., PloS one 2012, 7:e43844).
In another aspect, this disclosure describes a genetically-modified C. bescii in which CbeI activity is reduced, resulting in a strain that is easily transformable with unmethylated heterologous DNA (e.g., from E. coli), eliminating the need for in vitro methylation by M.CbeI (Cbes2437).
Restriction Digestion Analysis of Chromosomal DNA from Caldicellulosiruptor Species.
The observation that restriction was an absolute barrier to DNA transformation of C. bescii prompted us to investigate the prevalence of functional restriction-modification (R-M) systems in other Caldicellulosiruptor species. M.CbeI-methylated DNA successfully transforms C. hydrothermalis (Example 3) suggesting that C. hydrothermalis and C. bescii might share similar R-M activities. Putative R-M systems with significant variation were detected in Caldicellulosiruptor species based on REBASE (Roberts et al., Nucleic acids research 2010, 38:D234-236) and GenBank (Benson et al., Nucleic acids research 2010, 38:D46-51) analysis. To investigate which, if any, of these R-M systems are functional, chromosomal DNA was isolated from seven Caldicellulosiruptor species and digested with each of nine different restriction endonucleases, all of which have commercially available cognate methyltransferases (Table 3 and FIG. 7). We found that all species tested contain at least three types of functional R-M systems (Table 3). DNA isolated from each of the seven species was resistant to digestion by BamHI and BspEI, indicating the presence of a cognate methyltransferase for these restriction endonucleases is common in this genus. Resistance to digestion by HaeIII was observed for C. bescii, C. hydrothermalis, C. kristjansonii, and C. saccharolyticus. Resistance to digestion by MboI was observed for C. kristjansonii, C. saccharolyticus, C. obsidiansis, C. lactoaceticus, and C. kronotskyensis. HaeIII (5′-GGCC-3′) and MboI (5′-GATC-3′) would be expected to act as a formidable barrier for DNA transformation from E. coli for these species, since both enzymes are four base cutters and are known to be absolute barrier to DNA transformation in other microorganisms (Chung et al., Journal of industrial microbiology & biotechnology 2011, 38:1867-1877; Grogan, Journal of bacteriology 2003, 185:4657-4661; Donahue et al., Molecular microbiology 2000, 37:1066-1074). C. kronotskyensis appears to be the most different from the other species in terms of R-M systems, as it has apparent methyltransferase activity specific to HpaII and MspI recognition sites. All seven tested species were sensitive to digestion by AluI, EcoRI, and HhaI (Table 3).
Construction of a cbeI Deletion in C. bescii. 
Transformation of C. bescii with heterologous DNA from E. coli involves in vitro methylation of the E. coli DNA with M.CbeI. (Chung et al., PloS one 2012, 7:e43844). More importantly, the degree of methylation in vitro affected transformation efficiency. To test whether a deletion of cbeI would alleviate restriction of DNA from E. coli in C. bescii and allow transformation of unmethylated DNA, we constructed a chromosomal deletion of cbeI (Cbes2438) in JWCB005 (FIG. 8A, Table 4, Example 3), using a targeted marker replacement strategy previously described (Chung et al., PloS one 2012, 7:e43844). The cbeI knock-out vector, pDCW88, contains a 927 bp DNA fragment that includes both the 5′ (440 bp) and 3′ (487 bp) flanking regions of cbeI, and the wild type pyrF cassette (Example 3) for uracil prototrophic selection of transformants (FIG. 8A, FIG. 9). This non-replicating vector in C. bescii was transformed into JWCB005 (ΔpyrFA) with selection for uracil prototrophy followed by counter-selection for 5-fluoroorotic acid (5-FOA) resistance.
Initial screening of 18 isolates by PCR revealed merodiploids with a mixture of wild type and cbeI deletion genomes. Three of these were further purified on solid medium without 5-FOA and analyzed by PCR amplification of the cbeI locus in the chromosome with primers DC277 and DC239 (FIG. 8). PCR amplification of this locus from the parent strain JWCB005 (dpyrFA) produced the expected wild type  ̃2.4 kb band, while amplification from JWCB018 produced a  ̃1.4 kb band indicating a deletion within this region (FIG. 8B). The site of the deletion was confirmed by DNA sequence analysis of the PCR product. The resulting strain, JWCB018 (ΔpyrF ΔcbeI) (Table 4) was used for further analysis.
The cbeI coding region is located in the chromosome adjacent to the coding region encoding M.CbeI, its cognate methyltransferase (Chung et al., PloS one 2012, 7:e43844; Chung et al., Journal of industrial microbiology & biotechnology 2011, 38:1867-1877). The two coding regions are separated by only 45 bases, and are likely to be transcriptionally coupled. The deletion of cbeI spanned the entire cbeI coding region, but left the potential regulatory region upstream intact, and deleted only 23 bases of the downstream flanking region leaving the entire M.CbeI coding region intact. Chromosomal DNA isolated from JWCB018 was completely protected from cleavage by HaeIII and CbeI in vitro, suggesting that M.CbeI is still functional in JWCB018. Growth of this cbeI deletion mutant was comparable to growth of the parent JWCB005 and the wild type strain.
the JWCB018 is Efficiently Transformed with Unmethylated DNA.
To assess the effect of the cbeI deletion on transformation of C. bescii with unmethylated DNA from E. coli, JWCB005 (ΔpyrFA) and JWCB018 (ΔpyrFA ΔcbeI) were transformed with unmethylated pDCW89 DNA, using a replicating shuttle vector (Example 1) containing a wild type copy of the pyrF allele for uracil prototrophic selection (FIG. 10). No transformants of the parent strain, JWCB005, were detected using unmethylated plasmid DNA isolated from E. coli (<10−8 transformants per μg plasmid DNA). The ΔcbeI strain, however, was readily transformed with unmethylated pDCW89 DNA isolated from E. coli ( ̃1.0×103 transformants per μg of plasmid DNA, FIG. 10A). Methylated plasmid DNA transformed into the parent strain (JWCB005) at a frequency ( ̃0.5×103 transformants per mg plasmid DNA) and the difference may reflect incomplete methylation of the plasmid DNA in vitro. Transformation of C. bescii was initially confirmed by PCR amplification of the pSC101 E. coli replication origin fragment present only in the plasmid. Total DNA isolated from JWCB018 transformants was used to “back-transform” E. coli and plasmid DNA isolated from these back-transformants was analyzed by restriction digestion (FIG. 10B). pDCW89 DNA isolated from the “back transformants” was indistinguishable from the pDCW89 used to transform C. bescii and showed no obvious signs of rearrangement or deletion through transformation into JWCH018, replication in C. bescii, or back-transformation to E. coli (FIG. 10B).
Plasmid DNA Isolated from C. hydrothermalis Readily Transforms Strain JWCB005 (ΔpyrFA) without In Vitro Methylation.
Plasmid DNA was isolated from C. hydrothermalis transformants and used to transform C. bescii. Transformants were obtained at frequencies comparable to M.CbeI-methylated plasmid ( ̃0.5×103 per mg of plasmid DNA). The presence of pDCW89 in transformants was confirmed using PCR amplification of the aac (apramycin resistance gene), pSC101 ori region, and pyrF cassette, contained only on the plasmid. The size of the PCR products obtained in this analysis were as expected and were generated from total DNA isolated from the JWCB005 transformants and plasmid DNA isolated from E. coli, but not from JWCB005 (FIG. 11). Total DNA, isolated from JWCB005 transformants, was back-transformed to E. coli for further analysis. Restriction analysis of plasmid DNA isolated from back-transformants showed that pDCW89 was structurally stable through transformation and replication in C. bescii. Thus, we have constructed a variant of C. bescii that possesses a deletion of cbeI (Cbes2438) sufficient to disrupt cbeI activity that is otherwise an absolute barrier to transformation of C. bescii with heterologous DNA. Disruption of cbeI activity is the first targeted genetic deletion demonstrated in a Caldicellulosiruptor spp. and the resulting variant, JWCB018 (ΔpyrFA ΔcbeI), is readily transformed by heterologous DNA without in vitro methylation. PCR amplification and sequencing suggested that this deletion left the adjacent methyltransferase (M.CbeI) intact and its function was confirmed by the fact that chromosomal DNA isolated from JWCB018 was protected from digestion by CbeI and HaeIII in vitro.
While exemplified in the context of a deletion of the entire cbeI coding region, the genetic manipulation that allows for transformation of C. bescii with heterologous DNA can include any modification that interferes with CbeI restriction activity. such modifications can include, for example, a partial deletion of the cbeI coding region sufficient to disrupt expression of the remaining cbeI coding region and/or disrupt CbeI restriction activity of any CbeI fragment polypeptide that may be expressed.
Also, while exemplified in the context of permitting transformation of C. bescii with heterologous DNA isolated from E. coli, the heterologous DNA used to transform the C. bescii variant can be isolated from any appropriate source or prepared synthetically.
The construction of this variant C. bescii strain removes a substantial barrier to transformation and chromosomal modification. Moreover, the variant C. bescii strain permits genetic manipulation without labor intensive such as, for example, modifying the vector prior to transformation, using engineered vectors containing no or fewer restriction sites recognized by restriction endonuclease in host, conditionally inactivating the R-M systems, and/or using group II intron insertion technology.
The ability to make targeted genetic deletions is itself a powerful and direct tool for the investigation of in vivo genetic function and the deletion of this endonuclease resulted in a strain that can provide the basis for further genetic manipulation. The combined efficiencies of transformation and homologous recombination (with as few as 450 bp of homology) in C. bescii allows one to use non-replicating plasmids for genetic manipulation. This is fortuitous and a significant benefit for the development of Caldicellulosiruptor species as consolidated bioprocessing (CBP) organisms. The proven CBP microbe, Clostridium thermocellum, for example, is genetically tractable but the efficiency of transformation and/or recombination does not permit the use of non-replicating plasmids for marker replacement, significantly extending the time required for mutant construction.
Thus, in one aspect, this disclosure describes a method for improving transformation efficiency of a microbe in which restriction is a barrier to transformation. Generally, the method includes genetically modifying the microbe to decrease restriction activity and introducing a heterologous polynucleotide into the genetically modified microbe. In some embodiments, the microbe may be a microbe in which restriction is an absolute barrier to transformation with a heterologous polynucleotide.
In some embodiments, the genetic modification can include a deletion of at least a portion of a coding region that encodes a restriction endonuclease sufficient to reduce—in some cases even eliminate—restriction activity of the endonuclease and, therefore, allow maintenance of the heterologous polynucleotide in the genetically modified and transformed host cell.
Isolation of JWCB005, a C. bescii Variant for Nutritional Selection of Transformants.
Attempts to use drug resistance markers for selection of transformants in C. bescii are often unsuccessful either because the genetic products are unstable at 75° C. and/or because of high levels of natural resistance in C. bescii. Orotidine monophosphate (OMP) decarboxylase, encoded by the pyrF coding region 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, a toxic product that kills growing cells that are synthesizing uracil. 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.
Certain pyrF mutants can be constructed by deleting most of the pyrBCF region. Such mutants thus require complementation of all three coding regions for successful transformation. Transformation efficiency generally decreases as plasmid size increases, so it can be difficult to efficiently transform such a mutant.
We isolated a different mutant that was complemented by the pyrF coding region alone. 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 6), had an 878 bp deletion that spans most of the pyrF open reading frame (Cbes1377), and part of the adjacent gene, pyrA (Cbes1378) (FIG. 12A). The extent of the deletion was defined by PCR amplification of the pyrFA region in the mutant and subsequent sequencing of the PCR product (FIG. 12B). We also PCR amplified and sequenced the pyrE region, also required for uracil biosynthesis, and found it to be wild type (FIG. 12C).
JWCB005 is a tight uracil auxotroph capable of growth in media supplemented with uracil, but not orotate, confirming that pyrF function was absent in this deletion. The function of pyrA does not seem to be affected by the deletion, because transformation with pDCW89, containing only the wild type pyrF allele, was able to complement the uracil auxotrophy without added orotate, the product of pyrA in uracil biosynthetic pathway. As with all such deletions, reversion to uracil prototrophy was not a concern making prototrophic selection possible no matter how low the frequency of transformation. Growth of this mutant (JWCB005) supplemented with uracil (40 μM) was comparable to that of the wild type, reaching a cell density of  ̃2×108 in 24 hours.
Construction of a Replicating Shuttle Vector Based on pBAS2.
C. bescii contains two native plasmids, pBAL and pBAS2, 8.3 kb and 3.7 kb, respectively (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. To avoid disrupting the replication functions of the pBAS2 plasmid, we linearized the plasmid DNA just upstream of the Cbes2777 ORF and inserted the aac coding region for selection of apramycin resistance in E. coli. The C. bescii pyrF gene, under the transcriptional control of the promoter of the ribosomal protein Cbes2105 (30S ribosomal protein S30EA), was used for selection of uracil prototrophy in the C. bescii pyrFA deletion mutant (JWCB005), and the pSC101 replication origin for replication in E. coli. 
The resulting plasmid, pDCW89 (FIG. 13A), was transformed into C. bescii by electroporation, and cells were plated onto defined medium without uracil as described (Chung et al. (2012) PLoS One 7: e43844). Uracil prototrophic colonies were selected and transformation was confirmed by PCR amplification of a portion of the pSC101 replication origin present only in pDCW89 (FIG. 13B). Total DNA isolated from two biologically independent transformants was used to back-transform E. coli. Restriction digestion analysis showed that the plasmid was unchanged during transformation and replication in C. bescii and/or subsequent back transformation to E. coli (FIG. 13C). This result suggests that it was replicating autonomously in both organisms. The resulting strain was designated JWCB011 (Table 6). The transformation frequency varied between experiments, but was typically about 500 transformants per μg of plasmid DNA. This efficiency was 10 times higher than the transformation efficiency observed with non-replicating plasmids in C. bescii (Chung et al. (2012) PLoS One 7: e43844).
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. (2013) Journal of industrial Microbiology & Biotechnology 40:41-49). Total DNA isolated from cells after each passage was used for Southern hybridization analysis (FIG. 16). To generate a probe for the detection of a sequence contained once on both the plasmid and the chromosome, primers JF396 and JF397 were used to amplify a fragment of the pyrF coding region remaining in the genome of JWCB005, and also contained on the plasmid. Relative copy number was determined as the ratio of band intensity of the plasmid derived copy of the pyrF locus (7.7 kb) compared to the chromosomal derived copy of the pyrF locus (3.7 kb) in JWCB005 (FIG. 16). The relative intensity was 0.8 to 1.1 suggesting that the shuttle vector exists as a single copy per chromosome (FIG. 16). 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 reflect the fact that it competes with the endogenous replicon as they share replication and maintenance functions. The 4.3 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 (FIG. 16).
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 (FIG. 16). A single passage in nonselective media (with 40 μM uracil) is enough for the plasmid to be lost from the majority of cells (FIG. 16).
Transformation of C. hydrothermalis with Shuttle Vector DNA Methylated with M.CbeI.
Restriction of transforming DNA is a barrier to transformation with heterologous DNA (e.g., from E. coli). Transformation of plasmid DNA from E. coli into wild-type 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: 10.1007/s10295-10013-11244-z), JWCH003 (Table 6). 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 that has been 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 the aac coding region contained exclusively on the plasmid. As shown in FIG. 17, restriction digestion analysis using HindIII and EcoRI of shuttle vector plasmid DNA isolated from C. hydrothermalis transformants was indistinguishable from that isolated from E. coli (FIG. 17B and FIG. 17C) suggesting that it is structurally stable in C. hydrothermalis. Thus, modification with M.CbeI may have utility in DNA transformation of a variety of Caldicellulosiruptor species. These data also provide evidence that the use of the wild type C. bescii pyrF allele under the control of the ribosomal protein S30EA promoter functions in at least one other species and may be a useful selection marker for many species. This shuttle vector may, therefore, facilitate extension of genetic methods to a number of other Caldicellulosiruptor species.
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. FIG. 17A shows that the uncut plasmid DNA isolated from C. hydrothermalis migrated slower than that isolated from E. coli. This may be due, at least in part, to differences in the degree of methylation of the DNA in these different hosts. These results suggest that C. hydrothermalis, like C. bescii, contains a functional CbeI/M.CbeI like restriction-modification system and is consistent with the observation that pDCW89 isolated from C. hydrothermalis was resistant to digestion by purified CbeI or HaeIII (NEB) endonucleases (FIG. 17D).
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 (FIG. 18, pDCW129). Methylated pDCW129 was successfully transformed into JWCB005 at a comparable transformation efficiency to pDCW89. Transformation of C. bescii with pDCW129 was initially confirmed by PCR amplification of the region spanning the pyrF cassette and only in the plasmid (FIG. 18B). Total DNA isolated from JWCB018 transformants was used to “back-transform” into E. coli and plasmid DNA isolated from these back-transformants was analyzed by restriction digestion by EcoRV (FIG. 18C) and EcoRI and AatII. pDCW129 DNA isolated from the “back transformants” was indistinguishable from the pDCW129 used to transform C. bescii and showed no obvious signs of rearrangement or deletion through transformation and replication in JWCB005.
Thus, JWCB005, which contains a small deletion within the pyrFA locus, allows complementation of uracil auxotrophy with a single gene, pyrF. We constructed an E. coli/C. bescii shuttle vector by combining a native plasmid, pBAS2, with an E. coli vector and a pyrF cassette for nutritional selection of transformants (FIG. 13A). Methylation with M.CbeI methyltransferase was required for transformation of plasmid DNA isolated from E. coli into C. bescii (Chung et al. (2012) PLoS One 7: e43844). While it is possible that there is another endonucleases present in C. bescii, modification of heterologous DNA by M.CbeI was sufficient for successful transformation, and rescued plasmid did not show any signs of rearrangement during transformation and replication in C. bescii (FIG. 13C and FIG. 18C).
The shuttle vector replicates autonomously in C. bescii in single copy per chromosome and is stably maintained under selection, but quickly lost without selection (FIG. 16). This feature of the plasmid could be advantageous for future genetic applications that require plasmid curing, eliminating the need for counter-selection with 5-FOA or other antimetabolites that are potentially mutagenic. Single copy plasmids have many additional advantages over high copy plasmids providing expression of genes at physiologically relevant levels, and complementation analysis.
This shuttle is selectable in a uracil auxotrophic mutant of C. hydrothermalis. The development of genetic system in C. hydrothermalis is important for a number of reasons. C. hydrothermalis contains fewer IS elements compared with other Caldicellulosiruptor species, and may exhibit fewer genome stability issues associated with stress conditions such as, for example, nutritional selections and counter-selections. C. hydrothermalis is one of the least cellulolytic species of the eight well-characterized Caldicellulosiruptor species (Blumer-Schuette et al. (2012) J Bacteriol 194: 4015-4028) and provides the opportunity to explore the mechanisms (or key enzymes) related to plant biomass degradation by heterologous expression of genes derived from the most cellulolytic Caldicellulosiruptor species, C. bescii and C. saccharolyticus (Blumer-Schuette et al. (2012) J Bacteriol 194: 4015-4028).
Moreover, the shuttle vector may be used to genetically modify pyrF mutant strains in other Caldicellulosiruptor species. For example, its use for cloning homologous proteins (e.g., Ce1A) will allow the study of enzymes predicted to be glycosylated in vivo making homologous expression essential.
In another aspect, this disclosure describes the direct conversion of plant biomass to ethanol by C. bescii that have been genetically modified using the molecular tools described above. While exemplified in the context of converting plant biomass to ethanol, the methods exemplified may be modified to genetically modify other Caldicellulosiruptor species and/or to modify the Caldicellulosiruptor microbes to produce other products such as, for example, any carbon-based product. Exemplary carbon-based products include, for example, carbon-based fuels (e.g., ethanol, jet fuel, etc.) and/or commodity chemicals.
Heterologous Expression of the Clostridium thermocellum adhE Gene in C. bescii. 
The C. thermocellum adhE coding region (Cthe0423) is a bifunctional acetaldehyde-CoA/alcohol dehydrogenase. The coding region was amplified from C. thermocellum (ATCC 27405) chromosomal DNA and cloned into pDCW144 (FIG. 19 and FIG. 20) under the transcriptional control of the C. bescii S-layer protein (Cbes2303) promoter (PS-layer). RNA profiling has shown that S-layer protein RNA is abundant and present throughout growth suggesting that the promoter may be strong and constitutive. A rho-independent transcription terminator derived from a region immediately downstream of Cbes2303 was fused to the end of adhE coding region (FIG. 20A and FIG. 19) and the vector adds a C-terminal His-tag to the AdhE protein. In addition, the final vector, pDCW144, contained a 2.025 kb DNA fragment from the intercistronic region between Cbes0863 and Cbes0864 (FIG. 20A and FIG. 19) to allow targeted integration into the C. bescii chromosome. The vector does not contain an origin of replication for C. bescii and cannot replicate autonomously. It also contained a wild type pyrF expression cassette (Example 3).
pDCW144 was transformed into JWCB018 (ΔpyrFA ldh::ISCbe4 Δcbe1), which contains a deletion of the CbeI restriction enzyme and an insertion into the ldh coding region. It also contains a deletion in the pyrF locus resulting in uracil auxotrophy and allowing for selection for uracil prototrophy followed by counter-selection for 5-fluoroorotic acid (5-FOA) resistance as described in Example 2 and depicted in FIG. 20A.
Two of the forty transformants analyzed by PCR amplification using primers DC477 and DC478 (FIG. 20B) contained segregated insertions of the PS-layer-adhE cassette at the targeted chromosome site. As shown in FIG. 20, the parent strain, JWCB018, produced the expected wild type  ̃2.44 kb band, while amplification from JWCB032 produced a  ̃5.04 kb band indicating a knock-in of expression cassette within this region (FIG. 20B).
Expression of the AdhE protein was detected in transformants containing the expression cassette by Western hybridization using commercially available His-tag monoclonal antibody (FIG. 20C). Cells were grown in LOD medium with 10 g/l cellobiose and 100 μM uracil to mid-log phase at 60° C., 65° C., or 70° C. The wild type AdhE protein was expressed and readily detected in cells grown at 60° C. and 65° C., but not at 70° C. Since the optimal temperature for growth for C. thermocellum is 60° C. (Brown et al. (2011) Proc Natl Acad Sci USA 108: 13752-13757), the AdhE protein or adhE mRNA may not be stable at 70° C.
We also expressed a variant of the AdhE protein, AdhE*, from C. thermocellum that has been shown to increase ethanol tolerance without losing functional activity for ethanol production (Brown et al. (2011) Proc Natl Acad Sci USA 108: 13752-13757). The construction of JWCB033 (ΔpyrFA/Δcbe1::PS-layerCthe-adhE*) was the same as that for JWCB032. Interestingly, the AdhE* was detected at 60° C., but not 65° C. or 70° C. suggesting that either the protein or its mRNA it is less thermostable than the wild type.
Analysis of Metabolic Products from C. bescii Containing the C. thermocellum adhE Coding Region.
High performance liquid chromatography (HPLC) was performed to determine the fermentation products from C. bescii mutant strains. Strain JWCB018 (ΔpyrFA ldh::ISCbe4 Δcbe1) is the background genetic strain used in these studies. Strain JWCB032 (ΔpyrFA ldh::ISCbe4 Δcbe1::PS-layerCthe-adhE) contains the C. thermocellum adhE coding region described above. FIG. 21 shows fermentation product profiles from batch cultures grown at 65° C. with different substrates, 10 g/liter cellobiose, 20 g/liter AVICEL, or 20 g/liter unpretreated switchgrass.
Wild type C. bescii, JWCB018, JWCB032 and JWCB033 were grown on 1% cellobiose, and fermentation products were analyzed by HPLC (FIG. 21). JWCB032 produced ethanol up to 14.8 mM (FIG. 21A). No lactate was detected in the mutants JWCB018, JWCB032, and JWCB033, compared to approximately 2.7 mM lactate from the wild-type (FIG. 21C). JWCB018 and JWCB033 show higher amounts of acetate produced compared other strains, wild-type and JWCB032 (FIG. 21B) because JWCB018 and JWCB033 produce only acetate and hydrogen as final products from pyruvate (FIG. 21). In contrast, wild-type C. bescii produces lactate, acetate, and hydrogen, while JWCB032 produces ethanol, acetate and hydrogen as final products.
To compare the final products of fermentations on different substrates, a model microcrystalline cellulosic substrate, AVICEL (2%, wt/vol), and a real world substrate, unpretreated switchgrass (2%, wt/vol), were used for batch fermentations by C. bescii strains. When grown on AVICEL, wild-type produced lactate (3.1 mM), acetate (5.4 mM) and no ethanol (FIG. 22). JWCB018 and JWCB033 show similar final product profiles: acetate (7.3 ̃8.2 mM), no lactate, and no ethanol (FIG. 22). The engineered strain, JWCB032, however, shows the direct production of ethanol from 2% AVICEL up to approximately 14 mM (FIG. 22A) and lowest acetate production (4.3 mM) (FIG. 22B). The maximum ethanol produced on 2% AVICEL (14.0 mM) was similar to that on 1% cellobiose (14.6 mM).
The genetically engineered strain, JWCB032, is able to produce ethanol directly from unpretreated switchgrass. All four C. bescii strains in this study were grown on 2% switchgrass, and the end product analysis shows similar profiles for ethanol (FIG. 23A), acetate (FIG. 23B) and lactate (FIG. 23C) productions compared to those on cellobiose (FIG. 21) and AVICEL (FIG. 22). JWCB032 produce 11.0 mM of ethanol at 40 hours of incubation and reached 12.8 mM of ethanol accumulation at 120 hours of incubation.
Growth Properties of Ethanol-Producing C. bescii Strains and Ethanol Tolerance of C. bescii. 
To determine the overall effect of heterologous expression of adhE and its derivative on C. bescii growth properties, growth of JWCB032 and JWCB033 were compared to the wild type and parental (JWCB018) strains in LOD medium supplemented with 1% cellobiose as carbon source and 1 mM uracil (FIG. 24). The expressed enzymes AdhE and AdhE* are only stable and active near or below 65° C. based on the Western-blot analysis shown in FIG. 20C and fermentation product analysis (FIGS. 22-24). Therefore, we performed analyses at both 65° C. (FIG. 24A) and 75° C. (FIG. 24B). Growth at 75° C. was faster than at 65° C. Based on growth curves shown in FIG. 24, C. bescii cells reached stationary phase after approximately 12 hours at 75° C., but it took approximately 20 hours at 65° C. C. bescii were growing up to an OD680nm of 0.5-0.6 with shaking at 150 rpm. These results indicate that the expression of AdhE and its derivative do not negatively affect growth compared to the parental strain (JWCB018) and wild type.
To determine the C. bescii ethanol tolerance, the wild-type strain was assayed for its ability to grow in LOD medium with 1% cellobiose as the sole carbon source and elevated levels of ethanol at both 65° C. and 75° C. with shaking (150 rpm) (FIG. 25). Growth of C. bescii in the presence of 200 mM ethanol concentration in LOD media was comparable with the no ethanol medium at both 65° C. and 75° C. However, C. bescii exhibits higher ethanol tolerance at 65° C. than at 75° C. The 450 mM ethanol concentration has a modest effect on growth at 65° C., but it significantly inhibits growth at 75° C. The growth of C. bescii was completely inhibited at 600 mM ethanol concentration. The ethanol tolerance of C. bescii is comparable to Clostridium thermocellum ATCC27405 (Brown et al. (2011) Proc Natl Acad Sci USA 108: 13752-13757).
EXEMPLARY EMBODIMENTS
Embodiment 1
A recombinant Caldicellulosiruptor microbe genetically modified to produce a greater amount of acetate than a comparable wild type control.
Embodiment 2
A recombinant Caldicellulosiruptor microbe genetically modified to produce a greater amount of H2 than a comparable wild type control.
Embodiment 3
A recombinant Caldicellulosiruptor microbe genetically modified to produce that produces a greater amount of ethanol than a comparable wild type control.
Embodiment 4
The recombinant Caldicellulosiruptor microbe of any preceding exemplary Embodiment wherein the Caldicellulosiruptor microbe is Caldicellulosiruptor bescii. 
Embodiment 5
The recombinant Caldicellulosiruptor microbe of any preceding exemplary Embodiment wherein the recombinant Caldicellulosiruptor microbe produces lactate in an amount less than a comparable wild type control.
Embodiment 6
The recombinant Caldicellulosiruptor microbe of any preceding exemplary Embodiment comprising a deletion of at least a portion of a lactate dehydrogenase coding region.
Embodiment 7
The recombinant Caldicellulosiruptor microbe of exemplary Embodiment 6 wherein the lactate dehydrogenase coding region is Cbes_1918.
Embodiment 8
The recombinant Caldicellulosiruptor microbe of any preceding exemplary Embodiment genetically modified to exhibit decreased CbeI endonuclease activity compared to a comparable wild-type control.
Embodiment 9
The recombinant Caldicellulosiruptor microbe of any preceding exemplary Embodiment genetically modified to exhibit increased efficiency of transformation with unmethylated heterologous DNA.
Embodiment 10
The recombinant Caldicellulosiruptor microbe of exemplary Embodiment 8 or exemplary Embodiment 9 comprising a deletion of at least a portion of a cbeI coding region.
Embodiment 11
A recombinant Caldicellulosiruptor microbe genetically modified to exhibit decreased restriction compared to a comparable wild-type control compared to a comparable wild-type control.
Embodiment 12
A recombinant Caldicellulosiruptor microbe genetically modified to exhibit increased efficiency of transformation with unmethylated heterologous DNA.
Embodiment 13
The recombinant Caldicellulosiruptor microbe of exemplary Embodiment 11 or exemplary Embodiment 12 comprising a deletion of at least a portion of a cbeI coding region.
Embodiment 14
The recombinant Caldicellulosiruptor microbe of any one of exemplary Embodiments 10-13 further comprising a heterologous DNA.
Embodiment 15
A method comprising:
growing the recombinant Caldicellulosiruptor microbe of any one of exemplary Embodiments 1 and 4-7 under conditions effective for the recombinant Caldicellulosiruptor microbe to produce acetate.
Embodiment 16
The method of exemplary Embodiment 15 further comprising collecting at least a portion of the acetate.
Embodiment 17
A method comprising:
growing the recombinant Caldicellulosiruptor microbe of any one of exemplary Embodiments 2 and 4-7 under conditions effective for the recombinant Caldicellulosiruptor microbe to produce H2.
Embodiment 18
The method of exemplary Embodiment 17 further comprising collecting at least a portion of the H2.
Embodiment 19
A method comprising:
growing the recombinant Caldicellulosiruptor microbe of any one of exemplary Embodiments 3-7 under conditions effective for the recombinant Caldicellulosiruptor microbe to produce ethanol.
Embodiment 20
The method of exemplary Embodiment 19 further comprising collecting at least a portion of the ethanol.
Embodiment 21 The method of any one of exemplary Embodiments 15-20 wherein the conditions comprise a carbon source that comprises napier grass, Bermuda grass, switchgrass, or a hardwood.
Embodiment 22
The method of exemplary Embodiment 20 wherein the carbon source comprises unpretreated switchgrass.
Embodiment 23
A method comprising:
introducing a heterologous polynucleotide into the Caldicellulosiruptor microbe of any one of exemplary Embodiments 11-14.
Embodiment 24
The method of exemplary Embodiment 23 wherein the heterologous polynucleotide is unmethylated.
Embodiment 25
A method for improving transformation efficiency of a microbe in which restriction is a barrier to transformation, the method comprising:
genetically modifying the microbe to decrease restriction activity; and
introducing a heterologous polynucleotide into the genetically modified microbe.
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.
EXAMPLES
Example 1
Metabolic Engineering of Caldicellulosiruptor bescii Yields Increased Hydrogen Production from Lignocellulosic Biomass
Strains, Growth Conditions and Molecular Techniques.
A spontaneous mutant containing a deletion within the pyrFA locus of C. bescii, JWCB005 (Chung et al., PLoS One 2013, 8:e62881 (Example 3); Chung et al., Biotech biofuels 2013, 6:82 (Example 2)), was used in this study to select transformants. C. bescii strains were grown in modified DSMZ516 medium or LOD (low osmolality defined growth medium) (Farkas et al., J Ind Microbiol Biotechnol 2013, 40:41-49) containing 0.5% maltose as carbon source, final pH 7.0. 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. A solid medium was prepared by mixing an equal volume of liquid medium at a 2× concentration with the same volume of (wt/vol) agar, 3.6% (Difco, Sparks, Md.) that had been previously autoclaved. Both solutions were maintained at 70° C. and poured into petri dishes immediately after mixing. A series of dilutions of this culture were mixed with 4 ml of soft top agar (1.5% of agar) and poured across the top of the solid agar medium. The plates were degassed with five cycles of vacuum and argon and incubated at 75° C. for 4 days in anaerobic jars. E. coli DH5α was used to prepare plasmid DNA. Cells were grown in LB broth supplemented with apramycin (50 μg/ml). Plasmid DNA was isolated using a Qiagen Mini-prep Kit (Qiagen; Valencia, Calif.). A complete list of strains, plasmids, and primers used in this study is shown in Tables 1 and 2.







TABLE 1







Plasmids and C. bescii strains (JWCB) used in Example 1.









Strains and

Source or


Plasmids
Description and/or relevant characteristics
reference





pDCW88
Non-replicating plasmid in C. bescii
A (Example 2)


pDCW121
ldh knock-out plasmid
This study


JWCB001
C. bescii wild-type DSM 6725
B


JWCB005
DSM 6725 ΔpyrF4
C (Example 3)


JWCB017
DSM 6725 ΔpyrF4 Δldh
This study





A: Chung et al., Biotech biofuels 2013, 6: 82


B: Svetlichnyi et al., Microbiol 1990, 59: 598-604


C: Chung et al., PLoS One 2013, 8: e62881













TABLE 2







Oligonucleotides used in Example 1.











SEQ


Primer
Sequence 5′-3′
ID No:





DC081
TCCAATGATCGAAGTTAGGCTGGT
1


 


DC348
GAATTCTCTGACGCTCAGTGGAACGAA
2


 


DC349
GAAAACAAATGGGCTTGGGAGGATAGGAGGCTGT
3


 


DC350
TGGGCTTGGGAGGATAGGAGGCTGTCTAAAAACAA
4


 


DC351
TGCCAAGATATGAAATGAGAACT
5


 


DC356
CGTCTCATCTGTGCATATGGACAGTTATAATCCCA
6



AAAGGAGGATTGGATCC









Construction of pDCW121.

To construct a plasmid for deletion of the ldh coding region (Cbes1918), three cloning steps including overlapping polymerase chain reactions were used. All PCR amplifications were performed using Pfu Turbo DNA polymerase (Agilent Technologies; Santa Clara, Calif.). A 1009 bp fragment containing a KpnI site upstream of the ldh coding region was amplified using primers DC348 and DC349. A 1,011 bp fragment containing an EcoRI site downstream of ldh, was amplified using primers DC350 and DC351. The two fragments were joined by overlapping PCR using primers DC348 and DC351 to generate a 2,020 bp product that was cloned into pDCW88 (Chung et al., Biotech biofuels 2013, 6:82 (Example 2)) using the Kpn1 and EcoRI sites. The resulting plasmid, pDCW121, was transformed into E. coli DH5α by an electrotransformation via a single electric pulse (1.8 kV, 25 μF and 200Ω) in a pre-chilled 1 mm cuvette using a Bio-Rad gene Pulser (Bio-Rad Laboratories; Hercules, Calif.). Transformants were selected on LB solid medium containing apramycin (50 μg/ml final).
Competent Cells, Transformation and Mutant Selection in C. bescii. 
To prepare competent cells, a 50 ml culture of JWCB005 was grown in LOD minimal medium at 75° C. for 18 hours (to mid exponential phase) and 25 ml of the culture was used to inoculate a 500 ml culture of LOD (low osmolarity defined growth medium) supplemented with 40 μM uracil and a mixture of 19 amino acids (5% inoculum, v/v) (Farkas et al., J Ind Microbiol Biotechnol 2013, 40:41-49). The 500 ml culture was incubated at 75° C. for 5 hours and cooled to room temperature for 1 hr. Cells were harvested by centrifugation (6000×g, 20 min) at 25° C. and washed three times with 50 ml of pre-chilled 10% sucrose. After the third wash, the cell pellet was resuspended in 50 μl of pre-chilled 10% sucrose in a microcentrifuge tube and stored at −80° C. until needed. Before transformation, plasmids from E. coli cells were methylated in vitro with C. bescii methyltransferase (M.CbeI, Chung et al., PLoS One 2012, 7:e43844) and methylated plasmid DNAs (0.5-1.0 μg) were added to the competent cells, gently mixed and incubated for 10 minutes in ice. Electrotransformation of the cell/DNA mixture was performed via single electric pulse (1.8 kV, 25 μF and 350Ω) in a pre-chilled 1 mm cuvette using a Bio-Rad gene Pulser (Bio-Rad Laboratories; Hercules, Calif.). After pulsing, cells were inoculated into 10 ml of LOC medium (low osmolarity complex growth medium; Farkas et al., J Ind Microbiol Biotechnol 2013, 40:41-49) and incubated for 4 hours at 75° C. 100 μl of the culture was transferred into 20 ml of defined medium without uracil. After 18 hours incubation at 75° C., cells were harvested by centrifugation (at 6000×g for 20 min) and resuspended in 1 ml of 1× basal salts. 100 microliters of the cell suspension was plated onto solid defined media with 40 μM uracil and 8 mM 5-FOA (5-fluoroorotic acid monohydrate).
Analytical Techniques for Determining Fermentation End Products.
Batch fermentations were conducted in stoppered 125 ml serum bottles containing 50 ml LOD medium with 5 g/1 maltose, cellobiose or switchgrass. Cultures of JWCB005 and JWCB017 were supplemented with 40 μM uracil. Triplicate bottles were inoculated with a fresh 2% (v/v) inoculum and incubated at 75° C. without shaking. Total cell dry weight (CDW) was determined by concentrating 25 ml of each culture on dried, preweighed 47 mm Supor membrane filters (0.45, Pall Corporation; Port Washington, N.Y.) and washed with 10 ml of ddH2O. Cell retentates were dried for 16 hours at 85° C. and weighed on an analytical balance. Culture supernatants were analyzed via HPLC using a Waters Breeze 2 system (Waters Chromatography; Milford, Mass.) operated under isocratic conditions at 0.6 ml/min with 5 mM H2SO4 as a mobile phase. Analytes were separated on an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, Calif.) at 60° C. and monitored via refractive index (RI) using a Waters 2414 RI detector. Total peak areas were integrated using Waters Breeze 2 software and compared against peak areas and retention times of known standards for each analyte of interest. H2 was measured using an Agilent Technologies 6850 Series II Gas Chromatograph equipped with a thermal conductivity detector at 190° C. with a N2 reference flow and a HP-PLOT U Column (30 m×0.32 mm). To measure organic acid production, Nuclear magnetic resonance (NMR) analysis was performed. One-dimensional 1H-NMR spectra were recorded at 298° K with a Varian Inova-NMR operating at 600 MHz for 1H and equipped with a 5-mm NMR cold probe. Samples (500 μL) of cell free culture media were mixed with 150 μL of D2O as internal lock and immediately analyzed. 128 scans were recorded for each sample using a pre-saturation method to suppress the water resonance. The amounts of the most abundant components in the samples were calculated by integration of the proton signals in the spectra. The data were normalized to the amount of acetic acid in each sample.
Biomass Preparation.
Air-dried switchgrass (Panicum virgatum, Alamo variety) was reduced to 60 mesh using a Wiley Mini-Mill (Thomas Scientific; Swedesboro, N.J.). The ground switchgrass was subjected to a hot water treatment similar to that described by Yang et al. (Appl Environ Microbiol 2009, 75:4762-2769) however the biomass was boiled in distilled H2O (2% w/v) for 1 hour rather than treating overnight at 75° C. The switchgrass was then washed and dried overnight at 50° C. before dispensing into serum bottles as previously described (Yang et al., Appl Environ Microbiol 2009, 75:4762-2769).
Example 2
Overcoming Restriction as a Barrier to DNA Transformation in Caldicellulosiruptor Species Results in Efficient Marker Replacement
Strains, Media and Growth Conditions.
Caldicellulosiruptor and E. coli strains used in this study are listed in Table 4. All Caldicellulosiruptor species were grown anaerobically in liquid or on solid surface in either modified DSMZ 516 medium (Chung et al., PloS one 2012, 7:e43844) or in low osmolarity defined (LOD) medium (Farkas et al., Journal of industrial microbiology & biotechnology 2013, 40:41-49) with maltose as the carbon source. C. bescii, C. kristjansonii, and C. obsidiansis were incubated at 75° C. C. hydrothermalis, C. kronotskyensis, C. lactoaceticus, and C. saccharolyticus were incubated at 68° C. For growth of auxotrophic mutants, the defined medium contained 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, 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 Qiagen Mini-prep Kit. Chromosomal DNA from Caldicellulosiruptor strains was extracted using the Quick-gDNATM MiniPrep (Zymo Research; Irvine, Calif.) or using the DNeasy Blood & Tissue Kit (Qiagen; Valencia, Calif.) according to the manufacturer's instructions. Plasmid DNA isolation from Caldicellulosiruptor species was performed as described (Chung et al., Journal of industrial microbiology & biotechnology 2011, 38:1867-1877).







TABLE 4







Strains and plasmids used in Example 2.









Strain or




plasmid
Strain and genotype/phenotype
Source










Caldicellulosiruptor









C. bescii
wild type/(ura+/5-FOAS)
DSMZ1


DSM6725


C. hydrothermalis
wild type/(ura+/5-FOAS)
DSMZ1


DSM18901


C. kristijansonii
wild type/(ura+/5-FOAS)
DSMZ1


DSM12137


C. saccharolyticus
wild type/(ura+/5-FOAS)
DSMZ1


DSM8903


C. obsidiansis
wild type/(ura+/5-FOAS)
DSMZ1


ATCC BAA-2073


C. lactoaceticus
wild type/(ura+/5-FOAS)
DSMZ1


DSM9545


C. kronotskyensis
wild type/(ura+/5-FOAS)
DSMZ1


DSM12137


JWCB005
C. bescii ΔpyrF4/(ura−/5-FOAR)
2


JWCB018
C. bescii ΔpyrFAΔcbeI/(ura−/5-FOAR)
This




study


JWCH003
C. hydrothermalis IScahyI insertion
2, 3



mutation in pryF coding region/



(ura−/5-FOAR)


JWCH005
JWCH003 transformed with M. CbeI
2



methylated pDCW89/(ura+/5-FOAS)







Escherichia coli









JW291
DH5α containing pDCW88 (ApramycinR)
This




study


JW292
DH5α containing pDCW89 (ApramycinR)
2







Plasmids









pDCW88
cbeI knock-out vector (ApramycinR)
This




study


pDCW89
E. coli/Caldicellulosiruptor species shuttle
2



vector (ApramycinR)





1German Collection of Microorganisms and Cell Cultures


2 Chung D et al., PLoS one, 2013, 8: e62881 (Example 3)


3 Chung et al., Journal of industrial microbiology & biotechnology 2013. 10.1007/s10295-013-1244-z Construction of pDCW88.






A 927 bp DNA fragment containing the 5′ flanking region (440 bp) and the 3′ flanking region (487 bp) of cbeI (Cbes2438) was generated by overlap extension polymerase chain reaction (OE-PCR) using primers DC265 (with KpnI site), DC266, DC267, and DC268 (with ApaLI site). All PCR reactions were performed using pfu turbo (Agilent Technologies; Santa Clara, Calif.), and C. bescii genomic DNA as a template. The DNA fragments containing the apramycin resistance cassette, pyrF cassette, and the E. coli pSC101 replication origin, were amplified from pDCW 89 (Example 3) using primers DC081 (with KpnI site) and DC262 (with ApaLI site). These two linear DNA fragments were digested with KpnI and ApaLI, and ligated to generate pDCW88 using Fast-link DNA Ligase kit (Epicentre Biotechnologies; Madison, Wis.) according to the manufacturer's instructions. DNA sequences of the primers are shown in Table 5. A diagram of pDCW88 is shown in FIG. 9. 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 sequence of pDCW88 was confirmed by Automatic sequencing (Macrogen, Inc.; Rockville, Md.). All plasmids are available on request.







TABLE 5







Primers used in Example 2.












SEQ



Primers
Sequences (5′ to 3′)
ID No:
Description





DC277 F
TCTACACTCTTGCTTACACAGGT
 7
amplify the cbeI (Cbes_2438) region


 


DC239 R
TCTCCTCGAGCAGACCAAGTGCGTATTTTTC
 8
amplify the cbeI (Cbes_2438) region


 


DC265 F
AGAGAGGTACCTGCAACATCCGGCTTAATGAC
 9
amplify 440 bp of 5′ flanking region 





of (Cbes_2438)


 


DC266 R
TGTTAAAACCACCTACCTAATCTTATCATGTT
10
amplify 440 bp of 5′ flanking region 



GGAAGGCAAATTGA

(Cbes_2438)


 


DC267 F
AGATTAGGTAGGTGGTTTTAACA
11
amplify 487 bp of 3′ flanking region 





(Cbes_2438)


 


DC268 R
TGTGTGGTGCACTCCTTGATAATTTCAGCTGCCT
12
amplify 487 bp of 3 ′flanking 





(Cbes_2438)


 


DC262 F
TGTGTGGTGCACTCTGACGCTCAGTGGAACGAA
13
amplify the E. coli features 





from pDCW 89


 


DC081 R
AGAGAGGTACCACCAGCCTAACTTCGATCATTGGA
14
amplify the E. coli features 





from pDCW 89


 


JF263 F
AGGTACCGGTTCATGTGCAGCTCCATC
15
amplify the aac coding region cassette


 


JF264 R
CTCCAACGTCATCTCGTTCTC
16
amplify the aac coding region cassette


 


DC100 F
TAGTCTTGATGCTTCACTGATAG
17
amplify the pSC101 E. coli replication 





origin


 


JF199 R
CGCTAACGGATTCACCACT
18
amplify the pSC101 E. coli replication 





origin


 


DC233 F
ATCCGTTGATCTTCCTGCAT
19
amplify the pyrF cassette


 


DC235 R
AGGATCTGAGGTTCTTATGGCTC
20
amplify the pyrF cassette









Screening, Purification, and Sequence Verification of Deletion Mutants.

To construct strain JWCB018, one microgram of M.CbeI methylated pDCW88 DNA was used to electrotransform JWCB005 (ΔpyrFA) as described (Chung et al., PloS one 2012, 7:e43844). Cells were then plated onto solid defined medium (without uracil or casein) and uracil prototrophic transformant colonies were inoculated into liquid medium for genomic DNA extraction and subsequent PCR screening of the targeted region. 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. The cultures were plated onto 5-FOA (8 mM) containing solid medium. After initial screening, transformants containing the expected deletion were further purified by three additional passages under selection on solid medium and screened a second time by PCR to check for segregation of the deleted allele. The deletions were then verified by PCR amplification and sequence analysis. A PCR product was generated from genomic DNA by using primers (DC277 and DC239) outside the homologous regions used to construct the deletion, and internal primers were used to sequence the PCR product. For PCR, the extension time was sufficient to allow amplification of the wild-type allele, if it were still present. Another set of primers, one located inside of the Cbes2438 open reading frame, and the other located outside of the flanking region were used for further verification. Growth of this strain, JWCB018, supplemented with uracil (40 μM) was comparable to wild type reaching a cell density of  ̃2×108 in 20 hours. Cells were counted in a Petroff Hausser counting chamber using a phase-contrast microscope with 40× magnification.
Transformation of C. bescii and Selection of Transformants.
Electrotransformations of JWCB005 and JWCB018 with unmethylated pDCW89 from E. coli or isolated plasmid DNA from C. hydrothermalis transformants were performed as described (Chung et al., PloS one 2012, 7:e43844). For selection of transformants, after electro-pulse the recovery cultures with pDCW89 DNA (0.5-1.0 μg) were plated onto the defined medium without casein or uracil. Uracil prototrophic transformants were inoculated into liquid medium for DNA isolation. The presence of plasmid sequences in C. bescii transformants was confirmed by PCR amplification of the aac (apramycin resistance cassette) coding region, the pSC101 ori region, and the pyrF cassette, present only on pDCW89. The transformation frequencies reported herein take into account the number of cells plated as determined by culture cell counts (this does not take into account the plating efficiency), and, where indicated, the total amount of DNA added (i.e., the number of transformants per microgram of DNA). E. coli strain DH5α cells were used for back-transformation.
Restriction Endonuclease Digestion of Caldicellulosiruptor Species Chromosomal DNA.
Chromosomal DNA isolated from seven Caldicellulosiruptor species was subjected to digestion with the REs AluI, BamHI, BspEI, EcoRI, HaeIII, HhaI, HpaII, MboI, and MspI. All enzymes were from New England Biolabs. For each reaction, 1 microgram of DNA was incubated with the enzyme and appropriate buffer for 1 hour according to the manufacturer's instructions. After incubation, digestion patterns were compared by electrophoresis on a 1.0% agarose gel.
Example 3
Construction of a Stable Replicating Shuttle Vector for Caldicellulosiruptor Species: Use for Extending Genetic Methodologies to Other Members of this Genus
Strains, Media and Growth Conditions.
C. bescii, C. hydrothermalis, and E. coli strains used in this study are listed in Table 6. 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. (2013) Journal of industrial Microbiology & Biotechnology 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. (2001) Molecular Cloning: A Laboratory Manual: Cold Spring Harbor Laboratory Press). 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 Qiagen Mini-prep Kit. Chromosomal DNA from Caldicellulosiruptor species was extracted using the Quick-gDNATM MiniPrep (Zymo Research; 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).
Isolation of 5-FOA Resistant/Uracil Auxotrophic Mutants.
A spontaneous deletion within the C. bescii DSM6725 pyrFA locus (FIG. 12A, Table 6) was isolated using the same approaches as previously described (Chung et al. (2012) PLoS One 7: e43844). Growth of this strain, JWCB005, supplemented with uracil (40 μM) was comparable to wild type reaching a cell density of −2×108 in 24 hours. Cells were counted in a Petroff-Housser counting chamber using a phase-contrast microscope with 40× magnification.
Construction of Plasmids.
Plasmids were generated using high fidelity pfu AD DNA polymerase (Agilent Technologies; Santa Clara, Calif.), restriction enzymes (New England Biolabs; Ipswich, Mass.), and Fast-LinkTM DNA Ligase (Epicentre Biotechnologies; Madison, Wis.) according to the manufacturer's instructions. Plasmid pDCW89 (FIG. 13A, FIG. 14) was constructed in three cloning steps. First, a 2.9 kb of DNA fragment, containing the PSC101 replication origin and an apramycin resistance cassette (AprR), was amplified by PCR using the primers DC080 and DC084 and pDCW68 (Chung et al. (2011) J Ind Microbiol Biotechnol 38: 1867-1877) as template. The 2.9 kb DNA fragment was then blunt-end ligated after treatment with T4 PNK (New England Biolabs; Ipswich, Mass.) to construct Intermediate vector I (FIG. 14). A cassette containing the wild-type pyrF coding region was constructed by overlap extension polymerase chain reaction (OE-PCR) placing the pyrF coding region under the transcriptional control of the Cbes2105 (30S ribosomal protein S30EA) promoter. A 199 bp portion of the regulatory region of Cbes2105 was amplified from wild-type genomic DNA using primers DC175 and DC174. The pyrF (Cbes1377, 918 bp) coding region was amplified using primers DC173 and DC232, and joined to the fragment containing the regulatory region by OE-PCR. The DC175 and DC232 primers were engineered to contain NheI and AatII sites, respectively. The 2.9 kb DNA fragment were amplified by PCR from intermediate vector 1 using primers DC176 and DC230 to add restriction sites. The DC176 and DC230 primers were also engineered to contain NheI and AatII sites, respectively. The two linear DNA fragments were digested with NheI and AatII, and ligated to generate 4.02 kb size of intermediate vector II (FIG. 14). In the last step, a 3.65 kb DNA fragment containing the entire sequence of pBAS2 (Dam et al. (2011) Nucleic Acids Res 39: 3240-3254; Clausen et al. (2004) Plasmid 52: 131-138) were amplified by PCR using DC283 and DC284, that contained restriction sites added a KpnI site at 5′ end and a XhoI site at 3′ end. A 4.02 kb linear fragment was amplified from intermediate vector II using DC285 and DC286, which contain engineered restriction sites, KpnI and XhoI, respectively. The two linear DNA fragments were digested with KpnI and XhoI, and ligated to generate pDCW 89. Further details of this construction are described in FIG. 14. Plasmid pDCW129 (FIG. 18A) was generated inserting a 0.68 kb DNA fragment containing the carbohydrate binding domain (CBM) and linker region derived from celA (Cbes1867) into pDCW89. A 0.68 kb DNA fragment was amplified by PCR using the DC397 and DC398 primers and total DNA isolated from C. bescii as template. The 7.75 kb of backbone DNA fragment was amplified by PCR using the DC365 and DC399 and pDCW89 as template. DC397 and DC365 primers were engineered to contain a BamHI site at the end. DC398 and DC399 primers were engineered to contain an SphI site at the end. The two linear DNA fragments were digested with BamHI and SphI and ligated to generate pDCW129. Plasmid pDCW154 and pDCW155 were generated to reduce the size of pDCW89 (FIG. 15). The 4.02 kb backbone DNA fragment was amplified by PCR using primers DC081 and DC507 and pDCW89 as template. The DC081 and DC507 primers were engineered to contain KpnI and PvuII sites, respectively. To generate pDCW154, a 531 bp DNA fragment derived from pBAS2 (Dam et al. (2011) Nucleic Acids Res 39: 3240-3254; Clausen et al. (2004) Plasmid 52: 131-138) was amplified by PCR using DC505 and DC506, primers with engineered restriction sites, KpnI and PvuII, respectively. Two linear DNA fragments were digested with KpnI and PvuII, and ligated to generate pDCW154 (FIG. 15A). Plasmid pDCW155 (FIG. 15B) is identical to pDCW154 except that the 851 bp DNA fragment was replaced with the 531 bp of fragment. The 851 bp DNA fragment derived from pBAS2 (Dam et al. (2011) Nucleic Acids Res 39: 3240-3254; Clausen et al. (2004) Plasmid 52: 131-138) was amplified by PCR using DC508 and DC 506, primers with engineered restriction sites, KpnI and PvuII, respectively. This 851 bp DNA fragment was subsequently ligated into a 4.02 kb backbone DNA fragment as described for pDCW154. DNA sequences of the primers are shown in Table 7. 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 confirmed by Automatic sequencing (Macrogen, Inc.; Rockville, Md.).
Transformation of Caldicellulosiruptor Species.
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.
Assessment of Relative Copy Number, Maintenance and Stability.
C. bescii transformants (JWCB011) were serially subcultured every 16 hours for 5 passages in selective (without Uracil) and non-selective (supplemented with 40 μM 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 6 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; Madison, Wis.). Primers JF396 and JF397 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; Madison, Wis.). The membrane was incubated with probe at 42° C. and washed at 65° C. Band intensities were determined by using a Storm 840 Phospolmager (GE Healthcare; Niskayuna, N.Y.) equipped with ImageQuant v.5.4 software (Molecular Dynamics). 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 5 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.
Determine the Relative Copy-Number of pBAS2.
Total DNA was isolated from JWCB001 (Table 6) and treated with RNase A (Qiagen; Valencia, Calif.). qPCR experiments were carried out with an LightCycler 480 Real-Time PCR instrument (Roche; Madison, Wis.) with the LightCycler 480 SYBR Green I master mix (Roche; Madison, Wis.). 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 7 lists the primers used in the qPCR experiment.







TABLE 6







Strains and plasmids used in Example 3.









Strain or




plasmid
Strain and genotype/phenotype
Source










Caldicellulosiruptor









JWCB001
C. bescii DSM6725 wild type (ura+/5-FOAS)
DSMZ1


JWCB005
C. bescii ΔpyrFA (ura−/5-FOAR)
This




study


JWCB011
C. bescii JWCB005 transformed with pDCW89
This



(ura+/5-FOAS)
study


JWCB014
C. bescii JWCB005 transformed with pDCW129
This



(ura+/5-FOAS)
study


JWCH003
C. hydrothermalis ISCahyI insertion mutation in
2



pyrF (ura−/5-FOAR)


JWCH005
JWCH003 transformed with pDCW89
This



(ura+/5-FOAS)
study







Escherichia coli









JW261
DH5α containing pDCW68 (ApramycinR)
3


JW292
DH5α containing pDCW89 (ApramycinR)
This




study


JW301
DH5α containing pDCW129 (ApramycinR)
This




study


JW319
DH5α containing pDCW154 (ApramycinR)
This




study


JW320
DH5α containing pDCW155 (ApramycinR)
This




study







Plasmids









pDCW68
6-8 copy plasmid DNA (ApramycinR)
3


pDCW89
E. coli/Caldicellulosiruptor species shuttle vector
This



(ApramycinR)
study


pDCW129
E. coli/Caldicellulosiruptor species shuttle vector
This



(ApramycinR)
study


pDCW154
6-8 copy plasmid DNA (ApramycinR)
This




study


pDCW155
6-8 copy plasmid DNA (ApramycinR)
This




study





1German Collection of Microorganisms and Cell Cultures


2 Chung et al. (2013) J Ind Microbiol Biotechnol: 10.1007/s10295-10013-11244-z


3 Chung et al. (2011) J Ind Microbiol Biotechnol 38: 1867-1877













TABLE 7







Primers used in Example 3.












SEQ



Primers
Sequences (5' to 3')
ID No:
Description





FJ298 F
ACCAGCCTAACTTCGATCATTGGA
21
amplify pyrF (Cbes1377) region


 


JH020 R
TCTGACGCTCAGTGGAACGAA
22
amplify pyrF (Cbes1377) region


 


DC326 F
TCTGCTAGCTCAGGTCCTGCTATAAAGCCAA
23
amplify pyrE (Cbes1382) region


 


DC331 R
TCACACGTACCAGAAGGCAGAC
24
amplify pyrE (Cbes1382) region


 


JF199 R
CGCTAACGGATTCACCACT
25
amplify pSC101 E. coli replication origin


 


DC080 F
TCATCTGTGCATATGGACAG
26
amplify E. coli features in pDCW68


 


DC084 R
TCCAACGTCATCTCGTTCTC
27
amplify E. coli features in pDCW68


 


DC230 F
AAGAGACGTCTCATCTGTGCATATGGACAG
28
construct intermediate vector II


 


DC176 R
TCTGCTAGCTCCAACGTCATCTCGTTCTC
29
construct intermediate vector II


 


DC175 F
AGAGCTAGCTTCAACAACCAGAGACACTTGGGA
30
amplify pyrF cassette


 


DC174 R
AGCCTATCAGAGAAGTTCAACAATCTAGAGACCATC
31
amplify the pyrF cassette



CTTTCTATGTAGAAA




 


DC173 F
TTTCTACATAGAAAGGATGGTCTCTAGATTGTTGAA 
32
amplify the pyrF cassette



CTTCTCTGATAGGCT




 


DC232 R
AGAGACGTCTTAAGAGATTGCTGCGTTGATA
33
amplify pyrF cassette


 


DC283 F
TCTGGTACCACCGTGAGCATTCTGGACAGGT
34
amplify entire pBAS2


 


DC284 R
AGACTCGAGATTCCCATGAGCCCACGAACAGT
35
amplify entire pBAS2


 


DC285 F
AGACTCGAGTCTTCTGACGCTCAGTGGAACGAA
36
construct pDCW89


 


DC286 R
TCTGGTACCACCAGCCTAACTTCGATCATTGGAC
37
construct pDCW89


 


DC399 F
AGAGCATGCGAAAACTTGTATTTCCAGGGCCATCAC C
38
construct pDCW129



CATCACCATCACTAATTTC




 


DC365 R
TCTGGATCCAATCCTCCTTTTGGGATTATAACTGTC 
39
construct pDCW129



CATATGCACAGATGAGACG




 


DC397 F
AGAGGATCCATGCAGATAAAGGTATTGTATGCTAAC AAG
40
construct pDCW129


 


DC398 R
AGACAGAGGTTTATGTGGTTATGGGCATGC
41
construct pDCW129


 


DC505 F
TCTGGTACCTCTTTATCTTCCATTATGAGTTTGATAG
42
construct pDCW154


 


DC506 R
ACGTTAGTCAGCTGTTGTTAGTTC
43
construct pDCW154 and 155


 


DC507 F
AGAAGAACAGCTGTCTGACGCTCAGTGGAACGAA
44
construct pDCW154 and 155


 


DC081 R
AGAGGTACCACCAGCCTAACTTCGATCATTGGA
45
construct pDCW154 and 155


 


DC508 F
TCTGGTACCAGTTCCTGCTTTGTTAACATTCCTTG
46
construct pDCW155


 


JF396 F
AGTGTTCTTATAGCTGGAATTGATACGAG
47
produce probe within pyrF for 





southern analysis


 


JF397 R
AGCGTTTGAGTATCCTTTTGCAG
48
produce the probe within pyrF region  





for southern analysis


 


DC233 F
ATCCGTTGATCTTCCTGCAT
49
confirm the transformation of pDCW129


 


DC255 R
AGGATCTGAGGTTCTTATGGCTC
50
confirm the transformation of pDCW129


 


Q1 F
TGGGAAAGCCGTCCATAATC
51
qPCR primer for pBAS2


 


Q2 R
TCTCCCGCTCTTCTCTCTTT
52
qPCR primer for pBAS2


 


Q3 F
GTGCGTCTACAGGACCTTATTT
53
qPCR primer for pBAS2


 


Q4 R
GGCAAGATTCTACAGGCAAGA
54
qPCR primer for pBAS2


 


Q5 F
TGAGCGCCAATCAGGTATAAG
55
qPCR primer for chromosome (2249000-2249090)


 


Q6 R
GGAAGGGAGATAGCGGATAGA
56
qPCR primer for chromosome (2249000-2249090)


 


Q7 F
GCATCTGGTGGCTATGGATATT
57
qPCR primer for chromosome (1303164-1303201)


 


Q8 R
ACCTTTGCTCCACACCTTAC
58
qPCR primer for chromosome (1303164-1303201)









Example 4
Direct Conversion of Plant Biomass to Ethanol by Caldicellulosiruptor 
Strains, Media and Culture Conditions.
C. bescii strains and plasmids used in this study are listed in Table 8. 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) with maltose (0.5% wt/v; catalog no. M5895, Sigma-Aldrich; St. Louis, Mo.) as the carbon source, final pH 7.0. 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. (2001) Molecular Cloning: A Laboratory Manual: Cold Spring Harbor Laboratory Press). E. coli cells were grown in LB broth supplemented with apramycin (50 μg/mL) and plasmid DNA was isolated using a Qiagen Mini-prep Kit. Chromosomal DNA from C. bescii strains was extracted using the Quick-gDNATM MiniPrep (Zymo Research; Irvine, Calif.) or using the DNeasy Blood & Tissue Kit (Qiagen; Valencia, Calif.) according to the manufacturer's instructions.







TABLE 8







Strains and plasmids used in Example 4.









Strains/




Plasmids
Strain and genotype/phenotype
Source










Caldicellulosiruptor bescii









JWCB001
C. bescii DSMZ6725 wild type/(ura+/5-FOAS)
DSMZ1


JWCB018
ΔpyrFA ldh::ISCbe4 Δcbe1/(ura−/5-FOAR)
4, 5


JWCB032
ΔpyrFA ldh::ISCbe4 Δcbe1::PS-layer Cthe-adhE2/
This



(ura−/5-FOAR)
study


JWCB033
ΔpyrFA ldh::ISCbe4 Δcbe1::PS-layer Cthe-adhE* 3/
This



(ura−/5-FOAR)
study







Plasmids









pDCW88
cbeI knock-out vector (ApramycinR)
4


pDCW139
6-8 copy plasmid DNA (ApramycinR)
This




study


pDCW140
6-8 copy plasmid DNA (ApramycinR)
This




study


pDCW142
6-8 copy plasmid DNA/Back-bone plasmid for
This



knock-in vector (ApramycinR)
study


pDCW144
Knock-in plasmid containing PS-layer Cthe-adhE2
This



expression cassette
study


pDCW145
Knock-in plasmid containing PS-layer Cthe-adhE*3
This



expression cassette
study





1German Collection of Microorganisms and Cell Cultures


2Cthe-athE (Cthe0423; Bifunctional acetaldebyde-CoA/alcohol dehydrogenase derived from Clostridium thermocellum ATCC27405)


3 Cthe-athE (EA: Bifunctional acetaldehyde-CoA/alcohol dehydrogenase derived from Clostridium thermocellum EtOH)(Brown et al. (2011) Proc Natl Acad Sci USA 108: 13752-13757)


4 Chung et al. (2013) Biotechnol Biofuels 6: 82 (Example 2)


5 Cha et al. (2013) “Isolation and Bioinformatic Analysis of a Novel Transposable Element, ISCbe4, from a Hyperthermophilic Bacterium, Caldicellulosiruptor bescii” JIMB submitted






Constructions of Vectors for Knock-in of the Cthe0423 and its Derivative into C. bescii. 

The plasmids described below were generated using high fidelity pfu AD DNA polymerase (Agilent Technologies; Santa Clara, Calif.) for PCR reactions, restriction enzymes (New England Biolabs; Ipswich, Mass.), and Fast-link DNA Ligase kit (Epicentre Biotechnologies; Madison, Wis.) according to the manufacturer's instructions. Plasmid pDCW144 (FIG. 20A, FIG. 19) was constructed in four cloning steps. First, a 2.31 kb DNA fragment containing the targeted insertion region sequences (intergenic space between convergent coding regions 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 a apramycin resistance cassette, pyrF cassette (Example 3), and the pSC101 replication origin, were amplified from pDCW88 (Example 2) 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 (FIG. 19). Plasmid pDCW140 (FIG. 19) 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. This DNA fragment was amplified using primers DC460 (with PvuI site) and DC461 (with NotI site) using C. bescii genomic DNA as a template. The 6.1 kb DNA fragment was amplified from pDCW139 using primers DC458 (with PvuI site) and DC459 (with NotI site) to be used as a back-bone fragment. These two linear DNA fragments were digested with PvuI and NotI, and ligated to construct 9.3 kb size of pDCW140. Resulting plasmid is containing the 5′ flanking region (1,013 bp) and the 3′ flanking region (1,012 bp) of targeted insertion site in C. bescii genome in addition to S-layer protein expression cassette (FIG. 19). Plasmid pDCW142, back-bone vector for knock-in plasmid, was constructed by adding restriction sites for cloning and C-terminal 6× Histidine-tag in front of stop codon, in addition to 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; Ipswich, Mass.) to construct pDCW142 (FIG. 19). In the last step, 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 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 (FIG. 19). Plasmid pDCW145 is identical to pDCW144 except for the cloning of Cthe0423* (Brown et al. (2011) Proc Natl Acad Sci USA 108: 13752-13757), which containing 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 Natl Acad Sci USA 108: 13752-13757) genomic DNA as a template. DNA sequences of the primers are shown in Table 9. E. coli strain DH5α cells were transformed by electroporation in a 2-mm-gap cuvette at 2.5 V and transfonnants were selected for apramycin resistance. The sequences of all plasmids were verified by Automatic sequencing (Macrogen, Inc.; 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 (Example 2). 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. The cultures were then plated onto 5-FOA (8 mM) containing solid medium. After initial screening, transformants containing the expected knock-in were further purified by one additional passage under selection on solid medium and screened a second time by PCR to check for segregation of the PS-layer-adhE insertion. The location of the insertion was verified by PCR amplification and sequence analysis. A PCR product was generated from genomic DNA using primers (DC477 and DC478) outside the homologous regions used to construct the knock-in, and internal primers (DC456, DC457, DC462 and DC463). PCR products were sequenced to confirm Construction of JWCB033 was the same as JWCB032 except that pDCW145 was used to electrotransform JWCB018. All primers used are listed in Table 9.







TABLE 9







Primers used in Example 4.












SEQ



Primer
Sequences (5′ to 3′)
ID No:
Description





DC081
TCCAATGATCGAAGTTAGGCTGGT
59
construct pDCW139


 


DC356
TCTGAATTCTCTGACGCTCAGTGGAACGAA
60
construct pDCW139


 


DC456
AGAGGTACCTGTGAGGGCATGTCAATTTACGA
61
construct pDCW139


 


DC457
AGAGAATTCTCTTTTCGATGGAATCTTCTTCGGA
62
construct pDCW139


 


DC458
AGAGAGCGATCGTCTATTGTAACTTTCACTTCAGTGCA
63
construct pDCW140


 


DC459
AGAAGAAGGCGGCCGCTGGAAGAACTTGAAAGCAGGCT
64
construct pDCW140


 


DC460
AGAGAGCGATCGACAGTTTGATTACAGTTTAGTCAGAGCT
65
construct pDCW140


 


DC461
AGAAGAAGGCGGCCGCTTGGTTCCTTAAATCTAAGAGGTATGA
66
construct pDCW140


 


DC462
TGCTGGCAGAGAAGAGCGAAA
67
Sequencing pDCW140


 


DC463
TCTTCATCCCAATCTTCAACTTC
68
Sequencing pDCW140


 


DC464
ACTGGATCCCTCACCAAACCTCCTTGTATGAT
69
construct pDCW142


 


DC466
AGAGCATGCCATCACCATCACCATCACTAATAATAAAGCT
70
construct pDCW142



GAAATAAAAGAGGGTGAGA




 


DC469
ACTGGATCCATGACGAAAATAGCGAATAAATACGAAGT
71
construct pDCW144 and 145


 


DC470
AGAGCATGCTTTCTTCGCACCTCCGTAATAAGCGTTCAGA
72
construct pDCW144 and 145


 


DC471
TGGTAATGAGAGAAGCAGATG
73
Sequencing pDCW144 and 145


 


DC472
TGATAAAAAGCACCCAGTTTGT
74
Sequencing pDCW144 and 145


 


DC477
TGGTTGACCAGGAGAATTTTACACA
75
verify insertion


 


DC478
AGCAACAATCCTGCATTTGTAAG
76
verify insertion









Preparation of Cell Lysates and Western Blotting.

A 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 min 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; Hercules, Calif.) and electrotransferred to PVDF membranes (ImmobilonTM-P; Millipore; Billerica, Mass.) using a Bio-Rad Mini-Protean 3 electrophoretic apparatus. The membranes were then probed with His-tag (6×His) monoclonal antibody (1:5000 dilution; Invitrogen; Grand Island, N.Y.) using the ECL Western Blotting substrate Kit (Thermo Scientific; Waltham, Mass.) as specified by the manufacturer.
Growth Curve Analysis, Measurement of Ethanol Tolerance, and Fermentation Conditions.
Analysis of growth and ethanol tolerance was conducted in stoppered 125 ml serum bottles containing 50 ml LOD medium supplemented with 10 g/1 cellobiose (catalog no.M5895, 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 Jenway Genova spectrophotometer, measuring absorbance at 680 nm. Batch fermentations were performed for 5 days in the same culture conditions except using 10 g/1 cellobiose, 20 g/1 AVICEL (catalog no. 11365, Fluka), or 10 g/l unpretreated switchgrass (sieved −20/+80-mesh fraction; Brian Davison, Oak Ridge National Laboratory, Oak Ridge, Tenn.) as carbon sources.
Analytical Techniques for Determining Fermentation End Products.
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 is provided 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.
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.).
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.

## Claims

1. A recombinant Caldicellulosiruptor microbe genetically modified to produce a greater amount of ethanol than a comparable wild type control, wherein the Caldicellulosiruptor microbe:
comprises a knock-out of the coding region of a lactate dehydrogenase and produces lactate in an amount less than a comparable wild type control;
comprises at least one heterologous polynucleotide that encodes an enzyme in a metabolic pathway that converts acetyl-CoA to ethanol, wherein the enzyme is an acetaldehyde dehydrogenase that can convert acetyl-CoA to acetaldehyde, an aldehyde dehydrogenase that can convert acetyl-CoA to acetaldehyde, or a combination thereof; and wherein the Caldicellulosiruptor microbe is C. bescii, C. kristjansonii, C. obsidiansis, C. hydrothermalis, C. kronotskvensis, C. lactoaceticus, or C. saccharolvticus.

2. The recombinant Caldicellulosiruptor microbe of claim 1 wherein the Caldicellulosiruptor microbe is Caldicellulosiruptor bescii.

3. The recombinant Caldicellulosiruptor microbe of claim 1 comprising a deletion of at least a portion of a lactate dehydrogenase coding region.

4. The recombinant Caldicellulosiruptor microbe of claim 3 wherein the lactate dehydrogenase coding region encodes an NADH-dependent lactate dehydrogenase (Cbes 1918).

5. The recombinant Caldicellulosiruptor microbe of claim 1 further genetically modified to exhibit decreased activity of an endonuclease that digests unmethylated DNA in a Caldicellulosiruptor spp. (CbeI) compared to a comparable wild-type control.

6. The recombinant Caldicellulosiruptor microbe of claim 1 further genetically modified to exhibit increased efficiency of transformation with unmethylated heterologous DNA.

7. A method comprising:
growing the recombinant Caldicellulosiruptor microbe of claim 1, wherein the Caldicellulosiruptor microbe is genetically modified to produce a greater amount of ethanol than a comparable wild type control under conditions effective for the recombinant Caldicellulosiruptor microbe to produce ethanol, wherein the Caldicellulosiruptor microbe:
comprises a knock-out of the coding region of a lactate dehydrogenase and produces lactate in an amount less than a comparable wild type control;
comprises at least one heterologous polynucleotide that encodes an enzyme in a metabolic pathway that converts acetyl-CoA to ethanol, wherein the enzyme is an acetaldehyde dehydrogenase that can convert acetate to acetaldehyde, an aldehyde dehydrogenase that can convert acetyl-CoA to acetaldehyde, or a combination thereof; and wherein the

Caldicellulosiruptor microbe is C. bescii, C. kristjansonii, C. obsidiansis, C. hydrothermalis, C. kronotskvensis, C. lactoaceticus, or C. saccharolvticus.

8. The method of claim 7 further comprising collecting at least a portion of the ethanol.

9. The method of claim 7 wherein the conditions comprise a carbon source that comprises napier grass, Bermuda grass, switchgrass, or a hardwood.

10. The method of claim 9 wherein the carbon source comprises unpretreated switchgrass.

11. The recombinant Caldicellulosiruptor microbe of claim 1 further comprising a knock-out of the coding region of an acetate kinase where the microbe further comprises an alcohol dehydrogenase that converts acetaldehyde to ethanol.