Patent Grant
8470592
Lignocellulosic biomass is widely recognized as a promising source of raw material for production of renewable fuels and chemicals. The primary obstacle impeding the more widespread production of energy from biomass feedstocks is the general absence of low-cost technology for overcoming the recalcitrance of these materials to conversion into useful fuels. Lignocellulosic biomass contains carbohydrate fractions (e.g., cellulose and hemicellulose) that can be converted into ethanol. In order to convert these fractions, the cellulose and hemicellulose must ultimately be converted or hydrolyzed into monosaccharides; it is the hydrolysis that has historically proven to be problematic.
Biologically mediated processes are promising for energy conversion, in particular for the conversion of lignocellulosic biomass into fuels. Biomass processing schemes involving enzymatic or microbial hydrolysis commonly involve four biologically mediated transformations: (1) the production of saccharolytic enzymes (cellulases and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose, and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose). These four transformations occur in a single step in a process configuration called consolidated bioprocessing (CBP), which is distinguished from other less highly integrated configurations in that it does not involve a dedicated process step for cellulase and/or hemicellulase production.
CBP offers the potential for lower cost and higher efficiency than processes featuring dedicated cellulase production. The benefits result in part from avoided capital costs, substrate and other raw materials, and utilities associated with cellulase production. In addition, several factors support the realization of higher rates of hydrolysis, and hence reduced reactor volume and capital investment using CBP, including enzyme-microbe synergy and the use of thermophilic organisms and/or complexed cellulase systems. Moreover, cellulose-adherent cellulolytic microorganisms are likely to compete successfully for products of cellulose hydrolysis with non-adhered microbes, e.g., contaminants, which could increase the stability of industrial processes based on microbial cellulose utilization. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer; and engineering non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase and hemicellulase system enabling cellulose and hemicellulose utilization.
Three major types of enzymatic activities are required for native cellulose degradation: The first type are endoglucanases (1, 4-β-D-glucan 4-glucanohydrolases; EC 3.2.1.4). Endoglucanases cut at random in the cellulose polysaccharide chain of amorphous cellulose, generating oligosaccharides of varying lengths and consequently new chain ends. The second type are exoglucanases, including cellodextrinases (1, 4-β-D-glucan glucanohydrolases; EC 3.2.1.74) and cellobiohydrolases (1,4-β-D-glucan cellobiohydrolases; EC 3.2.1.91). Exoglucanases act in a processive manner on the reducing or non-reducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase) as major products. Exoglucanases can also act on microcrystalline cellulose, presumably peeling cellulose chains from the microcrystalline structure. The third type are (β-glucosidases (β-glucoside glucohydrolases; EC 3.2.1.21). β-Glucosidases hydrolyze soluble cellodextrins and cellobiose to glucose units.
A variety of plant biomass resources are available as lignocellulosics for the production of biofuels, notably bioethanol. The major sources are (i) wood residues from paper mills, sawmills and furniture manufacturing, (ii) municipal solid wastes, (iii) agricultural residues and (iv) energy crops. Pre-conversion of particularly the cellulosic fraction in these biomass resources (using either physical, chemical or enzymatic processes) to fermentable sugars (glucose, cellobiose and cellodextrins) would enable their fermentation to bioethanol, provided the necessary fermentative micro-organism with the ability to utilize these sugars is used.
On a world-wide basis, 1.3×1010 metric tons (dry weight) of terrestrial plants are produced annually (Demain, A. L., et al., Microbiol. Mol. Biol. Rev. 69, 124-154 (2005)). Plant biomass consists of about 40-55% cellulose, 25-50% hemicellulose and 10-40% lignin, depending whether the source is hardwood, softwood, or grasses (Sun, Y. and Cheng, J., Bioresource Technol. 83, 1-11 (2002)). The major polysaccharide present is water-insoluble, cellulose that contains the major fraction of fermentable sugars (glucose, cellobiose or cellodextrins).
Bakers' yeast (Saccharomyces cerevisiae) remains the preferred micro-organism for the production of ethanol (Hahn-Hägerdal, B., et al., Adv. Biochem. Eng. Biotechnol. 73, 53-84 (2001)). Attributes in favor of this microbe are (i) high productivity at close to theoretical yields (0.51 g ethanol produced/g glucose used), (ii) high osmo- and ethanol tolerance, (iii) natural robustness in industrial processes, (iv) being generally regarded as safe (GRAS) due to its long association with wine and bread making, and beer brewing. Furthermore, S. cerevisiae exhibits tolerance to inhibitors commonly found in hydrolyzaties resulting from biomass pretreatment. The major shortcoming of S. cerevisiae is its inability to utilize complex polysaccharides such as cellulose, or its break-down products, such as cellobiose and cellodextrins.
Genes encoding cellobiohydrolases in T. reseei (cbh1 and cbh2), A. niger (cbhA and cbhB) and P. chrysosporium (cbh1-4) have been cloned and described. The proteins encoded by these genes are all modular enzymes containing a catalytic domain linked via a flexible liner sequence to a cellulose-binding molecule. Cbh1, CbhB and Cbh1-4 are family 7 glycosyl hydrolases. Glycosyl hydrolases are a widespread group of enzymes that hydrolyse the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. A classification system for glycosyl hydrolases, based on sequence similarity, has led to the definition of 85 different families (Henrissat, B. et al., Proc. Natl. Acad. Sci. 92:7090-7094 (1995); Davies, G. and Henrissat, B., Structure 3: 853-859 (1995)). Glycoside hydrolase family 7 (GHF7) comprises enzymes with several known activities including endoglucanase (EC:3.2.1.4) and cellobiohydrolase (EC:3.2.1.91). These enzymes were formerly known as cellulase family C.
Exoglucanases such as cellobiohydrolases play a role in the conversion of cellulose to glucose by cutting a dissaccharide cellobiose from the reducing or nonreducing end of the cellulose polymer chain. Structurally, cellulases and xylanases generally consist of a catalytic domain joined to a cellulose-binding domain (CBD) via a linker region that is rich in proline and/or hydroxy-amino acids. In type I exoglucanases, the CBD domain is found at the C-terminal extremity of these enzyme (this short domain forms a hairpin loop structure stabilised by 2 disulphide bridges).
Glycosyl hydrolase family 7 enzymes usually have at least 50 to 60% homology at the amino acid level, but the homology between any of these enzymes and the glycosyl hydrolase family 6 CBH2 is less than about 15%.
With the aid of recombinant DNA technology, several of these heterologous cellulases from bacterial and fungal sources have been transferred to Saccharomyces cerevisiae, enabling the degradation of cellulosic derivatives (Van Rensburg, P., et al., Yeast 14, 67-76 (1998)), or growth on cellobiose (Van Rooyen, R., et al., J. Biotech. 120, 284-295 (2005)); McBride, J. E., et al., Enzyme Microb. Techol. 37, 93-101 (2005)).
Related work was described by Fujita, Y., et al., (Appl. Environ. Microbiol. 70, 1207-1212 (2004)) where cellulases immobilised on the yeast cell surface had significant limitations. First, Fujita et al. were unable to achieve fermentation of amorphous cellulose using yeast expressing only recombinant BGL1 and EGII. A second limitation of the Fujita et al. approach was that cells had to be pre-grown to high cell density on standard carbon sources before the cells were useful for ethanol production using amorphous cellulose (e.g., Fujita et al. teach high biomass loadings of ˜15 g/L to accomplish ethanol production).
As noted above, ethanol producing yeast such as S. cerevisiae require addition of external cellulases when cultivated on cellulosic substrates such as pre-treated wood because this yeast does not produce endogenous cellulases. Expression of fungal cellulases such as T. reesei Cbh1, Cbh2 in yeast S. cerevisiae have been shown to be functional (Den Haan, R., et al., Enzyme and Microbial Technology 40:1291-1299 (2007)). However current levels of expression and specific activity of cellulases heterologously expressed in yeast are still not sufficient to enable growth and ethanol production by yeast on cellulosic substrates without externally added enzymes. While studies have shown that perhaps recombinant fungal Cbh1 has specific activity comparable to that of the native protein, there remains a significant need for improvement in the amount of Cbh activity expressed in order to attain the goal of achieving a consolidated bioprocessing (CBP) system capable of efficiently and cost-effectively converting cellulosic substrates to ethanol.
Therefore it would be very beneficial to isolate other cellulases from cellulolytic organisms with higher specific activity and higher expression levels in host organisms, such as the yeast S. cerevisiae. Since Cbh1 activity seems to be the most limiting in terms of expression level in yeast (Pennilä M E et al., Gene 63:103-12 (1988)), it would be advantageous to isolate a novel cbh1 gene and demonstrate its functional expression in yeast.
In order to address the limitations of heterologous Cbh1 expression in consolidated bioprocessing systems, the present invention provides for the identification of novel cellulases that facilitate cellulose digestion and ethanol production in host cells. In particular, the present invention is directed to the isolation of novel cellulases that are capable of being heterologously expressed in yeast, e.g., Saccharomyces cerevisiae.
The present invention provides for the isolation and characterization of the cbh1 gene from Schizochytrium aggregatum. In particular, the present invention provides for the nucleic acid and amino acid sequences of Schizochytrium aggregatum cbh1, and domains, variants and derivatives thereof. The present invention further provides for the heterologous expression of Schizochytrium aggregatum Cbh1 in host cells, including yeast, e.g., Saccharomyces cerevisiae. Expression of Schizochytrium aggregatum Cbh1 in host cells augments cellulose digestion and facilitates ethanol production by those host cells on cellulosic substrates. In certain embodiments, heterologous expression in Saccharomyces cerevisiae is in coordination with heterologous expression of other known, or newly identified saccharolytic enzymes. Therefore, the present invention also provides that the novel Schizochytrium aggregatum cbh1 gene can be utilized in a consolidated bioprocessing system.
The present invention relates to, inter alia, the isolation and use of a novel cellulase gene, cbh1 from Schizochytrium aggregatum, a cellulolytic marine fungoid organism.
The invention further relates to expression systems in yeast, such as Saccharomyces cerevisiae, using this novel gene. The present invention provides important tools to enable growth of host such cells such as yeast on cellulosic substrates for ethanol production.
Definitions
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. Preferably, 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.
The term “heterologous” as used herein refers to an element of a vector, plasmid or host cell that is derived from a source other than the endogenous source. Thus, for example, 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). The term “heterologous” is also used synonymously herein with the term “exogenous.”
The term “domain” as used herein refers to a part of a molecule or structure that shares common physical or chemical features, for example hydrophobic, polar, globular, helical domains or properties, e.g., a DNA binding domain or an ATP binding domain. Domains can be identified by their homology to conserved structural or functional motifs. Examples of cellobiohydrolase (CBH) domains include the catalytic domain (CD) and the cellulose binding domain (CBD).
A “nucleic acid,” “polynucleotide,” or “nucleic acid molecule” 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.
An “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 analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term 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. In discussing the structure of particular double-stranded DNA molecules, 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.
A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified, e.g., in Sambrook, J., Fritsch, E. F. and Maniatis, T. MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein (hereinafter “Maniatis”, entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. For more stringent conditions, washes are performed at higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS are increased to 60° C. Another set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of highly stringent conditions are defined by hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS.
Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see, e.g., Maniatis at 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see, e.g., Maniatis, at 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “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.
As known in the art, “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.
Suitable nucleic acid sequences or fragments thereof (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% to 75% identical to the amino acid sequences reported herein, at least about 80%, 85%, or 90% identical to the amino acid sequences reported herein, or at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments are at least about 70%, 75%, or 80% identical to the nucleic acid sequences reported herein, at least about 80%, 85%, or 90% identical to the nucleic acid sequences reported herein, or at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequences reported herein. Suitable nucleic acid fragments not only have the above identities/similarities but typically encode a polypeptide having at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, or at least 250 amino acids.
The term “probe” refers to a single-stranded nucleic acid molecule that can base pair with a complementary single stranded target nucleic acid to form a double-stranded molecule.
The term “complementary” is used to describe the relationship between nucleotide bases that are capable to hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the instant invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences.
As used herein, the term “oligonucleotide” refers to a nucleic acid, generally of about 18 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule. Oligonucleotides can be labeled, e.g., with 32P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. An oligonucleotide can be used as a probe to detect the presence of a nucleic acid according to the invention. Similarly, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of a nucleic acid of the invention, or to detect the presence of nucleic acids according to the invention. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
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. The boundaries of the coding region are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. 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.
“Open reading frame” is abbreviated ORF and means a length of 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. In general, 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”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. 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 S1), 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. In eukaryotic cells, polyadenylation signals are control regions.
The term “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. For example, 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.
The term “expression,” as used herein, 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.
Polynucleotides of the Invention
The present invention is directed to a novel cbh1 nucleic acid sequence isolated from the celluloytic marine fungoid organism Schizochytrium aggregatum. The Schizochytrium aggregatum cbh1 gene is contained within a 7422 base pair region corresponding to a fragment of the Schizochytrium aggregatum chromosomal DNA, represented below as SEQ ID NO:1. The ATG start site and TAG termination site of the Schizochytrium aggregatum cbh1 gene are indicated in bold, with introns of cbh1 indicated by double underlining.
TAAGCAAAGGCAATAGGTGGTCTCAACGGCGCTCTGTACTTCGTGTCC
CCTATCTGTCCAATTTTTACTACTCTCCATGTATACTGACTCGCGTGACAGGGCCTGAC
The start codon, ATG, begins at position 3872 of SEQ ID NO: 1, followed by exon 1 (extending from position 3872 to 4345 of SEQ ID NO:1), intron 1 (extending from position 4346 to 4488 of SEQ ID NO:1), exon 2 (extending from position 4489 to 5542 of SEQ ID NO:1), intron 2 (extending from position 5543 to 5593 of SEQ ID NO:1), exon 3 (extending from position 5594 to 5626 of SEQ ID NO:1), and the termination codon TAG, ending at position 5626 of SEQ ID NO:1.
Also encompassed by the invention is the corresponding cDNA encoding the Schizochytrium aggregatum cbh1, 1638 base pairs in length, represented by SEQ ID NO:2, and encoding for a protein of 546 amino acid residues:
The present invention also encompasses an isolated polynucleotide comprising a nucleic acid at least about 60%, 65%, 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 SEQ ID NO:1, an intron or exon of SEQ ID NO:1, SEQ ID NO:2, or fragments, variants, or derivatives thereof.
In certain aspects, the present invention encompasses a polynucleotide comprising a nucleic acid encoding a functional or structural domain of Schizochytrium aggregatum cbh1, e.g., as represented schematically in
The present invention also encompasses an isolated polynucleotide comprising a nucleic acid that is about 60%, 65%, 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 a nucleic acid encoding a Cbh1 Schizochytrium aggregatum domain, as described above.
By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the 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 S. aggregatum polypeptide. In other words, to obtain a 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 SEQ ID NO:2 or any fragment specified as described herein.
As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 60%, 65%, 70%, 75%, 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. (1990) 6:237-245.) In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, 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.
For example, 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%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the 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. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only 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 SEQ ID NO:1, SEQ ID NO:2, or fragments thereof. In certain aspects, fragments of SEQ ID NO:1 or SEQ ID NO:2 encode a Cbh1 Schizochytrium aggregatum domain, as set forth above.
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 NO:3 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 SEQ ID NO:2.
In certain embodiments, 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 NO:3.
The polynucleotide encoding for the mature polypeptide of SEQ ID NO:3 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.
Thus, the term “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.
In further aspects of the invention, nucleic acid molecules having sequences at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences disclosed herein, encode a polypeptide having Schizochytrium aggregatum Cbh1 functional activity. By “a polypeptide having Schizochytrium aggregatum Cbh1 functional activity” is intended polypeptides exhibiting activity similar, but not necessarily identical, to a functional activity of the Schizochytrium aggregatum Cbh1 polypeptides of the present invention, as measured, for example, in a particular biological assay. For example, a Schizochytrium aggregatum Cbh1 functional activity can routinely be measured by determining the ability of a Schizochytrium aggregatum Cbh1 polypeptide with respect to cellulase activity.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large portion of the nucleic acid molecules having a sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or fragments thereof, will encode polypeptides “having Schizochytrium aggregatum Cbh1 functional activity.” In fact, since 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. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having Schizochytrium aggregatum Cbh1 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 Schizochytrium aggregatum cbh1 gene, or a gene 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.
In certain embodiments, 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 hereinabove-described sequences if there is at least 60%, at least 70%, at least 80%, 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. As herein used, the term “stringent conditions” means hybridization will occur only if there is at least 95% or at least 97% identity between the sequences. In certain aspects of the invention, the 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 SEQ ID NO:1, SEQ ID NO:2.
Alternatively, 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. For example, such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:1, or SEQ ID NO:2, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR 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.
For example, 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). Moreover, 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.
In certain aspects of the invention, 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 PCR 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 PCR primer design are common and well known in the art. Generally two short segments of the instant sequences may be used in polymerase chain reaction (PCR) protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. 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. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).
In addition, specific 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.
Therefore, the 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)).
The polynucleotides of the present invention also comprise nucleic acids encoding Schizochytrium aggregatum Cbh1, a domain of Schizochytrium aggregatum Cbh1, or a fragment of Schizochytrium aggregatum Cbh1 fused in frame to a marker sequence which allows for detection of the polypeptide of the present invention. The marker sequence may be a yeast selectable marker selected from the group consisting of URA3, HIS3, LEU2, TRP1, LYS2 or ADE2. Additional marker sequences include other auxotrophic markers or dominant markers known to one of ordinary skill in the art such as ZEO (zeocin), NEO (G418), hygromycin, arsenite, HPH, NAT, and the like.
The present invention also encompasses variants of the Schizochytrium aggregatum cbh1 gene. 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. In certain embodiments, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. In further embodiments, Schizochytrium aggregatum cbh1 polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the bacterial mRNA to those preferred by a lower eukaryotic host such as the yeast Saccharomyces cerevisiae).
Also provided in the present invention are allelic variants, orthologs, and/or species homologs. 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 SEQ ID NO:1 or SEQ ID NO:2, 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.
Codon Optimization
As used herein the term “codon optimized coding region” means a nucleic acid coding region that has been adapted for expression in the cells of a given vertebrate by replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that vertebrate.
In general, highly expressed genes in an organism are biased towards codons that are recognized by the most abundant tRNA species in that organism. One measure of this bias is the “codon adaptation index” or “CAI,” which measures the extent to which the codons used to encode each amino acid in a particular gene are those which occur most frequently in a reference set of highly expressed genes from an organism.
The CAI of codon optimized sequences of the present invention corresponds to between about 0.8 and 1.0, between about 0.8 and 0.9, or about 1.0. A codon optimized sequence may be further modified for expression in a particular organism, depending on that organism's biological constraints. For example, large runs of “As” or “Ts” (e.g., runs greater than 4, 4, 5, 6, 7, 8, 9, or 10 consecutive bases) can be removed from the sequences if these are known to effect transcription negatively. Furthermore, specific restriction enzyme sites may be removed for molecular cloning purposes. Examples of such restriction enzyme sites include PacI, AscI, BamHI, BglII, EcoRI and XhoI. Additionally, the DNA sequence can be checked for direct repeats, inverted repeats and mirror repeats with lengths of ten bases or longer, which can be modified manually by replacing codons with “second best” codons, i.e., codons that occur at the second highest frequency within the particular organism for which the sequence is being optimized.
Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The “genetic code” which shows which codons encode which amino acids is reproduced herein as Table 1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.
Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. Codon usage tables are readily available, for example, at http://phenotype.biosci.umbc.edu/codon/sgd/index.php (visited May 7, 2008) or at http://www.kazusa.or.jp/codon/ (visited Mar. 20, 2008), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Codon usage tables for yeast, calculated from GenBank Release 128.0 [15 Feb. 2002], are reproduced below as Table 2. This Table uses mRNA nomenclature, and so instead of thymine (T) which is found in DNA, the Table uses uracil (U) which is found in RNA. The Table has been adapted so that frequencies are calculated for each amino acid, rather than for all 64 codons.
By utilizing this or similar tables, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide, but which uses codons optimal for a given species. Codon-optimized coding regions can be designed by various different methods.
In one method, a codon usage table is used to find the single most frequent codon used for any given amino acid, and that codon is used each time that particular amino acid appears in the polypeptide sequence. For example, referring to Table 2 above, for leucine, the most frequent codon is UUG, which is used 27.2% of the time. Thus all the leucine residues in a given amino acid sequence would be assigned the codon UUG.
In another method, the actual frequencies of the codons are distributed randomly throughout the coding sequence. Thus, using this method for optimization, if a hypothetical polypeptide sequence had 100 leucine residues, referring to Table 2 for frequency of usage in the S. cerevisiae, about 5, or 5% of the leucine codons would be CUC, about 11, or 11% of the leucine codons would be CUG, about 12, or 12% of the leucine codons would be CUU, about 13, or 13% of the leucine codons would be CUA, about 26, or 26% of the leucine codons would be UUA, and about 27, or 27% of the leucine codons would be UUG.
These frequencies would be distributed randomly throughout the leucine codons in the coding region encoding the hypothetical polypeptide. As will be understood by those of ordinary skill in the art, the distribution of codons in the sequence can vary significantly using this method; however, the sequence always encodes the same polypeptide.
Codon-optimized sequences of the present invention include SEQ ID NO: 4 as follows, where the Schizochytrium aggregatum cbh1 cDNA sequence has been codon optimized for Saccharomyces cerevisiae. The native Schizochytrium aggregatum cbh1 signal sequence is exchanged by replacing it with a slightly modified (one amino acid different) Saccharomyces cerevisiae alpha mating factor pre signal sequence (underlined); STOP-codon is double underlined:
ATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTCGCAGCCTCATC
GAGTCTAGCTCAACAGGCCGGTACTCTAACGCCTGAGAAACATCCCG
In one method, a codon usage table is used to find the single most frequent codon used for any given amino acid, and that codon is used each time that particular amino acid appears in the polypeptide sequence. For example, referring to Table 2 above, for leucine, the most frequent codon is UUG, which is used 27.2% of the time. Thus all the leucine residues in a given amino acid sequence would be assigned the codon UUG. The Saccharomyces cerevisiae codon-optimized nucleotide sequence encoding Schizochytrium aggregatum cbh 1 which has been optimized using this method is presented herein as SEQ ID NO 5:
In another method, the actual frequencies of the codons are distributed randomly throughout the coding sequence. Thus, using this method for optimization, if a hypothetical polypeptide sequence had 100 leucine residues, referring to Table 2 for frequency of usage in the S. cerevisiae, about 5, or 5% of the leucine codons would be CUC, about 11, or 11% of the leucine codons would be CUG, about 12, or 12% of the leucine codons would be CUU, about 13, or 13% of the leucine codons would be CUA, about 26, or 26% of the leucine codons would be UUA, and about 27, or 27% of the leucine codons would be UUG.
These frequencies would be distributed randomly throughout the leucine codons in the coding region encoding the hypothetical polypeptide. As will be understood by those of ordinary skill in the art, the distribution of codons in the sequence can vary significantly using this method, however, the sequence always encodes the same polypeptide. A different Saccharomyces cerevisiae codon-optimized nucleotide sequences encoding Schizochytrium aggregatum cbh1 which has been optimized using this method is presented herein as SEQ ID NO: 6:
When using the latter method, the term “about” is used precisely to account for fractional percentages of codon frequencies for a given amino acid. As used herein, “about” is defined as one amino acid more or one amino acid less than the value given. The whole number value of amino acids is rounded up if the fractional frequency of usage is 0.50 or greater, and is rounded down if the fractional frequency of use is 0.49 or less. Using again the example of the frequency of usage of leucine in human genes for a hypothetical polypeptide having 62 leucine residues, the fractional frequency of codon usage would be calculated by multiplying 62 by the frequencies for the various codons. Thus, 7.28 percent of 62 equals 4.51 UUA codons, or “about 5,” i.e., 4, 5, or 6 UUA codons, 12.66 percent of 62 equals 7.85 HUG codons or “about 8,” i.e., 7, 8, or 9 UUG codons, 12.87 percent of 62 equals 7.98 CUU codons, or “about 8,” i.e., 7, 8, or 9 CUU codons, 19.56 percent of 62 equals 12.13 CUC codons or “about 12,” i.e., 11, 12, or 13 CUC codons, 7.00 percent of 62 equals 4.34 CUA codons or “about 4,” i.e., 3, 4, or 5 CUA codons, and 40.62 percent of 62 equals 25.19 CUG codons, or “about 25,” i.e., 24, 25, or 26 CUG codons.
Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence, can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly. Additionally, various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the “EditSeq” function in the Lasergene Package, available from DNAstar, Inc., Madison, Wis., the backtranslation function in the VectorNTI Suite, available from InforMax, Inc., Bethesda, Md., and the “backtranslate” function in the GCG—Wisconsin Package, available from Accelrys, Inc., San Diego, Calif. In addition, various resources are publicly available to codon-optimize coding region sequences, e.g., the “backtranslation” function at http://www.entelechon.com/bioinformatics/backtranslation.php?lang=eng (visited Apr. 15, 2008) and the “backtranseq” function available at http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited Jul. 9, 2002). Constructing a rudimentary algorithm to assign codons based on a given frequency can also easily be accomplished with basic mathematical functions by one of ordinary skill in the art.
A further Saccharomyces cerevisiae codon-optimized polynucleotide sequence of the present invention, corresponding to SEQ ID NO: 7 is as follows:
A number of options are available for synthesizing codon optimized coding regions designed by any of the methods described above, using standard and routine molecular biological manipulations well known to those of ordinary skill in the art. In one approach, a series of complementary oligonucleotide pairs of 80-90 nucleotides each in length and spanning the length of the desired sequence are synthesized by standard methods. These oligonucleotide pairs are synthesized such that upon annealing, they form double stranded fragments of 80-90 base pairs, containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region that is complementary to the other oligonucleotide in the pair. The single-stranded ends of each pair of oligonucleotides is designed to anneal with the single-stranded end of another pair of oligonucleotides. The oligonucleotide pairs are allowed to anneal, and approximately five to six of these double-stranded fragments are then allowed to anneal together via the cohesive single stranded ends, and then they ligated together and cloned into a standard bacterial cloning vector, for example, a TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif. The construct is then sequenced by standard methods. Several of these constructs consisting of 5 to 6 fragments of 80 to 90 base pair fragments ligated together, i.e., fragments of about 500 base pairs, are prepared, such that the entire desired sequence is represented in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector, and sequenced. Additional methods would be immediately apparent to the skilled artisan. In addition, gene synthesis is readily available commercially.
In certain embodiments, an entire polypeptide sequence, or fragment, variant, or derivative thereof is codon optimized by any of the methods described herein. Various desired fragments, variants or derivatives are designed, and each is then codon-optimized individually. In addition, partially codon-optimized coding regions of the present invention can be designed and constructed. For example, the invention includes a nucleic acid fragment of a codon-optimized coding region encoding a polypeptide in which at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the codon positions have been codon-optimized for a given species. That is, they contain a codon that is preferentially used in the genes of a desired species, e.g., a yeast species such as Saccharomyces cerevisiae, in place of a codon that is normally used in the native nucleic acid sequence.
In additional embodiments, a full-length polypeptide sequence is codon-optimized for a given species resulting in a codon-optimized coding region encoding the entire polypeptide, and then nucleic acid fragments of the codon-optimized coding region, which encode fragments, variants, and derivatives of the polypeptide are made from the original codon-optimized coding region. As would be well understood by those of ordinary skill in the art, if codons have been randomly assigned to the full-length coding region based on their frequency of use in a given species, nucleic acid fragments encoding fragments, variants, and derivatives would not necessarily be fully codon optimized for the given species. However, such sequences are still much closer to the codon usage of the desired species than the native codon usage. The advantage of this approach is that synthesizing codon-optimized nucleic acid fragments encoding each fragment, variant, and derivative of a given polypeptide, although routine, would be time consuming and would result in significant expense.
The codon-optimized coding regions can be versions encoding a cbh1 gene from any strain of Schizochytrium aggregatum, or fragments, variants, or derivatives thereof. The present invention therefore encompasses nucleic acid fragments of codon-optimized coding regions encoding the Schizochytrium aggregatum Cbh1, the nucleic acid fragments encoding the complete polypeptide, as well as various fragments, variants, and derivatives thereof, although other Cbh1-encoding nucleic acid sources are not excluded.
Codon optimization is carried out for a particular vertebrate species by methods described herein, for example, in certain embodiments codon-optimized coding regions encoding polypeptides of Schizochytrium aggregatum Cbh1, or nucleic acid fragments of such coding regions encoding fragments, variants, or derivatives thereof are optimized according to yeast codon usage, e.g., Saccharomyces cerevisiae. In particular, the present invention relates to codon-optimized coding regions encoding polypeptides of Schizochytrium aggregatum Cbh1, or nucleic acid fragments of such coding regions fragments, variants, or derivatives thereof which have been optimized according to yeast codon usage, for example, Saccharomyces cerevisiae codon usage. For example, yeast codon-optimized coding regions encoding polypeptides of Schizochytrium aggregatum Cbh1, or nucleic acid fragments of such coding regions encoding fragments, variants, or derivatives thereof, are prepared by incorporating codons preferred for use in yeast genes into the DNA sequence encoding the Schizochytrium aggregatum Cbh1 polypeptide. Also provided are polynucleotides, vectors, and other expression constructs comprising codon-optimized coding regions encoding polypeptides of Schizochytrium aggregatum Cbh1, or nucleic acid fragments of such coding regions encoding fragments, variants, or derivatives thereof, and various methods of using such polynucleotides, vectors and other expression constructs.
The present invention is further directed towards polynucleotides comprising codon-optimized coding regions encoding polypeptides of Schizochytrium aggregatum Cbh1. The invention is also directed to polynucleotides comprising codon-optimized nucleic acid fragments encoding fragments, variants and derivatives of these polypeptides.
The present invention provides isolated polynucleotides comprising codon-optimized coding regions of Schizochytrium aggregatum Cbh1, or fragments, variants, or derivatives thereof. In certain embodiments described herein, a codon-optimized coding region encoding SEQ ID NO:3 is optimized according to codon usage in yeast (Saccharomyces cerevisiae). Alternatively, a codon-optimized coding region encoding SEQ ID NO:3 may be optimized according to codon usage in any plant, animal, or microbial species.
Polypeptides of the Invention
The present invention is further directed to a novel isolated Schizochytrium aggregatum Cbh1 polypeptide. The Schizochytrium aggregatum Cbh1 polypeptide is 546 amino acid residues in length. The amino acid sequence of the Schizochytrium aggregatum Cbh1 polypeptide corresponds to SEQ ID NO: 3 as follows:
The Schizochytrium aggregatum Cbh1 protein exhibits the highest degree of homology to a Botryotinia fuckeliana Cbh1 cellulase (SEQ ID NO:77) (Accession No. XP—001555330), with 57% identity over the 546 amino acid stretch.
The present invention further encompasses polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the polypeptide sequence shown in SEQ ID NO:3 and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein, or domains of SEQ ID NO:3).
By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the 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. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a 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.
As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of SEQ ID NO:3 can be determined conventionally using known computer programs. As discussed above, 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. 6:237-245 (1990)). In a sequence alignment 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. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter. Also as discussed above, manual corrections may be made to the results in certain instances.
In certain aspects of the invention, the 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 a polypeptide comprising the amino acid sequence of SEQ ID NO:3, and to portions of such a polypeptide, with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art “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 fragment, variant, derivative, or analog of the polypeptide comprising the amino acid sequence of SEQ ID NO:3.
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 Schizochytrium aggregatum Cbh1 polypeptides of the present invention encompass domains, proteolytic fragments, deletion fragments and in particular, fragments of Schizochytrium aggregatum Cbh1 polypeptides which retain any specific biological activity of the Schizochytrium aggregatum Cbh1 protein. Polypeptide fragments further include any portion of the polypeptide which comprises a catalytic activity of the Cbh1 protein.
The variant, derivative or analog of the polypeptide comprising the amino acid sequence of SEQ ID NO:3, can 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, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide or (v) one in which a fragment of the polypeptide is soluble, i.e., not membrane bound, yet still binds ligands to the membrane bound receptor. Such variants, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The present invention also encompasses variants of the polynucleotide sequence disclosed in SEQ ID NO:1 or SEQ ID NO:2, the complementary strand thereto, and variants of the polypeptide sequence disclosed in SEQ ID NO:3. Variants include one or several nucleic acid/amino acid deletions, substitutions and/or additions, where the variant retains cellobiohydrolase activity. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions and/or additions and/or deletions, less than 40 amino acid substitutions and/or additions and/or deletions, less than 30 amino acid substitutions and/or additions and/or deletions, less than 25 amino acid substitutions and/or additions and/or deletions, less than 20 amino acid substitutions and/or additions and/or deletions, less than 15 amino acid substitutions and/or additions and/or deletions, less than 5 amino acid substitutions and/or additions and/or deletions, less than 4 amino acid substitutions and/or additions and/or deletions, less than 3 amino acid substitutions and/or additions and/or deletions or less than 2 amino acid substitutions and/or additions and/or deletions relative to the reference Cbh1 polypeptide, domain, fragment or derivative thereof.
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. As used herein, 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. For example, the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, 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.
A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e.g., cellobiohydrolase activity).
By 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 Schizochytrium aggregatum Cbh1 protein.
The allelic variants, the conservative substitution variants, and members of the Cbh1 protein family, will have an amino acid sequence having at least 75% amino acid sequence identity with the Schizochytrium aggregatum Cbh1 amino acid sequence set forth in SEQ ID NO:3, at least 80%, at least 90%, at least 95%. 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.
Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the Schizochytrium aggregatum Cbh1 amino acid sequence of the present invention.
Thus, the proteins and peptides of the present invention include molecules comprising the amino acid sequence of SEQ ID NO: 3 or fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or more amino acid residues of the Schizochytrium aggregatum Cbh1 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 PCR mutagenesis, and the corresponding proteins of other animal species, including but not limited to rabbit, rat, porcine, bovine, ovine, equine and non-human primate species, the alleles or other naturally occurring variants of the family of proteins; 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).
Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the Schizochytrium aggregatum Cbh1 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.
Thus, the invention further includes Schizochytrium aggregatum Cbh1 polypeptide variants which show substantial biological activity. Such 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 skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.
For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
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.
As the authors state, these two strategies have revealed that proteins are often surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, 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.
The terms “derivative” and “analog” refer to a polynucleotide or polypeptide differing from the Schizochytrium aggregatum Cbh1 polynucleotide or polypeptide, but retaining essential properties thereof. Generally, derivatives and analogs are overall closely similar, and, in many regions, identical to the Schizochytrium aggregatum Cbh1 polynucleotide or polypeptide. The term “derivative” and “analog” when referring to Schizochytrium aggregatum polypeptides of the present invention include any polypeptides which retain at least some of the activity of the corresponding native polypeptide, e.g., the endoglucanase activity, or the activity of the its catalytic domain.
Derivatives of Schizochytrium aggregatum Cbh1 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 a Schizochytrium aggregatum Cbh1 polypeptide 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 Schizochytrium aggregatum Cbh1. 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.
Tethered and Secreted Schizochytrium aggregatum Cbh1 Polypeptides
In certain aspects of the invention, the Schizochytrium aggregatum Cbh1 is prepared as a tethered fusion polypeptide.
As used herein, a protein is “tethered” to an organism's cell surface if at least one terminus of the protein is bound, covalently and/or electrostatically for example, to the cell membrane or cell wall. It will be appreciated that a tethered protein may include one or more enzymatic regions that may be joined to one or more other types of regions at the nucleic acid and/or protein levels (e.g., a promoter, a terminator, an anchoring domain, a linker, a signaling region, etc.). While the one or more enzymatic regions may not be directly bound to the cell membrane or cell wall (e.g., such as when binding occurs via an anchoring domain), the protein is nonetheless considered a “tethered enzyme” according to the present specification.
Tethering can, for example, be accomplished by incorporation of an anchoring domain into a recombinant protein that is heterologously expressed by a cell, or by prenylation, fatty acyl linkage, glycosyl phosphatidyl inositol anchors or other suitable molecular anchors which may anchor the tethered protein to the cell membrane or cell wall of the host cell. A tethered protein can be tethered at its amino terminal end or optionally at its carboxy terminal end.
Such tethered polypeptides can provide an advantage for cell growth, where saccharified substrate is unable to diffuse away from the cell before being metabolized. In addition, a portion of a population of cells expressing a Schizochytrium aggregatum tethered Cbh1 can exhibit enhanced expression of the tethered enzyme relative to the overall population. This portion may exhibit enhanced binding to the substrate and improved growth characteristics.
In other aspects of the invention, the Schizochytrium aggregatum Cbh1 is a secreted polypeptide.
As used herein, “secreted” means released into the extracellular milieu, for example into the media. Although tethered proteins may have secretion signals as part of their immature amino acid sequence, they are maintained as attached to the cell surface, and do not fall within the scope of secreted proteins as used herein.
As used herein, “flexible linker sequence” refers to an amino acid sequence which links a cell wall anchoring amino acid sequence with an amino acid sequence that contains the desired enzymatic activity. The flexible linker sequence allows for necessary freedom for the amino acid sequence that contains the desired enzymatic activity to have reduced steric hindrance with respect to proximity to the cell and may also facilitate proper folding of the amino acid sequence that contains the desired enzymatic activity.
Homology of Schizochytrium aggregatum Cbh1 Polypeptides to GHF7 Family Members
Using BLAST analysis, SEQ ID NO:3, corresponding to the amino acid sequence of the Schizochytrium aggregatum Cbh1, was found to be homologous to several members of the glycosyl hydrolase, family 7 (
Glycoside hydrolase family 7 (also referred to as GH7 or GHF7) comprises enzymes with several known activities including endoglucanase activity and cellobiohydrolase (CBH) activity. These enzymes were formerly known as cellulase family C. Exoglucanases such as cellobiohydrolases play a role in the conversion of cellulose to glucose by cutting the dissaccharide cellobiose from the reducing or nonreducing end of the cellulose polymer chain. Cbh1 s generally cut reducing ends, while Cbh2s cut nonreducing ends. See http://www.cazy.org/fam/GH7.html (last updated May 23, 2008). Structurally, cellulases and xylanases generally consist of a catalytic domain (CD) joined to a cellulose-binding domain (CBD) via a linker region that is rich in proline and/or hydroxy-amino acids. In type I exoglucanases, the CBD domain is found at the C-terminal extremity of these enzyme (this short domain forms a hairpin loop structure stabilised by 2 disulphide bridges).
The “catalytic domain” is also referred to as the active site. The structure and chemical properties of the active site allow the recognition and binding of the substrate. The active site is usually a small pocket at the surface of the enzyme that contains residues responsible for the substrate specificity (charge, hydrophobicity, steric hindrance) and catalytic residues which often act as proton donors or acceptors or are responsible for binding a cofactor such as PLP, TPP or NAD. The active site is also the site of inhibition of enzymes. In the case of Schizochytrium aggregatum Cbh1, the catalytic domain contains residues that allow the recognition and binding of the Cbh1 exoglucanase to the cellulose substrate. Examples of such amino acid residues include the proton donor, probable secondary nucleophile, and catalytic nucleophile as shown in
A “cellulose-binding domain” is a domain that naturally binds to cellulose. It is thought that CBDs concentrate the catalytic domains on the surface of the insoluble cellulose substrate. The CBD of the present invention is comprised of three folded anti-parallel β-sheets. It is wedge-shaped, with a hydrophobic flat face that interacts with the cellulose surface via two tyrosine residues and a glutamine, and a hydrophilic face. The CBD binds to both amorphous and crystalline cellulose.
“Cellulose” is an unbranched homopolymer of β(1-4) linked glucose subunits. Crystalline cellulose presents a surface array of parallel, closely-packed cellulose chains to a CBD. Amorphous cellulose presents antiparallel or disordered chains to a CBD. The binding site of a CBD is adapted to binding to a surface.
Vectors and Host Cells
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 can be, for example, a cloning vector or an expression vector. The vector can 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 transformants 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. Thus, for example, 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. However, any other appropriate vector known to one of ordinary skill in the art may be used.
The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, 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. Representative examples of such promoters are as follows:
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
Additionally, promoter sequences from stress and starvation response genes are useful in the present invention. In some embodiments, promoter regions from the S. cerevisiae genes GAC1, GET3, GLC7, GSH1, GSH2, HSF1, HSP12, LCB5, LRE1, LSP1, NBP2, PIL1, PIM1, SGT2, SLG1, WHI2, WSC2, WSC3, WSC4, YAP1, YDC1, HSP104, HSP26, ENA1, MSN2, MSN4, SIP2, SIP4, SIP5, DPL1, IRS4, KOG1, PEP4, HAP4, PRB1, TAX4, ZPR1, ATG1, ATG2, ATG10, ATG11, ATG12, ATG13, ATG14, ATG15, ATG16, ATG17, ATG18, and ATG19 can be used. Any suitable promoter to drive gene expression in the host cells of the invention can be used.
Additionally, the E. coli, lac or trp, and other promoters known to control expression of genes in prokaryotic or lower eukaryotic cells can be used. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector can also include appropriate sequences for amplifying expression, or can include additional regulatory regions.
In addition, the expression vectors may contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as URA3, HIS3, LEU2, TRP1, LYS2 or ADE2, dihydrofolate reductase, neomycin (G418) resistance or zeocin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli.
The vector containing the appropriate DNA sequence as herein, as well as an appropriate promoter or control sequence, can be employed to transform an appropriate host to permit the host to express the protein.
Thus, in certain aspects, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, e.g., Saccharomyces cerevisiae, or the host cell can be a prokaryotic cell, such as a bacterial cell.
Representative examples of appropriate hosts include bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; thermophilic or mesophlic bacteria; fungal cells, such as yeast; and plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
Appropriate fungal hosts include yeast. In certain aspects of the invention the yeast is Saccharomyces cerevisiae, Kluveromyces lactus, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schwanniomyces occidentalis, Issatchenkia orientalis, or Kluveromyces marxianus.
Major groups of 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. In certain embodiments, 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. Some examples of thermophilic microorganisms (including bacteria, prokaryotic microorganisms such as fungi), which may be suitable for the present invention 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, Desulfovibrio thermophilus, Thermoplasma acidophilum, Hydrogenomonas thermophilus, Thermomicrobium roseum, Thermus flavas, Thermus ruber, Pyrococcus furiosus, Thermus aquaticus, Thermus thermophilus, Chlorofiexus aurantiacus, Thermococcus litoralis, Pyrodictium abyssi, Bacillus stearothermophilus, Cyanidium caldarium, Mastigocladus laminosus, Chlamydothrix calidissima, Chlamydothrix penicillata, Thiothrix carnea, Phormidium tenuissimum, Phormidium geysericola, Phormidium subterraneum, Phormidium bijahensi, Oscillatoria filiformis, Synechococcus lividus, Chloroflexus aurantiacus, Pyrodictium brockii, Thiobacillus thiooxidans, Sulfolobus acidocaldarius, Thiobacillus thermophilica, Bacillus stearothermophilus, Cercosulcifer hamathensis, Vahlkampfia reichi, Cyclidium citrullus, Dactylaria gallopava, Synechococcus lividus, Synechococcus elongatus, Synechococcus minervae, Synechocystis aquatilus, Aphanocapsa thermalis, Oscillatoria terebriformis, Oscillatoria amphibia, Oscillatoria germinata, Oscillatoria okenii, Phormidium laminosum, Phormidium parparasiens, Symploca thermalis, Bacillus acidocaldarias, Bacillus coagulans, Bacillus thermocatenalatus, Bacillus licheniformis, Bacillus pamilas, Bacillus macerans, Bacillus circulars, Bacillus laterosporus, Bacillus brevis, Bacillus subtilis, Bacillus sphaericus, Desulfotomaculum nigrificans, Streptococcus thermophilus, Lactobacillus thermophilus, Lactobacillus bulgaricus, Bifidobacterium thermophilum, Streptomyces fragmentosporus, Streptomyces thermonitrificans, Streptomyces thermovulgaris, Pseudonocardia thermophila, Thermoactinomyces vulgaris, Thermoactinomyces sacchari, Thermoactinomyces candidas, Thermomonospora curvata, Thermomonospora viridis, Thermomonospora citrina, Microbispora thermodiastatica, Microbispora aerata, Microbispora bispora, Actinobifida dichotomica, Actinobifida chromogena, Micropolyspora caesia, Micropolyspora faeni, Micropolyspora cectivugida, Micropolyspora cabrobrunea, Micropolyspora thermovirida, Micropolyspora viridinigra, Methanobacterium thermoautothropicum, variants thereof, and/or progeny thereof.
In certain embodiments, 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.
In certain embodiments, 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 stearothermophilus, Saccharococcus caldoxylosilyticus, Saccharoccus thermophilus, Paenibacillus campinasensis, Bacillus flavothermus, Anoxybacillus kamchatkensis, Anoxybacillus gonensis, variants thereof, and progeny thereof.
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. In one aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably associated to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example only.
Yeast: Yeast vectors include those of five general classes, based on their mode of replication in yeast: YIp (yeast integrating plasmids), YRp (yeast replicating plasmids), YCp (yeast replicating plasmids with centromere (CEN) elements incorporated), YEp (yeast episomal plasmids), and YLp (yeast linear plasmids). With the exception of the YLp plasmids, all of these plasmids can be maintained in E. coli as well as in Saccharomyces cerevisiae and thus are also referred to as yeast shuttle vectors.
In certain aspects, these plasmids contain types of selectable genes including plasmid-encoded drug-resistance genes and/or cloned yeast genes, where the drug resistant gene and/or cloned yeast gene can be used for selection. Drug-resistance genes include, e.g., ampicillin, kanamycin, tetracycline, neomycin, hygromycin, zeocin, NAT, arsentied and sulfometuron methyl. Cloned yeast genes include HIS3, LEU2, LYS2, TRP1, URA3, TRP1 and SMR1. Other yeast genes that may be used correspond to different appropriate auxotrophic or dominant markers known to one of ordinary skill in the art. pYAC vectors may also be utilized to clone large fragments of exogenous DNA on to artificial linear chromosomes.
In certain aspects of the invention, YCp plasmids, which have high frequencies of transformation and increased stability to due the incorporated centromere elements, are utilized. In certain other aspects of the invention, YEp plasmids, which provide for high levels of gene expression in yeast, are utilized. In additional aspects of the invention, YRp plasmids are utilized.
In particular embodiments, the vector of the present invention is a plasmid selected from the group consisting of the pMU506 or the pMU562 plasmid.
Representative examples of bacterial plasmids include pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223 3, pKK233-3, pDR540, pRIT5 (Pharmacia).
However, any other appropriate plasmid or vector known to one of ordinary skill in the art may be used.
Promoter regions can be selected from any desired gene. Particular named yeast promoters include the constitute promoter ENO1, the PGK1 promoter, the TEF1 promoter and the HXT7 promoter. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
Introduction of the construct into a host yeast cell, e.g., Saccharomyces cerevisiae, can be effected by, e.g., lithium acetate transformation, spheroplast transformation, or transformation by electroporation, as described in Current Protocols in Molecular Biology, 13.7.1-13.7.10.
Introduction of the construct in host cells can also be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation. (Davis, L., et al., Basic Methods in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Following creation of a suitable host cell and growth of the host cell to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
Yeast cells, e.g., Saccharomyces cerevisiae, employed in expression of proteins can be manipulated as follows. The Cbh polypeptides can be recovered and purified from recombinant cell cultures by methods including spheroplast preparation and lysis, cell disruption using glass beads, and cell disruption using liquid nitrogen for example.
Various mammalian cell culture systems can also be employed to express recombinant protein. Expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences.
Additional methods include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
The Cbh polypeptides can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
Cbh polypeptides are provided in an isolated form, and, in certain aspects, are substantially purified. A recombinantly produced version of a Cbh polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Cbh polypeptides also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art.
The Cbh polypeptides of the present invention can be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.
Secretion of desired proteins into the growth media has the advantages of simplified and less costly purification procedures. It is well known in the art that secretion signal sequences are often useful in facilitating the active transport of expressible proteins across cell membranes. The creation of a transformed host capable of secretion may be accomplished by the incorporation of a DNA sequence that codes for a secretion signal which is functional in the host production host. Methods for choosing appropriate signal sequences are well known in the art (see for example EP 546049; WO 9324631). The secretion signal DNA or facilitator may be located between the expression-controlling DNA and the instant gene or gene fragment, and in the same reading frame with the latter.
Heterologous Expression of Schizochytrium aggregatum Cbh1 in Host Cells
In order to address the limitations of the previous systems, the present invention provides a novel cbh1 gene and Cbh1 polypeptide that can be effectively and efficiently utilized in a consolidated bioprocessing system.
One aspect of the invention is thus related to the efficient production of saccharolytic enzymes (cellulases and hemicellulases) to aid in the digestion of cellulose and generation of ethanol.
A “saccharolytic enzyme” is also referred to as a cellulase, and can correspond to any enzyme involved in cellulase digestion, metabolism and/or hydrolysis, including an endoglucananse, exoglucanase, or β-glucosidase. An exoglucanase can be, for example, a cellobiohydrolase.
In particular, the invention relates to the production of Schizochytrium aggregatum Cbh1 in a host organism. In certain embodiments, this host organism is yeast, such as Saccharomyces cerevisiae.
In certain embodiments of the present invention, a host cell comprising a vector which encodes and expresses Schizochytrium aggregatum Cbh1 that is utilized for consolidated bioprocessing is co-cultured with additional host cells expressing one or more additional endoglucanases, cellobiohydrolases and/or β-glucosidases. In other embodiments of the invention, a host cell transformed with Schizochytrium aggregatum Cbh1 is transformed with and expresses one or more heterologous endoglucanases, cellobiohydrolases or β-glucosidases. The endoglucanase, cellobiohydrolase and/or β-glucosidase can be any suitable endoglucanase, cellobiohydrolase and β-glucosidase derived from, for example, a fungal or bacterial source. Furthermore, the endoglucanase, cellobiohydrolase and/or β-glucosidase can be either tethered or secreted.
In certain embodiments of the invention, the endoglucanase(s) can be an endoglucanase I or an endoglucanase II isoform, paralogue or orthologue. In another embodiment, the endoglucanase expressed by the host cells of the present invention can be recombinant endo-1,4-β-glucanase. In certain embodiments of the present invention, the endoglucanase is an endoglucanase I from Trichoderma reesei. In another embodiment, the endoglucanase is encoded by the polynucleotide sequence of SEQ ID NO:59 (Accession No. M15665), as follows:
In certain embodiments of the present invention the (β-glucosidase is derived from Saccharomycopsis fibuligera. In certain embodiments, the β-glucosidase is a β-glucosidase I or a β-glucosidase II isoform, paralogue or orthologue. In certain other embodiments, the β-glucosidase expressed by the cells of the present invention can be recombinant β-glucanase I from a Saccharomycopsis fibuligera source, corresponding to SEQ ID NO: 60 (Accession No. M22475), as follows:
In certain embodiments of the invention, the cellobiohydrolase(s) can be an cellobiohydrolase I and/or an cellobiohydrolase II isoform, paralogue or orthologue. In certain embodiments of the present invention the cellobiohydrolases are cellobiohydrolase I and II, or a domain of a Cbh1 or Cbh2 as set forth in the Table below:
Humicola
grisea cbh1
Thermoascus
aurantiacus
Talaromyces
emersonii
Talaromyces
emersonii
Trichoderma
ATGGTCTCCTTCACCTCCCTGCTGGCCGGCGTTGCCGCTATCTCTGGTGTCCTAGCAGC
reesei cbh1
Trichoderma
ATGGTCTCCTTCACCTCCCTGCTGGCCGGCGTTGCCGCTATCTCTGGTGTCCTAGCAGC
reesei cbh2
CCATCTTTCTTGTAA
In other embodiments, the cellobiohydrolases can be encoded by the polynucleotide sequences of SEQ ID NOs: 47-52.
In further embodiments, the one or more additional endoglucanases, cellobiohydrolases and/or βglucosidases can be from a termite or termite-associated symbiont. For example, the endogluconase can be a Coptotermes formosanus endogluconase (SEQ ID NO: 78) (Accession No. AB058671) as follows:
The nucleic acids encoding the termite or termite-associated symbiont cellulases can be codon-optimized for expression in a yeast strain.
The transformed host cells or cell cultures, as described above, are measured for endoglucanase, cellobiohydrolase and/or β-glucosidase protein content. Protein content can be determined by analyzing the host (e.g., yeast) cell supernatants. In certain embodiments, 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 cellulase activity (cellulose utilization), e.g., by measuring the individual cellulase (endoglucanase, cellobiohydrolase or β-glucosidase) or by measuring total cellulase activity. Endoglucanase activity can be measured based on a reduction in cellulosic substrate viscosity and/or an increase in reducing ends determined by a reducing sugar assay. Cellobiohydrolase activity can be measured, for example, by using Avicel as a substrate and determining its hydrolysis. β-glucosidase activity can be measured by a variety of assays, e.g., using cellobiose.
A total cellulase activity, which includes the activity of endoglucanase, cellobiohydrolase and β-glucosidase, will hydrolyze crystalline cellulose synergistically. Total cellulase activity can thus be measured using insoluble substrates including pure cellulosic substrates such as Whatman No. 1 filter paper, cotton linter, microcrystalline cellulose, bacterial cellulose, algal cellulose, and cellulose-containing substrates such as dyed cellulose, alpha-cellulose or pretreated lignocellulose.
It will be appreciated that 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. In various embodiments, the lignocellulosic biomass comprises, for example, wood, corn, corn stover, sawdust, bark, leaves, agricultural and forestry residues, grasses such as switchgrass, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard or combinations thereof.
Specific activity of cellulases can also be detected by methods known to one of ordinary skill in the art, such as by the Avicel assay (described supra) that would be normalized by protein (cellulase) concentration measured for the sample. To accurately measure protein concentration, Schizochytrium aggregatum Cbh1 can be expressed with a His-taq or HA-tag and purified by a standard nickel resin purification technique or similar approach.
In additional embodiments, the transformed host cells or cell cultures are assayed for ethanol production. Ethanol production can be measured by techniques known to one of ordinary skill in the art, e.g., by a standard HPLC refractive index method.
The isolation of genes for saccharolytic enzymes from cellulolytic marine fungoid organisms of the Thraustochytrid family was considered. Thraustochytrids are important mangrove decomposers on decaying cellulose-rich materials such as macroalgae and mangrove leaves. Thus, Thraustochytrid family organisms are potential candidates from which novel cellulases can be isolated. Cellulase production has been detected in the Thraustochytrid family marine fungoid protist Schizochytrium aggregatum (Bremer GB, 1995). Given the production of cellulase in this organism, it was determined that isolation of a cellobiohydrolase I (cbh1) gene from Schizochytrium aggregatum would be advantageous, as the encoded protein could serve as a potential exoglucanase for use in a consolidated bioprocessing system.
Secreted and cell associated cellulase activity was measured for three cellulolytic marine fungoid Thraustochytrid strains, obtained from ATCC. The three marine fungoid strains utilized are as follows: (1) Schizochytrium aggregatum 16, ATCC #28209; (2) unidentified Thraustochytrid ATCC #PRA-147; and (3) Schizochytrium limacinum SR21, ATCC #MYA-1381. PCR analysis of 18S ribosomal RNA was carried out on these strains to confirm their identity. A Table summarizing the three strains is as follows:
Schizochytrium
Schizochytrium
Aggre-
aggregatum
gatum
Aurantiochytrium
Schizochytrium
The three strains were grown in shaker flasks for 10 days in media using six different carbon sources: glucose, Sigma cell, Avicel, Solka floc, lactose, and glycerol. The media composition was as follows: every 1 Liter (L) of media contained the following: yeast extract—1 g; peptone—1 g; carbon source—5 g; bacto agar—20 g; and sea water—to 1 L. The sea water was prepared by combining the following ingredients: every 2 Liters of sea water contained the following: NaCl—40 g; MgCl2×6H2O—6 g; CaCl2×2 H2O—0.3 g; KCl—1 g; and, water—to 2 L.
Cellulase activity of the three strains was measured by the resorufin-cellobioside assay (MarkerGene Fluorecent Cellulase Assay Kit, MGT Inc.). As shown in
As an initial step to isolate the cbh1 gene from the Schizochytrium aggregatum strain, thirty four degenerate primers having homology to conservative regions of fungal and protist Cbh's were designed. The fungal and protist Cbh sequences that were analyzed to find conservative regions within Cbh sequences are set forth in Table 5 as follows:
Neosartorya
fischeri
Gibberella
zeae
Penicillium
janthinellum
Nectria
haematococca
Fusarium
poae
Chaetomium
thermophilum
Aspergillus
terreus
Penicillium
chrysogenum
Neurospora
crassa
Trichoderma
viride
Humicola
grisea
Thermoascus
aurantiacus
Talaromyces
emersonii
Trichoderma
reesei
Phanerochaete
chrysosporium
Aspergillus
niger
Aspergillus
niger
The thirty four degenerate primers designed to have homology to conservative regions of the fungal and protist Cbh's list above are presented in Table 6 below. These primers were ordered from Integrated DNA Technologies (IDT) and utilized to clone the cbh1 gene as discussed further below.
Next, genomic DNA from Schizochytrium aggregatum was isolated as follows: Schizochytrium aggregatum was grown in 25 ml of cultivation media (as described above) in shaker flasks for 5-10 days. After 5-10 days, S. aggregatum culture was harvested and centrifuged to isolate the cells. The cells were washed with H2O and resuspended in 1 volume of “Smash and Grab” buffer. The “Smash and Grab” buffer contains 1% SDS (10% stock), 2% Triton X-100 (20% stock); 100 mM NaCl (4M stock); 1 mM EDTA (0.5M stock); and 10 mM Tris-Hl pH 8.0 (1M stock).
An equal volume of phenol-chloroform mix with glass beads was then added (2.5 ml cell pellet+2.5 ml buffer+5 ml phenol/chloroform+3 g beads). The resuspended cells were vortexed for 7 minutes, and then spun at 13,200 rpm in a 1.5 ml tubes for 10 minutes. The supernatant was then transferred to eppendorf tubes (450 μl/tube). 1 ml of cold ethanol was added to the supernatant, and this mixture was then spun down for 15 minutes. After this spinning step, the supernatant was discarded and 100 μl of TE was added to the pellet in each tube.
The resuspended pellets were combined and 1 μl RNAase (QIAGEN®) was added. This solution was next incubated at 37° C. for 5 minutes. After this, 1 μl of 4M NaCl, 10 μl of proteinase K, 20 μl of 10% SDS were added, and the mixture further incubated at 37° C. for 30 minutes. 400 μl of phenol-chloroform mix was then added, the mixture vortexed, and then spun down for 5 minutes. The resulting supernatant was transferred into new tubes, after which 400 μl of phenol-chloroform mix was again added, the mixture vortexed, and spun again for 5 minutes. To this final supernatant was added 40 μA 3M sodium acetate and 1 ml of cold ethanol. This final mixture was vortexed, spun for 15 minutes, and the supernatant discarded. The resulting pellet was washed with 70% ethanol twice and the pellet air-dried. The air-dried pellet was resuspended in 200 μl TE.
A GenomeWalker kit (Clontech) and protocol were used according to the manufacturer's instructions to perform PCR amplification using the Schizochytrium aggregatum genomic DNA as template. PCR amplification was performed using primer pairs, where each primer pair included one primer from Table 1 in combination with a primer homologous to the adapters that were used to make the template library according to the GenomeWalker kit manufacturer's protocol. Primers #4, 6, 8 (reverse primers for conservative region EMDIWEA (SEQ ID NO: 38)) and #22 (reverse primer for conservative region GYCDAQC (SEQ ID NO: 40)) yielded PCR products that had homology to fungal Cbh1s, as described infra (Example 2). The corresponding forward primers were primers #3, 5, 7, and 21. The sequencing analysis of the generated PCR products revealed that all of the isolated products were fragments of the same gene. The full length gene sequence including coding and flanking regions was obtained as described herein. First, a PCR fragment was isolated from a library (with reverse primer #4) corresponding to a fragment containing the 5′ flanking region and a portion of the 5′ end of the Schizochytrium aggregatum cbh1 gene. Based on the sequence of this PCR fragment, Schizochytrium aggregatum cbh1-specific primers were designed (as indicated below):
S. aggregatum
S. aggregatum
S. aggregatum
S. aggregatum
Primers 39 and 40 are complementary to each other as well as primers 41 and 42 are complementary to each other. Primers 39 and 40 generated distinct and abundant PCR products. Each of the primer pairs generated a PCR product about 2.5 to about 4 kb in length using the library template (four library templates were prepared according manufacture instructions). PCR products obtained with primers 39 and 40 were sequenced. The PCR fragment from primer 39 contained a portion of the 3′ end of the Schizochytrium aggregatum cbh1 gene and additional 3′ flanking region. The PCR fragment from primer 40 contained a portion of the 5′ end of the Schizochytrium aggregatum cbh1 gene and additional 5′ flanking region. The two sequences described above (the 5′ and 3′ portions of cbh1) were combined into one sequence about 7400 base pairs in length. This sequence containing the Schizochytrium aggregatum cbh1 is presented above and corresponds to SEQ ID NO:1.
As described above, the start codon, ATG, begins at position 3872 of SEQ ID NO: 1, followed by exon 1 (extending from position 3872 to 4345 of SEQ ID NO:1), intron 1 (extending from position 4346 to 4488 of SEQ ID NO:1), exon 2 (extending from position 4489 to 5542 of SEQ ID NO:1), intron 2 (extending from position 5543 to 5593 of SEQ ID NO:1), exon 3 (extending from position 5594 to 5626 of SEQ ID NO:1), and the termination codon TAG, ending at position 5626 of SEQ ID NO:1.
The cDNA was isolated as described further herein. Total RNA was isolated from Schizochytrium aggregatum as follows: 50-100 μl of cells or tissue were collected in a 2 ml tube with screw cap. 200 μl acid-washed 0.45-0.55 mm glass beads were added (beads were soaked in nitric acid for 1 hour, washed with water, and dried in a baking oven before utilization). 1 ml of TRIzol reagent (Invitrogen cat #15596-018) was added and the mixture homogenized in a homogenizer (Precellys 24, Bertin Technologies) for 2-3 minutes. The mixture was then incubated at room temperature for 5 minutes. Next, 0.2 ml of chloroform was added, the mixture shaken and then spun down for 10 minutes at 12,000 g at 2-8° C. The clear supernatant was removed into a new tube and 1 volume of 70% ethanol was added. The final sample was applied to a column from the RNaesy kit (Qiagen cat #74104). The yeast protocol from the kit manual was followed starting with the column step. cDNA was prepared using the Invitrogen TermoScript RT-PCR System (cat #11146-016).
The prepared cDNA was utilized to isolate the cbh1 cDNA. A DNA fragment containing the coding sequence of Schizochytrium aggregatum cbh1 cDNA was obtained by PCR with Schizochytrium aggregatum cDNA as a template and primers specific for the Schizochytrium aggregatum cbh1 gene (forward primer: 5′ acttaattaaaATGTCTGCCATTACCCTCGCCC (SEQ ID NO: 92), where the lower case letters represent the restriction enzyme sites; reverse primer: 5′ acggcgcgccCTACAAGCACTGCGAGTAGTAGTC (SEQ ID NO: 93)). Sequence analysis of the DNA fragment yielded the complete cDNA sequence of the Schizochytrium aggregatum cbh1 gene, corresponding to SEQ ID NO:2. The cDNA sequence encodes for the Schizochytrium aggregatum Cbh1 polypeptide, also presented above, and corresponding to SEQ ID NO:3.
The isolated Schizochytrium aggregatum Cbh1 is novel. No identical sequences can be found in the public protein databases (PubMed). The translated sequence, based on sequence homology, belongs to the Glycosyl Hydrolase Family 7 (GHF7) of proteins and contains tunnel forming loops. Therefore, the sequence was predicted to encode a exogluconase type I or Cbh1 based on previous data demonstrating that exoglucanases have tunnel forming loops, whereas endoglucanases do not (see Zhou X et al., Gene 395:29-39 (2007). The isolated cbh1 gene is predicted to contain a N-terminal 19 amino acid signal sequence, and thus encodes a secreted protein. A schematic diagram of Schizochytrium aggregatum Cbh1 is shown in
Results of BLAST analysis between PCR products obtained from reactions using Primers #39 and 40 as described supra (Example 1) and other fungal Cbh1 's indicates that Schizochytrium aggregatum Cbh1 has substantial homology to these fungal sequences. The alignment between these sequences is shown in
A comparison of the full translated Cbh1 sequence with other fungal Cbh1 s reveals that the novel Cbh1 contains an N-terminal exogluconase catalytic domain and C-terminal cellulose binding domain. Alignment between the predicted amino acid sequence of Schizochytrium aggregatum and the Cbh1 amino acid sequences of various Cbh1 source organisms is shown in
Trichoderma reesei
Humicola grisea
Thermoascus aurantiacus*
Talaromyces emersonii*
Botryotinia fuckeliana
Phanerochaete chrysosporium
Chaetomidium pingungium
Genes encoding cellobiohydrolases in T. reseei (cbh1 and cbh2), A. niger (cbhA and cbhB) and P. chrysosporium (cbh1-4) have been cloned and described. The proteins encoded for by these genes are all modular enzymes containing a catalytic domain linked via a flexible liner sequence to a cellulose-binding molecule, similar to the isolated Schizochytrium aggregatum cbh1 gene described above. Cbh1, CbhB and Cbh1-4 are family 7 glycosyl hydrolases (GHF7) and have at least 50 to 60% homology at the amino acid level, but the homology between any of these enzymes and the glycosyl hydrolase family 6 CBH2 is less than 15%.
As shown above, the Schizochytrium aggregatum Cbh1 full length polypeptide sequence shares a 52% identity with the T. reesei Cbh1, and a 56% identity with the P. chrysosporium Cbh1, both of which have been previously identified and characterized. In addition, as shown above in Table 7, there is a higher percent identity shared between the individual domains of the Schizochytrium aggregatum Cbh1, and the corresponding domains in each of the various organisms identified in the table above. For example, the Schizochytrium aggregatum Cbh1 catalytic domain shares about 55% identity with the T. reesei Cbh1 catalytic domain. The Schizochytrium aggregatum Cbh1 shares the highest percent identity with the full length Botryotinia fuckeliana Cbh1, and shares about 62% identity with the Botryotinia fuckeliana Cbh1 catalytic domain.
Thus, based on the percent identity between the full length sequence of S. aggregatum Cbh1 and several GHF7 family members discussed above, the S. aggregatum Cbh1 is predicted to be a novel cellobiohydrolase I (Cbh1) of the GHF7 group, and is predicted to function similarly to the Cbh1 proteins of the organisms discussed above.
To evaluate expression and activity of the novel Schizochytrium aggregatum Cbh1, the cbh1 cDNA, with its native signal sequence, was inserted into an episomal yeast expression vector under control of the ENO1 promoter and terminator to generate the pMU506 construct. The pMU506 construct is depicted in
Yeast Transformation
A lithium acetate transformation (LiOAc) protocol was utilized for transformation of yeast with episomal plasmids containing the cbh1 sequence. Yeast were grown in 2 ml YPD media at 30° C. overnight. In the morning, 50 ml of YPD were inoculated with 0.5 ml of the overnight culture. This 50 ml culture was grown at 30° C. with shaking. After 4-5 hours, the yeast cells were spun down in a clinical centrifuge at 13,200 rpm for 5 minutes. The cells were suspended in sterile water and spun again. The cells were then suspended in 1 ml 100 mL LiOAc and transferred to a microfuge tube. The microfuge tube was spun at top speed for 15 seconds. The LiOAc was pipetted off. The remaining pellet was suspended in a tranformation mix, where 150 μl of the transformation mix was added to each sample. The 150 μl transformation mix contained 15 μl water, 15 μl 1M LiOAc, 20 μl DNA carrier (Ambion, cat #AM9680) and 100 μl 50% PEG 3350.
For the transformation, 1 μl of the DNA sample (the plasmid containing the cbh1 gene) was placed into a microfuge tube. 150 μl of the cells resuspended in transformation mix was added to the DNA sample. This sample with the DNA was incubated at 30° C. for 30 minutes in an incubator. Subsequently, the sample was heat-shocked in a water bath at 42° C. for 15 minutes. The cells were next spun down for 15 seconds and the transformation mix was pipetted off. Then 50 μl of sterile water was added to the cells, the cells were resuspended and then plated on selective plates. The plates were incubated at 30° C. for 2-3 days. In this way, cells transformed with the S. aggregatum cbh1 expression vector were generated.
Initially, the Y294 yeast strain (genotype: α leu2-3,112 ura3-52 his3 trp1-289; ATCC No. 201160), also referred to as MO013, was transformed with Saccharomyces cerevisiae His3 and Trp1 polymerase chain reaction (PCR) products to rescue the His3 and Trp1 auxotrophies. This rescued yeast strain is referred to as MO375. The pMU506 vector construct containing the S. aggregatum cbh1 sequence was then transformed into the MO375 yeast strain to generate the MO430 yeast strain.
Measurement of Cellulase Activity
Studies to determine the cellulase activity of the transformed MO430 yeast strain were next conducted. The MO430 strain and a Y294-derived control strain (MO375) transformed with empty vector (MO419) were inoculated in 50 ml YPD media and grown with shaking for three days.
After three days, cell supernatants were concentrated 100-fold by incubating with the cellulosic substrate phosphoric acid swollen cellulose (PASC). For each 50 ml of supernatant, 100 μl of 2.4% PASC was added. The supernatant-PASC combination was mixed with a stir bar for five to six hours at 4° C. The PASC and attached cellulose was then collected by centrifugation, washed with 50 mM NaAc pH 5.0, using microspin columns with filters for the final spin, and resuspended in 0.5 ml of 50 mM NaAc pH 5.0 in 1.5 ml tubes. The mixture in the 1.5 ml tubes was then incubated with shaking at 35° C. for 24-48 hours.
The accumulation of reducing sugars was measured at 0, 20 and 44 hours with 1% Dinitrosalicylic Acid Reagent Solution (DNS). The DNS includes the following: (1) 3,5-dinitrosalicylic acid: 10 g; (2) Sodium sulfite: 0.5 g; (3) Sodium hydroxide: 10 g; (4) Water to 1 liter total volume. The DNS was calibrated by glucose (using glucose samples with a concentration of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 g/l. Samples, 100 ul, were spun down at highest speed for 1 min, 50 ul of supernatant was then mixed with 100 ul DNS in PCR tubes, heated at 99° C. for 5 min and cooled down to 4° C. in a PCR machine. Next, 50 μl from each well of the cooled supernatant-DNS mixture was transferred to a microtiter plate. The absorbance of each sample was measured at 565 nm by a plate reader and reducing sugars concentration was calculated based on DNS glucose calibration slope.
As shown in
The above-described experiments were also performed utilizing yeast strains (e.g., Saccharomyces cerevisiae) transformed with a vector containing the Schizochytrium aggregatum cbh1 that has been codon-optimized for Saccharomyces cerevisiae.
The codon-optimized Schizochytrium aggregatum CBH1 gene having an Saccharomyces cerevisiae alpha mating factor pre signal sequence (SEQ ID NO:4) was inserted into PacI/AscI sites of pMU451 episomal yeast expression vector (
The resulting strains are summarized in Table 8 below. The strains from Table 8 were inoculated in 10 ml YPD in 50 ml tubes and were grown with shaking for 2 days. Secreted cellulase activity in culture supernatants was analyzed by the Avicel conversion assay (as described above). The results of the assay are shown in
The expression level of Schizochytrium aggregatum Cbh1 is further optimized by screening for optimal signal sequence and/or mutagenesis of the protein sequence. Additional codon-optimized sequences that are utilized include those corresponding to SEQ ID NOs: 5-7.
The construct used to transform Saccharomyces cerevisiae and express Schizochytrium aggregatum Cbh1 can also include a variant, fragment or derivative thereof of a native or codon-optimized version of Schizochytrium aggregatum cbh1. A fragment of cbh1 includes a sequence encoding any domain of the Schizochytrium aggregatum Cbh1, e.g., the CD. The expression construct is optionally constructed to include an anchoring or tethering domain.
Additional gene sequences for one or more saccharolytic enzymes can optionally be included in a cbh1 vector construct using techniques well known in the art. For example, constructs for expressing two or three cellulases simultaneously (Schizochytrium aggregatum Cbh1, Eg1, Bgl1, and/or Cbh2) are constructed.
Schizochytrium
aggregatum
Neosartorya fischeri
Chaetomium
thermophilum
Aspergillus terreus
Penicillium
chrysogenum
Schizochytrium
aggregatum
To further evaluate expression and activity of the novel Schizochytrium aggregatum Cbh1, the cbh1 cDNA is inserted into an yeast integrative expression vector. The yeast integrative expression vector, pMU562 is depicted in
These examples illustrate possible embodiments of the present invention. While the invention has been particularly shown and described with reference to some embodiments thereof, it will be understood by those skilled in the art that they have been presented by way of example only, and not limitation, and various changes in form and details can be made therein without departing from the spirit and scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.
All documents cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued or foreign patents, or any other documents, are each entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited documents.
This is the U.S. National Phase of International Appl. No. PCT/US2009/003972, filed Jul. 7, 2009, which claims the benefit of U.S. Provisional Appl. No. 61/129,585, filed Jul. 7, 2008, each of which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2009/003972 | 7/7/2009 | WO | 00 | 9/1/2011 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2010/005553 | 1/14/2010 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20020138878 | Sticklen et al. | Sep 2002 | A1 |
| Number | Date | Country |
|---|---|---|
| 1 482 033 | Dec 2004 | EP |
| WO 03070939 | Aug 2003 | WO |
| WO 2007115201 | Oct 2007 | WO |
| Entry |
|---|
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| Number | Date | Country | |
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| 20110312054 A1 | Dec 2011 | US |
| Number | Date | Country | |
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| 61129585 | Jul 2008 | US |
