WO2011071829A2 - Expression hétérologue de l'uréase dans des hôtes thermophiles anaérobies - Google Patents

Expression hétérologue de l'uréase dans des hôtes thermophiles anaérobies Download PDF

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WO2011071829A2
WO2011071829A2 PCT/US2010/059120 US2010059120W WO2011071829A2 WO 2011071829 A2 WO2011071829 A2 WO 2011071829A2 US 2010059120 W US2010059120 W US 2010059120W WO 2011071829 A2 WO2011071829 A2 WO 2011071829A2
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urease
host cell
anaerobic
thermophilic
sequence
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PCT/US2010/059120
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WO2011071829A3 (fr
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Arthur J. Shaw, Iv
Sean Covalla
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Mascoma Corporation
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Priority to US13/514,519 priority patent/US20130171708A1/en
Publication of WO2011071829A2 publication Critical patent/WO2011071829A2/fr
Publication of WO2011071829A3 publication Critical patent/WO2011071829A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01005—Urease (3.5.1.5)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • Urease (EC 3.5.1.5 ) catalyzes the hydrolysis of urea to C0 2 and ammonia.
  • Ureases have been heterologously expressed in E. coli. Maeda et al., J. Bacteriol. 776:432-442 (1994).
  • urea as a nitrogen source has several benefits for a consolidated bioprocessing (CBP) or simultaneous saccharification and fermentation (SSF) configuration.
  • CBP consolidated bioprocessing
  • SSF simultaneous saccharification and fermentation
  • Urea is a low cost nitrogen source that has favorable handling and safety qualities compared to ammonia gas or ammonium hydroxide.
  • the use of urea does not require active base addition to maintain neutral pH, as is true with ammonium salts. This has benefits for both the large (process) and small (laboratory) scale, where pH control can be technically challenging.
  • the hydrolysis of urea to ammonia in laboratory media tends to keep the pH at or above 6, which is favorable for a co-culture of certain CBP microorganisms, such as Clostridium thermocellum (C.
  • thermocellum and Thermoanaerobacterium saccharolyticum (T. saccharolyticum).
  • C. thermocellum carries an active urease enzyme.
  • urease enzymes appear to be absent from all known Thermoanaerobacter and Thermoananerbacterium strains.
  • the present invention is directed to a recombinant anaerobic, thermophilic host cell, where the anaerobic, thermophilic host heterologously expresses two or three catalytic subunits ( ⁇ , ⁇ and/or ⁇ ) and four accessory proteins (D, E, F, and G) of a urease enzyme; where the host cell is capable of catalyzing the hydrolysis of urea to carbon dioxide and ammonia.
  • the host is of the genus Thermoanaerobacter or Thermoananerbacterium.
  • the host is T. saccharolyticum.
  • the urease catalytic subunits and accessory proteins are derived from an anaerobic, thermophilic organism that natively expresses the urease enzyme.
  • the urease catalytic subunits and accessory proteins are derived from Clostridium thermocellum (C. thermocellum).
  • nickel is properly captured by the metallochaperone ureE and/or the urease apo-enzyme is properly activated by ureD, ureF, and ureG.
  • the invention is further directed to a method of producing ethanol comprising: (a) culturing the recombinant anaerobic, thermophilic host cell of the invention in the presence of urea as the sole nitrogen source; (b) contacting the anaerobic, thermophilic host cell with lignocellulosic biomass; and (c) recovering the ethanol from the host cell culture.
  • the host cell is of the genus Thermoanaerobacter or Thermoananer bacterium, hi particular embodiments, the host is T. saccharolyticum.
  • the host cell is co-cultured with a second anaerobic, thermophilic host strain.
  • the second anaerobic, thermophilic host strain is C. thermocellum.
  • the host is cultured in a medium having a pH range of 6 to 9, ideally suited for growth of certain anaerobic thermophilic organisms, such as C. thermocellum as well as species of the genera Thermoanaerbacter or Thermanaerobacterium, such as T. saccharolyticum .
  • the host cell produces increased ethanol titers with utilization of urea as a sole nitrogen source as compared to the levels of ethanol produced with utilization of complex additives or ammonium salts as a nitrogen source.
  • Figure 1 depicts a schematic diagram of the plasmid constructs used to create the urease " T. saccharolyticum strains M l 051 (Fig. 1 A) and M 1 151 (Fig. I B).
  • Figure 2 depicts a graph showing pressure measurements over time for urease and urease " strains of T. saccharolyticum using different nitrogen sources.
  • Figure 3 depicts two bar graphs showing the fermentation performance of urease " and urease * T. saccharolyticum strains in various growth media.
  • a "vector,” e.g., a "plasmid” or “YAC” (yeast artificial chromosome) refers to an extrachromosomal element often carrying one or more genes that are not part of the central metabolism of the cell, and is usually in the form of a circular double-stranded DNA molecule.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
  • the plasmids or vectors of the present invention are stable and self-replicating.
  • An "expression vector” is a vector that is capable of directing the expression of genes to which it is operably associated.
  • heterologous refers to an element of a vector, plasmid or host cell that is derived from a source other than the endogenous source.
  • a heterologous sequence could be a sequence that is derived from a different gene or plasmid from the same host, from a different strain of host cell, or from an organism of a different taxonomic group (e.g., different kingdom, phylum, class, order, family genus, or species, or any subgroup within one of these classifications).
  • taxonomic group e.g., different kingdom, phylum, class, order, family genus, or species, or any subgroup within one of these classifications.
  • heterologous is also used synonymously herein with the term “exogenous.”
  • nucleic acid is a polymeric compound comprised of covalently linked subunits called nucleotides.
  • Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded.
  • DNA includes cDNA, genomic DNA, synthetic DNA, and semi -synthetic DNA.
  • isolated nucleic acid molecule or “isolated nucleic acid fragment” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester anologs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.
  • RNA molecules phosphate ester polymeric form of ribonucleosides
  • deoxyribonucleosides deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine
  • DNA molecules or any phosphoester anologs thereof, such as phosphorothioates and thioesters
  • Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a “gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to a nucleic acid fragment that expresses a specific protein, including intervening sequences (introns) between individual coding segments (exons), as well as regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences.
  • identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • a DNA or RNA “coding region” is a DNA or RNA molecule which is transcribed and/or translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • Suitable regulatory regions refer to nucleic acid regions located upstream (5' non-coding sequences), within, or downstream (3' non- coding sequences) of a coding region, and which influence the transcription, RNA processing or stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, RNA processing site, effector binding site and stem-loop structure.
  • a coding region can include, but is not limited to, prokaryotic regions, cDNA from mRNA, genomic DNA molecules, synthetic DNA molecules, or RNA molecules. If the coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding region.
  • ORF Open reading frame
  • a length o nucleic acid either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.
  • Promoter refers to a DNA fragment capable of controlling the expression of a coding sequence or functional RNA.
  • a coding region is located 3' to a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.
  • a promoter is generally bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S I ), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding region is "under the control" of transcriptional and translational control elements in a cell when RNA polymerase transcribes the coding region into mRNA, which is then trans-RNA spliced (if the coding region contains introns) and translated into the protein encoded by the coding region.
  • Transcriptional and translational control regions are DNA regulatory regions, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding region in a host cell.
  • polyadenylation signals are control regions.
  • operably associated refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably associated with a coding region when it is capable of affecting the expression of that coding region (i.e., that the coding region is under the transcriptional control of the promoter).
  • Coding regions can be operably associated to regulatory regions in sense or antisense orientation.
  • expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
  • Nitrogen composes approximately ten percent of a dry cell mass, the largest element mass fraction after carbon and oxygen. Lignocellulosic biomass is a low nitrogen substrate, and to support microorganism growth, nitrogen must be added to the medium during fermentation. The cost of nitrogen supplementation is a significant factor of the overall medium expense. Nitrogen can be supplied in several forms, including complex additives (proteins), ammonium salts, ammonium hydroxide, ammonia gas, or urea. Complex additives are often prohibitively expensive to serve as a nitrogen source in an industrial medium.
  • Ammonium salts and ammonium hydroxide offer lower cost alternatives, but their use impacts the medium pH - either by decreasing pH upon utilization of ammonium salts, or by increasing the pH upon addition to the media by ammonium hydroxide.
  • a neutralizing agent must be used at additional cost.
  • Ammonia gas is a low cost chemical that does not impact pH; however, it is a hazardous chemical that must be stored at high pressure which is undesirable from a process safety standpoint.
  • Urea offers a low cost, safe nitrogen source that does not require additional pH neutralization when used as a medium additive, and as such, is attractive for an industrial process.
  • urease enzyme which converts urea to ammonium and carbon dioxide.
  • Urease activity is a common but not ubiquitous phenotype of bacteria. Studies have indicated that between 8- 20% of cultured microorganisms from human feces and 0-50% of cultured organisms from cow rumens displayed urease activity. See Wozny et al., Appl. Environ. Microbiol. 33: 1097-1104 (1977).
  • the saccharo lytic, thermophilic, anaerobic eubacteria including species belonging to the genera Thermoanaerobacter, Thermoanaerobium, Thermobacterioides, and Clostridium are highly useful for use in consolidated bioprocessing (CBP) systems. Particular species belonging to these genera have certain advantageous functionalities for CBP systems over others.
  • CBP consolidated bioprocessing
  • Plant biomass is composed of a heterogeneous matrix whose primary components are cellulose, hemicellulose (xylan), and lignin.
  • cellulose and hemicellulose can be degraded by anaerobic metabolism, while lignin requires oxygen to be degraded into more basic components.
  • thermophilic anaerobic bacteria the fermentation of cellulose and hemicellulose is largely divided among different species, with cellulose fermentation proceeding primarily through cellulolytic organisms such as Clostridium thermocellum or Clostridium straminisolvens, while hemicellulose fermentation is carried out primarily by xylanolytic species of Thermoanaerobacterium, Thermoanaerobacter, or other related genera.
  • Other distinguishing characteristics of these two organism types include the fermentation of monosaccharides, the minimum pH tolerated for growth, and the ability to use urea as a nitrogen source.
  • thermophilic Xylanolytic No Yes Yes 4-5 No thermophilic
  • the present invention is directed to the heterologous expression of at least two or three three catalytic subunits of urease together with four accessory genes comprising the urease operon in an anaerobic, thermophilic host for use in a consolidated bioprocessing system.
  • the urease enzyme contains an active site with two Ni 2+ ions, which requires the transport of nickel into the cell, proper capture of nickel by the metallochaperone ureE, and activation of the urease apo-enzyme by ureD, ureF, and ureG. See Remaut et al., J. Biol. Chem. 27(5:49365-49370 (2001).
  • the invention is directed to an anaerobic thermophilic host, such as a Thermoanaerobacterium or Thermoanaerobacter host capable of utilizing urea by expression of a urease enzyme.
  • the urease genes (a, ⁇ , ⁇ , D, E, F, G) that are heterologously expressed in a Thermoanaerobacterium or Thermoanaerobacter host are derived from a microorganism that natively expresses the urease enzyme, such as Clostridium thermocellum (C. thermocellum).
  • the urease genes are under the control of an appropriate promoter, such as the C. thermocellum cbp promoter, or the native C. thermocellum urease promoter as part of a synthetic operon.
  • the present invention provides for the use of urease genes ( ⁇ , ⁇ , ⁇ , D, E, F, G) polynucleotide sequences from anaerobic, thermophilic organisms that natively express the urease enzyme, such as C. thermocellum.
  • thermocellum urease gene ( ⁇ , ⁇ . ⁇ , D, E, F. G) nucleic acid sequences are available in GenBank (Accession Numbers YP 001038230, YP 00103823 1 ,
  • urea protein sequence is:
  • the urea protein is encoded by the following sequence:
  • TTTAA SEQ ID NO: 8
  • urep protein sequence is:
  • 3 protein is encoded by the following sequence:
  • the urey protein sequece is:
  • the urey protein is encoded by the following sequence:
  • the iireD protein sequence is: MKNKFGKESRLYIRAKVSDGKTCLQDSYFTAPFKIA PFYEGHGGFMNL
  • the ureD protein is encoding by the following sequence:
  • GATTAGAAATATCCTCTATTAG SEQ ID NO: 11
  • ureE protein sequence is:
  • the ureE protein is encoded by the following sequence:
  • ureF protein sequence is:
  • the ureF protein is encoded by the following sequence:
  • TAG (SEQ ID NO: 13 ).
  • ureG protein sequence is:
  • the ureG protein is encoded by the following sequence:
  • the present invention also provides for the use of an isolated polynucleotide comprising a nucleic acid at least about 70%, 75%, or 80% identical, at least about 90% to about 95% identical, or at least about 96%, 97%, 98%, 99% or 100% identical to any of SEQ ID NOs: 8-14, or fragments, variants, or derivatives thereof.
  • the present invention also encompasses the use of variants of the urease gene (a, ⁇ , ⁇ , D, E, F, G) genes, as described above.
  • Variants may contain alterations in the coding regions, non-coding regions, or both. Examples are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide.
  • nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code.
  • urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host ⁇ e.g., change codons in the C. thermocellum urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) mRNAs to those preferred by a host such as T. saccharolyticum).
  • allelic variants, orthologs, and/or species homologs are also provided in the present invention. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to any of SEQ ID NOs: 8-14, using information from the sequences disclosed herein. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
  • nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the particular polypeptide.
  • nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • the query sequence may be an entire sequence shown of any of SEQ ID NOs: 8-14, or any fragment or domain specified as described herein.
  • whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence or polypeptide of the present invention can be determined conventionally using known computer programs.
  • a method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.
  • RNA sequence can be compared by converting U's to T's.
  • the result of said global sequence alignment is in percent identity.
  • the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.
  • This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
  • a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity.
  • the deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end.
  • the 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
  • a 90 base subject sequence is compared with a 100 base query sequence.
  • deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query.
  • percent identity calculated by FASTDB is not manually corrected.
  • bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
  • Some embodiments of the invention encompass a nucleic acid molecule comprising at least 10, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, or 800 consecutive nucleotides or more of any of SEQ ID NOs: 8-14, or domains, fragments, variants, or derivatives thereof.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the mature polypeptide may be identical to the coding sequence encoding SEQ ID NOs: 1-7 or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of any one of SEQ ID NOs: 8-14.
  • the present invention provides an isolated polynucleotide comprising a nucleic acid fragment which encodes at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 95, or at least 100 or more contiguous amino acids of SEQ ID NOs: 1-7.
  • the polynucleotide encoding for the mature polypeptide of SEQ ID NOs: 1-7 or the mature polypeptide encoded by the deposited clone may include: only the coding sequence for the mature polypeptide; the coding sequence of any domain of the mature polypeptide; and the coding sequence for the mature polypeptide (or domain-encoding sequence) together with non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only sequences encoding for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences.
  • nucleic acid molecules having sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences disclosed herein encode a polypeptide having functional urease gene ( , ⁇ , ⁇ , D, E, F, G) activity.
  • a polypeptide having urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) functional activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to a functional activity of the urease ( ⁇ , ⁇ , ⁇ , D, E, F, G) polypeptides of the present invention, as measured, for example, in a particular biological assay.
  • a urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) functional activity can routinely be measured by determining the ability of the encoded urease enzyme to utilize nitrogen, or by measuring the level of urease activity.
  • nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any of SEQ ID NOs: 8-14, or fragments thereof, will encode polypeptides "having urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) functional activity.”
  • degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay.
  • nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) functional activity.
  • Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the urease genes ( ⁇ , ⁇ , ⁇ , D, E, F, G) of the present invention, or genes encoding for a protein with similar biological activity.
  • the probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.
  • a hybridization probe may have at least 30 bases and may contain, for example, 50 or more bases.
  • the probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promoter regions, exons, and introns.
  • An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of bacterial or fungal cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • the present invention further relates to polynucleotides which hybridize to the herein above-described sequences if there is at least 70%, at least 90%, or at least 95% identity between the sequences.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% or at least 97% identity between the sequences.
  • polynucleotides which hybridize to the hereinabove described polynucleotides encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the DNAs of any of SEQ ID NOs: 8-14, or the deposited clones.
  • polynucleotides which hybridize to the hereinabove-described sequences may have at least 20 bases, at least 30 bases, or at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity.
  • such polynucleotides may be employed as probes for the polynucleotide of any of SEQ ID NOs: 8-14, or the deposited clones, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PGR primer.
  • Hybridization methods are well defined and have been described above. Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions.
  • a primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.
  • genes encoding similar proteins or polypeptides to those of the instant invention could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired bacteria using methodology well known to those skilled in the art.
  • Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (see, e.g., Maniatis, 1989).
  • the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primers DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems.
  • polynucleotides which hybridize to the hereinabove-described sequences having at least 20 bases, at least 30 bases, or at least 50 bases which hybridize to a polynucleotide of the present invention may be employed as PGR primers.
  • the primers typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PGR primer design are common and well known in the art.
  • PCR polymerase chain reaction
  • the polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding microbial genes.
  • the second primer sequence may be based upon sequences derived from the cloning vector.
  • primers can be designed and used to amplify a part of or full- length of the instant sequences.
  • the resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length DNA fragments under conditions of appropriate stringency.
  • nucleic acid sequences and fragments thereof of the present invention may be used to isolate genes encoding homologous proteins from the same or other fungal species or bacterial species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, Mullis et al, U.S. Pat. No. 4,683,202; ligase chain reaction (LCR) (Tabor, S. et al, Proc. Acad. Sci. USA 82, 1074, (1985)); or strand displacement amplification (SDA), Walker, et al, Proc. Natl. Acad. Sci. U.S.A., 89, 392, (1992)).
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • the present invention further relates to the expression of an urease enzyme from an anaerobic, thermophilic organism that natively expresses such an enzyme.
  • the urease enzyme is composed of C. thermocellum urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) polypeptides and is expressed in a host cell, such as a Thermoanaerobacterium or Thermoanaeorobatcter strain, e.g., T. saccharolyticum.
  • the present invention further encompasses polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, the polypeptide sequence shown in SEQ ID NOs: 1- 7, and/or domains, fragments, variants, or derivative thereof, of any of these polypeptides ⁇ e.g., those fragments described herein, or domains of any of SEQ ID NOs: 1-7).
  • polypeptide having an amino acid sequence at least, for example, 95%
  • amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (5:237-245(1990)).
  • the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
  • the result of said global sequence alignment is in percent identity.
  • polypeptides and polynucleotides of the present invention are provided in an isolated form, e.g., purified to homogeneity.
  • the present invention also encompasses polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% similar to the polypeptide of any of SEQ ID NOs: 1-7, and to portions of such polypeptide with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
  • similarity between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide.
  • the present invention further relates to a domain, fragment, variant, derivative, or analog of the polypeptide of any of SEQ ID NOs: 1-7.
  • fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length polypeptides.
  • Fragments of urease ( ⁇ , ⁇ . ⁇ , D, E, F, G) polypeptides of the present invention encompass domains, proteolytic fragments, deletion fragments and in particular, fragments of C. thermocellum urease ( ⁇ , ⁇ , ⁇ , D, E, F, G) polypeptides which retain any specific biological activity of the urease ( ⁇ , ⁇ , ⁇ , D, E, F, G) protein.
  • Polypeptide fragments further include any portion of the polypeptide which comprises a catalytic activity of the urease enzyme.
  • the variant, derivative or analog of the polypeptide of any of SEQ ID NOs: 1-7 may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • substituted amino acid residue may or may not be one encoded by the genetic code
  • amino acid residues includes a substituent group.
  • the polypeptides of the present invention further include variants of the polypeptides.
  • a "variant' of the polypeptide can be a conservative variant, or an allelic variant.
  • a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the protein.
  • a substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the protein.
  • the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity.
  • the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the protein.
  • an "allelic variant” is intended alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will still have the same or similar biological functions associated with the C. thermocellum urease enzyme.
  • allelic variants, the conservative substitution variants, and members of the urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) family will have an amino acid sequence having at least 75%, at least 80%, at least 90%, at least 95% amino acid sequence identity with a C. thermocellum urease gene ( , ⁇ , ⁇ , D, E, F, G) amino acid sequence set forth in any one of SEQ ID NOs: 1-7.
  • Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity.
  • N terminal, C terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.
  • the proteins and peptides of the present invention include molecules comprising the amino acid sequence of SEQ ID NOs: 1-7 or fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues of the C. thermocellum urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) polypeptide sequence; amino acid sequence variants of such sequences wherein at least one amino acid residue has been inserted N- or C terminal to, or within, the disclosed sequence; amino acid sequence variants of the disclosed sequences, or their fragments as defined above, that have been substituted by another residue.
  • Contemplated variants further include those containing predetermined mutations by, e.g., homologous recombination, site-directed or PGR mutagenesis; and derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example, a detectable moiety such as an enzyme or radioisotope).
  • variants may be generated to improve or alter the characteristics of the urease polypeptides. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function.
  • the invention further includes C. thermocellum urease gene ( ⁇ , ⁇ , ⁇ , D, E, F,
  • G polypeptide variants which show substantial biological activity.
  • variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
  • the first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
  • the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244: 1081 -1085 (1989).) The resulting mutant molecules can then be tested for biological activity.
  • tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and lie; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
  • derivatives and analogs refer to a polypeptide differing from the C. thermocellum urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) polypeptides, but retaining essential properties thereof. Generally, derivatives and analogs are overall closely similar, and, in many regions, identical to the C. thermocellum urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) polypeptides.
  • derivatives and analogs when referring to C.
  • thermocellum urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) polypeptides of the present invention include any polypeptides which retain at least some of the activity of the corresponding native polypeptide, e.g., the hydrolysis of urea to C0 2 and ammonia.
  • Derivatives of C. thermocellum urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) polypeptides of the present invention are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide.
  • Derivatives can be covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example, a detectable moiety such as an enzyme or radioisotope). Examples of derivatives include fusion proteins.
  • An analog is another form of C. thermocellum urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) polypeptides of the present invention.
  • An “analog” also retains substantially the same biological function or activity as the polypeptide of interest, i.e., functions as a component of an enzyme that hydrolyzes urea to C0 2 and ammonia.
  • An analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • C. thermocellum urease gene ( ⁇ , ⁇ , ⁇ , I), E, F, G) polypeptides in host cells
  • the present invention provides C. thermocellum urease gene ( ⁇ , ⁇ , ⁇ , D, E, F, G) polypeptides, or domains, variants, or derivatives thereof that can be effectively and efficiently expressed in a consolidated bioprocessing system.
  • a host cell comprising a vector which expresses the urease enzyme encoded by C. thermocellum urease genes ( ⁇ , ⁇ , ⁇ , D, E, F, G) is utilized for consolidated bioprocessing and is optionally co-cultured with additional host cells capable of utilizing urea.
  • the host cell can be an anaerobic, thermophilic host, such as T. saccharolyticum, and the additional host cell can be a different anaerobic, thermophilic host, such as C. thermocellum expressing native urease.
  • the transformed host cells or cell cultures, as described above, are measured for urease protein content.
  • Protein content can be determined by analyzing the host cell supernatants.
  • the high molecular weight material is recovered from the yeast cell supernatant either by acetone precipitation or by buffering the samples with disposable de-salting cartridges.
  • the analysis methods include the traditional Lowry method or protein assay method according to BioRad's manufacturer's protocol. Using these methods, the protein content of saccharolytic enzymes can be estimated.
  • the transformed host cells or cell cultures, as described above, can be further analyzed for hydrolysis of urea ⁇ e.g., by measuring carbon dioxide and ammonia levels).
  • suitable lignocellulosic material can be any feedstock that contains soluble and/or insoluble cellulose, where the insoluble cellulose can be in a crystalline or non-crystalline form.
  • the lignocellulosic biomass comprises, for example, wood, corn, corn cobs, corn stover, corn fiber, sawdust, bark, leaves, agricultural and forestry residues, grasses such as switchgrass, cord grass, rye grass or reed canary grass, miscanthus, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard, miscanthus, sugar-processing residues, sugarcane bagasse, agricultural wastes, rice straw, rice hulls, barley straw, cereal straw, wheat straw, canola straw, oat straw, oat hulls, stover, soybean stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood or combinations thereof.
  • Vectors and Host Vectors and Host
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transform ants or amplifying the genes of the present invention.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques.
  • the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; and yeast plasmids.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively associated with an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoters include the E. coli, lac or trp, and other promoters known to control expression of genes in prokaryotic or lower eukaryotic cells, the cbp promoter of C. thermocellum, or other promoters for gene expression in anaerobic, thermophilic organisms.
  • the C. thermocellum cbp promoter can have the following sequence:
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression, or may include additional regulatory regions.
  • the expression vectors may contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as the aph3 gene from the S. facealis plasmid p D102 conferring thermostable kanamycin resistance (Mai et al, FEMS Microbio. Let. 745:163-167(1997)).
  • the vector containing the appropriate DNA sequence as herein, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be an anaerobic thermophilic host, such as a Thermoanaerobacterium or Thermoanaerobacter host.
  • a representative example of such a host is T. saccharolyticum. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • thermophilic bacteria include eubacteria and archaebacteria.
  • Thermophilic eubacteria include: phototropic bacteria, such as cyanobacteria, purple bacteria, and green bacteria; Gram-positive bacteria, such as Bacillus, Clostridium, Lactic acid bacteria, and Actinomyces; and other eubacteria, such as Thiobacillus, Spirochete, Desulfotomaculum, Gram-negative aerobes, Gram-negative anaerobes, and Thermotoga. Within archaebacteria are considered Methanogens, extreme thermophiles (an art- recognized term), and Thermoplasma.
  • the present invention relates to Gram-negative organotrophic thermophiles of the genera Thermus, Gram- positive eubacteria, such as genera Clostridium, and also which comprise both rods and cocci, genera in group of eubacteria, such as Thermosipho and Thermotoga, genera of Archaebacteria, such as Thermococcus, Thermoproteus (rod-shaped), Thermofilum (rod- shaped), Pyrodictium, Acidianus, Sulfolobus, Pyrobaculum, Pyrococcus, Thermodiscus, Staphylothermus, Desulfurococcus, Archaeoglobus, and Methanopyrus.
  • thermophilic microorganisms including bacteria, prokaryotic microorganism, and fungi
  • thermophilic microorganisms include, but are not limited to: Clostridium thermosulfurogenes, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium thermohydrosulfuricum, Clostridium thermoaceticum, Clostridium thermosaccharolyticum, Clostridium tartarivorum, Clostridium thermocellulaseum, Thermoanaerobacterium thermosaccarolyticum, Thermoanaerobacterium saccharolyticum, Thermobacteroides acetoethylicus, Thermoanaerobium brockii, Methanobacterium thermoautotrophicum, Pyrodictium occultum, Thermoproteus neutrophilus, Thermofilum librum, Thermothrix thioparus, Desulfovibri
  • the present invention relates to thermophilic bacteria of the genera Thermoanaerobacterium or Thermoanaerobacter, including, but not limited to, species selected from the group consisting of: Thermoanaerobacterium thermosulfurigenes, Thermoanaerobacterium aotearoense, Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium saccharolyticum, Thermoanaerobium brockii, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacter thermohydrosulfuricus , Thermoanaerobacter ethanolicus, Thermoanaerobacter brockii, variants thereof, and progeny thereof
  • the present invention relates to microorganisms of the genera Geobacillus, Saccharococcus, Paenibacillus, Bacillus, and Anoxybacillus, including, but not limited to, species selected from the group consisting of: Geobacillus thermoglucosidasius, Geobacillus stear other mophilus, Saccharococcus caldoxylosilyticus, Saccharoccus thermophilus, Paenibacillus campinasensis, Bacillus flavothermus, Anoxybacillus kamchatkensis, Anoxybacillus gonensis, variants thereof, and progeny thereof.
  • species selected from the group consisting of: Geobacillus thermoglucosidasius, Geobacillus stear other mophilus, Saccharococcus caldoxylosilyticus, Saccharoccus thermophilus, Paenibacillus campinasensis, Bacillus flavothermus, Anoxybacillus kamchatkensis, Anoxybacillus gone
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably associated to the sequence.
  • suitable vectors and promoters are known to those of skill in the art, and are commercially available.
  • Two examples of vectors of the present application include pDest-Ct-Urease (pMU1336) and pMetE urease fixA (pM Li 1728) (as shown in Figs. 1 A and B).
  • Promoter regions can be selected from any desired gene.
  • Particular named bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda PR, PL and trp.
  • Other promoters include those that regulate gene expression in anaerobic, thermophilic organisms, such as the cbp promoter from C. thermocellum. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • Introduction of the construct in other host cells can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or cleetroporation. (Davis, L., et al., Basic Methods in Molecular Biology, (1986)).
  • constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • the selected promoter is induced by appropriate means ⁇ e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • the host cell can be cultured in a medium having a particular pH.
  • the host cell can be cultured in medium having a pH range from about 4 to about 9, from about 5 to about 8, or from about 6 to about 8.
  • the host cell can also be cultured in medium having a pH range from about 5 to about 7, from about 6 to about 7, or from about 6.2 to about 6.8.
  • the host cell can also be cult ured in presence of a particular concentration of urea.
  • the concentration of urea can be at least about 0.5 g L, at least about 1.0 g/L, at least about 1.5 g/L, at least about 2.0 g/L, at least about 2.5 g/L, at least about 3.0 g/L, at least about 3.5 g/L, at least about 4.0 g/L, at least about 4.5 g/L, or at least about 5.0 g/L.
  • Example 1 Heterologous cloning of urease operon into T. sacch arol ticum
  • the urease genes ( ⁇ , ⁇ , ⁇ , D, E, F, G) (SEQ ID NO: 8 through SEQ ID NO: 14, respectively) from Clostridium thermocellum were heterologously cloned into the genome of T. saccharolyticum under the control of the C. thermocellum cbp promoter (SEQ ID NO: 17).
  • These urease genes include the catalytic subunits of the urease enzyme (typically three ureafiy subunits, but in some species only two subunits) and the accessory proteins wreDEFG that facilitate protein folding and nickel activation.
  • T. saccharolyticum JW/SL-YS485, strain M0863 carrying deletion of L-lactate dehydrogenase (L-ldh), phosphoacetyltransferase (pta), and acetate kinase (ack) was used as the host strain for this work.
  • T. saccharolyticum transformed with pDest-Ct-urease (pMU1336) (SEQ ID NO: 15) is refered to as strain M 105 1.
  • Plasmid pMU1366 is a non-replicating plasmid which integrates into the chromosome a the AL-ldh locus.
  • the Gateway® cloning system (Invitrogen) was used according to the manufacturer's instructions in the creation of the Ml 051 strain.
  • T. saccharolyticum transformed with pMetEjTx A (pMU1728) (SEQ ID NO: 16) is refered to as strain Ml 151.
  • Plasmid pMU1728 is a non-replicating plasmid which integrates into the chromosome at the orf796 locas.
  • Strains M1051 ATCC deposit designation PTA-10494
  • Ml 151 ATCC deposit designation PTA- 10495 were deposited at the ATCC on November 24, 2009.
  • TSD1 media formulations (as shown in Table 2) were used. 1.85 g/L ammonium sulfate was replaced with 2 g/L urea to make urea containing media as required in each experiment.
  • M0863 cells using urea as a nitrogen source never exceeded 10 psig over the same period.
  • Ml 051 cells using urea as a nitrogen source peaked at over 35 psig during the period of measurement.
  • Table 4 depicts measurements of the fermentation indicator cthanol
  • M0863 cells tested with urea as a nitorgen source only produced 2.0 g/L of EtOH, whereas Ml 051 cells, in contrast, produced 11.5 g/L of EtOH.
  • the final pH of ammonium contains M0863 and Ml 051 fermentations was 3.58 and 3.48, respectively, while the final pH of urea containing fermentations was 4.37 and 5.45 for M0863 and Ml 051.
  • Figure 3A depicts the fermentation performance of strains M0863 (L-ldh- pta ack) and M 1 151 ( L-ldh- pta/ack-, urease+, metE+, or796-) in high yeast extract (i.e. 8.5 g/L) rich medium, cellobiose (about 75 g/L), and maltodextrin (about 75 g/L).
  • the strains were grown with different nitrogen sources and presence or absence of CaC0 3 buffering. Fermentation performance was measured by the amount of ethanol ( EtOH), Cellobiose (CB), Glucose, and Xylose present after 96 hours o fermentation.
  • AH cultures were grown at 55 °C with shaking at 150 rpm. Fermentations were performed in 150 mL serum bottles with a 20mL culture volume, and bottles were sealed with butyric rubber stoppers after evacuation of air and replacement with an atmosphere containing 95% nitrogen and 5% carbon dioxide.
  • M0863 converted the most cellobiose into EtOH when ammonium sulfate and
  • M0863 cells converted the least amount of cellobiose into EtOH when urea was added to the growth media.
  • the Ml 151 strain converted cellobiose and maltodextrin into EtOH at a final titer of 56 g/L when urea and CaC(3 ⁇ 4 buffer were added to the growth media. Without the CaC0 3 buffer, Ml 151 cells were slightly less efficient at converting cellobiose into EtOH. Using ammonium sulfate as a nitrogen source, the Ml 151 strain's efficiency at cellobiose fermentation into EtOH was equivalent to that of the M0863 strain, at 43-45 g/L EtOH.
  • FIG. 3B depicts ethanol (EtOH) production by M0863 and Ml 151 grown in low yeast extract (i.e. 0.5 g/L) rich medium with cellobiose (about 75 g/L), maltodextrin (about 75 g/L), and vitamins.
  • the strains were grown with different nitrogen sources and presence or absence of CaC0 3 buffering, as discussed below.
  • M0863 cells produced the most EtOH when grown in the above-described media with ammonium sulfate as a nitrogen source and the presence of CaC0 3 buffer.
  • M0863 cells produced the least EtOH when grown in media supplemented with urea only. The addition of methionine had very little effect on the production of EtOH by M0863 cells grown under either condition.
  • Ml 151 cells produced the most EtOH when grown in media with urea and methionine. EtOH production by these cells was slightly less when urea, methionine and a buffer were included in the growth media. The addition of urea allowed for the production of over 30 g/L of EtOH by Ml 151 cells. When the ammonium sulfate was used as a nitrogen source, the production of EtOH was equivalent between the M0863 and Ml 151 strains.
  • Example 4 Expression of urease genes in a T. saccharolyticum strain producing organic acids
  • Plasmid pMU1728 was transformed into wildtype T. saccharolyticum cells, creating a stain carrying the urease operon, the MetE gene, and two copies of the pta and ack genes (the wildtype copy and a recombinant copy).
  • this strain, M1447 is also able to produce lactic acid and ethanol. Utilization of urea allows for a higher pH during ethanol and organic acid production, as well as a final higher product titer in the urea utilizing strain. Batch fermentations were run in 15 mL falcon tubes with a 5 mL working volume for 7 days at 55°C without shaking in an anaerobic chamber.
  • TSC4 media 29.99 0.19 4.91 0.00 0.00 0.21 5.80 100
  • TSD1 media 13.1 1 0.00 4.04 0.00 0.00 0.00 6.10 100
  • TSC4 media used in these experiments was prepared as described in Table 6.
  • Solution 1 is prepared at l .lx final concentration and autoclaved, while solution 2 is prepared at l Ox concentration and filter sterilized. Solutions 1 and 2 are then combined under an anaerobic atmosphere.

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  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne l'expression hétérologue de l'uréase dans des hôtes thermophiles anaérobies, tels que Thermoanaerobacterium, Thermoanaerobacter, et d'autres genres apparentés. Par exemple, l'hôte thermophile anaérobie peut être T. saccharolyticum. Les cellules hôtes expriment les sous-unités catalytiques de l'enzyme uréase conjointement avec les protéines facultatives ureDEFG facilitant le repliement de la protéine et l'activation du nickel. L'invention concerne en outre l'utilisation de l'urée comme source d'azote dans la croissance des microorganismes impliqués dans les systèmes consolidés de biotraitement.
PCT/US2010/059120 2009-12-07 2010-12-06 Expression hétérologue de l'uréase dans des hôtes thermophiles anaérobies WO2011071829A2 (fr)

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CA2783533A CA2783533A1 (fr) 2009-12-07 2010-12-06 Expression heterologue de l'urease dans des hotes thermophiles anaerobies
US13/514,519 US20130171708A1 (en) 2009-12-07 2010-12-06 Heterologous Expression of Urease in Anaerobic, Thermophilic Hosts

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US26727309P 2009-12-07 2009-12-07
US61/267,273 2009-12-07

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WO2011071829A2 true WO2011071829A2 (fr) 2011-06-16
WO2011071829A3 WO2011071829A3 (fr) 2011-09-29

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US8309324B2 (en) * 2004-11-10 2012-11-13 University Of Rochester Promoters and proteins from Clostridium thermocellum and uses thereof
JP2009513145A (ja) * 2005-10-31 2009-04-02 ザ トラスティーズ オブ ダートマウス カレッジ リグノセルロース系バイオマスをエタノールに変換する好熱性生物
ES2413482T3 (es) * 2007-11-10 2013-07-16 Joule Unlimited Technologies, Inc. Organismos hiperfotosintéticos

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US20130171708A1 (en) 2013-07-04
CA2783533A1 (fr) 2011-06-16

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