Patent Application
20150082691
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 23, 2011, is named “19578_US_Sequence_Listing.txt”, lists 25 sequences, and is 91.4 kb in size.
The present disclosure relates to methods for conferring fatty acid and fatty acid ester-producing properties to a heterotrophic or photoautotrophic host, such that the modified host can be used in the commercial production of fuels and chemicals.
Many existing photoautotrophic organisms (i.e., plants, algae, and photosynthetic bacteria) are poorly suited for industrial bioprocessing and have therefore not demonstrated commercial viability. Such organisms typically have slow doubling times (3-72 hrs) compared to industrialized heterotrophic organisms such as Escherichia coli (20 minutes), reflective of low total productivities. A need exists, therefore, for engineered photosynthetic microbes which produce increased yields of fatty acids and esters.
In one embodiment, the invention provides a method for producing fatty acid esters, comprising: (i) culturing an engineered photosynthetic microorganism in a culture medium, wherein said engineered photosynthetic microorganism comprises a recombinant thioesterase, a recombinant acyl-CoA synthetase, and a recombinant wax synthase; and (ii) exposing said engineered photosynthetic microorganism to light and carbon dioxide, wherein said exposure results in the incorporation of an alcohol into a fatty acid ester produced by said engineered photosynthetic microorganism. In a related embodiment, the engineered photosynthetic microorganism is an engineered cyanobacterium. In another related embodiment, at least one of said fatty acid esters produced by the engineered cyanobacterium is selected from the group consisting of a tetradecanoic acid ester, a hexadecanoic acid ester, a heptadecanoic acid ester, a Δ9-octadecenoic acid ester, and an octadecanoic acid ester. In another related embodiment, the amount of said fatty acid esters produced by said engineered cyanobacterium is increased relative to the amount of fatty acid produced by an otherwise identical cell lacking said recombinant thioesterase, acyl-CoA synthetase or wax synthase. In certain embodiments, the incorporated alcohol is an exogenously added alcohol selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, hexanol, cyclohexanol, and isoamyl alcohol.
In another related embodiment, the esters produce by the engineered cyanobacteria include a hexadecanoic acid ester and an octadecanoic acid ester. In another related embodiment, the amount of hexadecanoic acid ester produced is between 1.5 and 10 fold greater than the amount of octadecanoic acid ester. In yet another related embodiment, the amount of hexadecanoic acid ester produced is between 1.5 and 5 fold greater than the amount of octadecanoic acid ester produced. In yet another related embodiment, at least 50% of the esters produced by said engineered cyanobacterium are hexadecanoic acid esters. In yet another related embodiment, between 65% and 85% of the esters produced by said engineered cyanobacterium are hexadecanoic acid esters.
In a related embodiment of the method for producing fatty acid esters described above, the exogenously alcohol is butanol and fatty acid butyl esters are produced. In yet another related embodiment, the yield of fatty acid butyl esters is at least 5% dry cell weight. In yet another related embodiment, the yield of fatty acid butyl esters is at least 10% dry cell weight. In yet another related embodiment, exogenously added butanol is present in said culture at concentrations between 0.01 and 0.2% (vol/vol). In yet another related embodiment, the concentration of exogenously added butanol is about 0.05 to 0.075% (vol/vol).
In another related embodiment of the method for producing fatty acid esters described above, the exogenously added alcohol is ethanol. In yet another related embodiment, the yield of ethyl esters is at least 1% dry cell weight.
In another related embodiment of the method for producing fatty acid esters described above, the exogenously added alcohol is methanol. In yet another related embodiment, the yield of methyl esters is at least 0.01% dry cell weight.
In another related embodiment, said engineered cyanobacterium further comprises a recombinant resistance nodulation cell division type (“RND-type”) transporter, e.g., a TolC-AcrAB transporter. In another related embodiment, the expression of TolC is controlled by a promoter separate from the promoter that controls expression of AcrAB. In another related embodiment, the genes encoding the recombinant transporter are encoded by a plasmid. In another related embodiment, the fatty acid esters are secreted into the culture medium at increased levels relative to an otherwise identical cyanobacterium lacking the recombinant transporter.
In certain embodiments of the methods for producing fatty acid esters described above, the recombinant thioesterase, wax synthase, and acyl-CoA synthetase are expressed as an operon under the control of a single promoter. In certain embodiments, the single promoter is an inducible promoter. In other embodiments of the methods described above, the expression of at least two of the genes selected from the group consisting of a recombinant thioesterase, wax synthase, and acyl-CoA synthetase is under the control of different promoters. One or more of the promoters can be an inducible promoter. In related embodiments, at least one of said recombinant genes is encoded on a plasmid. In yet other related embodiments, at least one of said recombinant genes is integrated into the chromosome of the engineered cyanobacteria. In yet other related embodiments, at least one of said recombinant genes is a gene that is native to the engineered cyanobacteria, but whose expression is controlled by a recombinant promoter. In yet other related embodiments, one or more promoters are selected from the group consisting of a cI promoter, a cpcB promoter, a lacI-Ptrc promoter, an EM7 promoter, an PaphII promoter, a NirA-type promoter, a PnrsA promoter, or a PnrsB promoter.
In another embodiment, the invention provides a method for producing fatty acid esters, comprising: (i) culturing an engineered cyanobacterium in a culture medium, wherein said engineered cyanobacterium comprises a recombinant acyl-CoA synthetase and a recombinant wax synthase; and (ii) exposing said engineered cyanobacterium to light and carbon dioxide, wherein said exposure results in the conversion of an alcohol by said engineered cyanobacterium into fatty acid esters, wherein at least one of said fatty acid esters is selected from the group consisting of a tetradecanoic acid ester, a hexadecanoic acid ester, a heptadecanoic acid ester, a Δ9-octadecenoic acid ester, and an octadecanoic acid ester, wherein the amount of said fatty acid esters produced by said engineered cyanobacterium is increased relative to the amount of fatty acid produced by an otherwise identical cell lacking said recombinant acyl-CoA synthetase or wax synthase. In a related embodiment, the alcohol is an exogenously added alcohol selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, hexanol, cyclohexanol, and isoamyl alcohol.
In another embodiment, the invention provides a method for producing a fatty acid ester, comprising: (i) culturing an engineered cyanobacterium in a culture medium, wherein said engineered cyanobacterium comprises a recombinant RND-type transporter; and (ii) exposing said engineered cyanobacterium to light and carbon dioxide, wherein said exposure results in the production of a fatty acid ester by said engineered cyanobacterium, and wherein said RND-type transporter secretes said fatty acid ester into said culture medium. In a related embodiment, said RND-type transporter is a TolC-AcrAB transporter.
In an embodiment related to the methods described above, the invention further comprises isolating said fatty acid ester from said engineered cyanobacterium or said culture medium.
In another embodiment, the invention also provides an engineered cyanobacterium, wherein said cyanobacterium comprises a recombinant thioesterase, a recombinant acyl-CoA synthetase, and a recombinant wax synthase. In certain embodiments, the engineered cyanobacterium additionally comprises a recombinant RND-type transporter, e.g., a TolC-AcrAB transporter.
In a related embodiment, at least one of said recombinant enzymes is heterologous with respect to said engineered cyanobacterium. In another embodiment, said cyanobacterium does not synthesize fatty acid esters in the absence of the expression of one or both of the recombinant enzymes. In another embodiment, at least one of said recombinant enzymes is not heterologous to said engineered cyanobacterium.
In yet another related embodiment, the recombinant thioesterase, acyl-CoA synthetase and wax synthase are selected from the enzymes listed in Table 3A, Table 3B and Table 3C, respectively. In yet another related embodiment, the recombinant thioesterase has an amino acid sequence that is identical to SEQ ID NO: 1. In yet another related embodiment, the recombinant thioesterase has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In yet another related embodiment, the recombinant acyl-CoA synthetase is identical to SEQ ID NO:2. In yet another related embodiment, the recombinant acyl-CoA synthetase has an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In yet another related embodiment, recombinant wax synthase is identical to SEQ ID NO: 3. In yet another related embodiment, the recombinant wax synthase has an amino acid sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In yet another related embodiment, the recombinant TolC transporter amino acid sequence is identical to SEQ ID NO: 7. In yet another related embodiment, the recombinant TolC transporter has an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7. In yet another related embodiment, the recombinant AcrA amino acid sequence is identical to SEQ ID NO: 8. In yet another related embodiment, the recombinant AcrA amino acid sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8. In yet another related embodiment, the recombinant AcrB amino acid sequence is identical to SEQ ID NO: 9. In yet another related embodiment, the recombinant AcrB amino acid sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9.
In related embodiments of the above-described embodiments, an engineered photosynthetic microorganism other than a cyanobacterium can be used. In other related embodiments, a thermophilic cyanobacterium can be used.
In another embodiment, the invention provides a methods and compositions for producing fatty acids using an engineered photosynthetic microorganism. For example, in one embodiment, the invention provides a method for producing fatty acids, comprising: (a) culturing an engineered photosynthetic microorganism, wherein said engineered photosynthetic microorganism comprises a modification which reduces the expression of said microorganism's endogenous acyl-ACP synthetase; and (b) exposing said engineered photosynthetic microorganism to light and carbon dioxide, wherein said exposure results in the production of fatty acids by said engineered cyanobacterium, wherein the amount of fatty acids produced is increased relative to the amount of fatty acids produced by an otherwise identical microorganism lacking said modification. In a related embodiment, the engineered microorganism is a thermophile. In another related embodiment, the engineered microorganism is a cyanobacterium. In yet another related embodiment, the engineered microorganism is a thermophilic cyanobacterium. In yet another related embodiment, the engineered microorganism is Thermosynechococcus elongatus BP-1. In yet another related embodiment of the method for producing fatty acids, the modification is a knock-out or deletion of the gene encoding said endogenous acyl-ACP synthetase. In yet another related embodiment, the gene encoding said acyl-ACP synthetase is the acyl-ACP synthetase or aas gene, e.g., GenBank accession number NP—682091.1. In yet another related embodiment, the increase in fatty acid production is at least a 2 fold increase. In yet another related embodiment, the increase in fatty acid production is between 2 and 4.5 fold. In yet another related embodiment, the increase in fatty acid production includes an increase in fatty acids secreted into a culture media. In yet another related embodiment, most of said increase in fatty acid production arises from the increased production of myristic and oleic acid. In yet another related embodiment of the method for producing fatty acids, the engineered photosynthetic microorganism further comprises a TolC-AcrAB transporter.
In another embodiment, the invention provides an engineered photosynthetic microorganism, wherein said microorganism comprises a deletion or knock-out of an endogenous gene encoding a acyl-ACP synthetase or long-chain fatty acid ligase. In a related embodiment, engineered photosynthetic microorganism is a thermophile. In yet another related embodiment, the engineered photosynthetic microorganism is a cyanobacterium or a thermophilic cyanobacterium. In yet another related embodiment, the cyanobacterium is Thermosynechococcus elongatus BP-1. In yet another related embodiment, the acyl-ACP synthetase is the aas gene of the thermophilic cyanobacterium, e.g., GenBank accession number NP—682091.1. In yet another related embodiment, the engineered photosynthetic microorganism further comprises a TolC-AcrAB transporter.
In yet another embodiment, the invention provides an engineered cyanbacterial strain selected from the group consisting of JCC723, JCC803, JCC1215, JCC803, JCC1132, and JCC1585. In yet another embodiment, the invention provides an engineered cyanobacterial strain selected from the group consisting of the engineered Synechococcus sp. PCC7002 strains JCC1648 (Δaas tesA, with tesA under control of P(nir07) on pAQ4), JCC1704 (Δaas fatB, with fatB inserted at aquI under the control of P(nir07)), JCC1705 (Δaas fatB1, with fatB1 inserted at aquI under the control of P(nir07)), JCC1706 (Δaas fatB2 with fatB2 inserted at aquI under the control of P(nir07)), JCC1751 (Δaas tesA, with tesA under control of P(nir07) on pAQ3), and JCC1755 (Δaas fatB_mat, with fatB_mat under control of P(nir07) on pAQ3). In yet another embodiment, the invention provides the engineered cyanobacterial strain JCC1862 (Thermosynechococcus elongatus BP-1 kanR Δaas).
These and other embodiments of the invention are further described in the Figures, Description, Examples and Claims, herein.
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.
The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).
All publications, patents and other references mentioned herein are hereby incorporated by reference in their entireties.
The following terms, unless otherwise indicated, shall be understood to have the following meanings:
The term “polynucleotide” or “nucleic acid molecule” refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation.
Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:”, “nucleic acid comprising SEQ ID NO:1” refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complementary to SEQ ID NO:1. The choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.
An “isolated” RNA, DNA or a mixed polymer is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated.
As used herein, an “isolated” organic molecule (e.g., a fatty acid or a fatty acid ester) is one which is substantially separated from the cellular components (membrane lipids, chromosomes, proteins) of the host cell from which it originated, or from the medium in which the host cell was cultured. The term does not require that the biomolecule has been separated from all other chemicals, although certain isolated biomolecules may be purified to near homogeneity.
The term “recombinant” refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and/or mRNAs encoded by such nucleic acids.
As used herein, an endogenous nucleic acid sequence in the genome of an organism (or the encoded protein product of that sequence) is deemed “recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become “recombinant” because it is separated from at least some of the sequences that naturally flank it.
A nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. A “recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.
As used herein, the phrase “degenerate variant” of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence. The term “degenerate oligonucleotide” or “degenerate primer” is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.
The term “percent sequence identity” or “identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (hereby incorporated by reference in its entirety). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference. Alternatively, sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).
The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 76%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.
Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions. “Stringent hybridization conditions” and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization.
In general, “stringent hybridization” is performed at about 25° C. below the thermal melting point (Tm) for the specific DNA hybrid under a particular set of conditions. “Stringent washing” is performed at temperatures about 5° C. lower than the Tm for the specific DNA hybrid under a particular set of conditions. The Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), page 9.51, hereby incorporated by reference. For purposes herein, “stringent conditions” are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours, followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65° C. will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing.
The nucleic acids (also referred to as polynucleotides) of this present invention may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in “locked” nucleic acids.
The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as “error-prone PCR” (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)); and “oligonucleotide-directed mutagenesis” (a process which enables the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57 (1988)).
The term “attenuate” as used herein generally refers to a functional deletion, including a mutation, partial or complete deletion, insertion, or other variation made to a gene sequence or a sequence controlling the transcription of a gene sequence, which reduces or inhibits production of the gene product, or renders the gene product non-functional. In some instances a functional deletion is described as a knockout mutation. Attenuation also includes amino acid sequence changes by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulation, expressing interfering RNA, ribozymes or antisense sequences that target the gene of interest, or through any other technique known in the art. In one example, the sensitivity of a particular enzyme to feedback inhibition or inhibition caused by a composition that is not a product or a reactant (non-pathway specific feedback) is lessened such that the enzyme activity is not impacted by the presence of a compound. In other instances, an enzyme that has been altered to be less active can be referred to as attenuated.
Deletion: The removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, the regions on either side being joined together.
Knock-out: A gene whose level of expression or activity has been reduced to zero. In some examples, a gene is knocked-out via deletion of some or all of its coding sequence. In other examples, a gene is knocked-out via introduction of one or more nucleotides into its open reading frame, which results in translation of a non-sense or otherwise non-functional protein product.
The term “vector” as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which generally refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).
“Operatively linked” or “operably linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.
The term “expression control sequence” as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
Promoters useful for expressing the recombinant genes described herein include both constitutive and inducible/repressible promoters. Examples of inducible/repressible promoters include nickel-inducible promoters (e.g., PnrsA, PnrsB; see, e.g., Lopez-Mauy et al., Cell (2002) v. 43:247-256, incorporated by reference herein) and urea repressible promoters such as PnirA (described in, e.g., Qi et al., Applied and Environmental Microbiology (2005) v. 71: 5678-5684, incorporated by reference herein). In other embodiments, a PaphII and/or a lacIq-Ptrc promoter can used to control expression. Where multiple recombinant genes are expressed in an engineered cyanobacteria of the invention, the different genes can be controlled by different promoters or by identical promoters in separate operons, or the expression of two or more genes may be controlled by a single promoter as part of an operon.
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.
The term “peptide” as used herein refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.
The term “polypeptide” encompasses both naturally-occurring and non-naturally-occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.
The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As thus defined, “isolated” does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment.
The term “polypeptide fragment” as used herein refers to a polypeptide that has a deletion, e.g., an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.
A “modified derivative” refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as 125I, 32P, 35S, and 3H, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods for labeling polypeptides are well known in the art. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002) (hereby incorporated by reference).
The term “fusion protein” refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusions that include the entirety of the proteins of the present invention have particular utility. The heterologous polypeptide included within the fusion protein of the present invention is at least 6 amino acids in length, often at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length. Fusions that include larger polypeptides, such as an IgG Fc region, and even entire proteins, such as the green fluorescent protein (“GFP”) chromophore-containing proteins, have particular utility. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.
As used herein, the term “antibody” refers to a polypeptide, at least a portion of which is encoded by at least one immunoglobulin gene, or fragment thereof, and that can bind specifically to a desired target molecule. The term includes naturally-occurring forms, as well as fragments and derivatives.
Fragments within the scope of the term “antibody” include those produced by digestion with various proteases, those produced by chemical cleavage and/or chemical dissociation and those produced recombinantly, so long as the fragment remains capable of specific binding to a target molecule. Among such fragments are Fab, Fab′, Fv, F(ab′).sub.2, and single chain Fv (scFv) fragments.
Derivatives within the scope of the term include antibodies (or fragments thereof) that have been modified in sequence, but remain capable of specific binding to a target molecule, including: interspecies chimeric and humanized antibodies; antibody fusions; heteromeric antibody complexes and antibody fusions, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies (see, e.g., Intracellular Antibodies: Research and Disease Applications, (Marasco, ed., Springer-Verlag New York, Inc., 1998), the disclosure of which is incorporated herein by reference in its entirety).
As used herein, antibodies can be produced by any known technique, including harvest from cell culture of native B lymphocytes, harvest from culture of hybridomas, recombinant expression systems and phage display.
The term “non-peptide analog” refers to a compound with properties that are analogous to those of a reference polypeptide. A non-peptide compound may also be termed a “peptide mimetic” or a “peptidomimetic.” See, e.g., Jones, Amino Acid and Peptide Synthesis, Oxford University Press (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: A Handbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry—A Practical Textbook, Springer Verlag (1993); Synthetic Peptides: A Users Guide, (Grant, ed., W. H. Freeman and Co., 1992); Evans et al., J. Med. Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger, Trends Neurosci., 8:392-396 (1985); and references sited in each of the above, which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to useful peptides of the present invention may be used to produce an equivalent effect and are therefore envisioned to be part of the present invention.
A “polypeptide mutant” or “mutein” refers to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a native or wild-type protein. A mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and/or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the naturally-occurring protein, and/or truncations of the amino acid sequence at either or both the amino or carboxy termini. A mutein may have the same but preferably has a different biological activity compared to the naturally-occurring protein.
A mutein has at least 85% overall sequence homology to its wild-type counterpart. Even more preferred are muteins having at least 90% overall sequence homology to the wild-type protein.
In an even more preferred embodiment, a mutein exhibits at least 95% sequence identity, even more preferably 98%, even more preferably 99% and even more preferably 99.9% overall sequence identity.
Sequence homology may be measured by any common sequence analysis algorithm, such as Gap or Bestfit.
Amino acid substitutions can include those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and (5) confer or modify other physicochemical or functional properties of such analogs.
As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nd ed. 1991), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand end corresponds to the amino terminal end and the right-hand end corresponds to the carboxy-terminal end, in accordance with standard usage and convention.
A protein has “homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences.) As used herein, homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) is interpreted as implying similarity in function.
When “homologous” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (herein incorporated by reference).
The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1.
A preferred algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).
Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.
Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62. The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated by reference herein). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference.
“Specific binding” refers to the ability of two molecules to bind to each other in preference to binding to other molecules in the environment. Typically, “specific binding” discriminates over adventitious binding in a reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold. Typically, the affinity or avidity of a specific binding reaction, as quantified by a dissociation constant, is about 10−7 M or stronger (e.g., about 10−8 M, 10−9 M or even stronger).
“Percent dry cell weight” refers to a production measurement of esters of fatty acids or fatty acids obtained as follows: a defined volume of culture is centrifuged to pellet the cells. Cells are washed then dewetted by at least one cycle of microcentrifugation and aspiration. Cell pellets are lyophilized overnight, and the tube containing the dry cell mass is weighed again such that the mass of the cell pellet can be calculated within ±0.1 mg. At the same time cells are processed for dry cell weight determination, a second sample of the culture in question is harvested, washed, and dewetted. The resulting cell pellet, corresponding to 1-3 mg of dry cell weight, is then extracted by vortexing in approximately 1 ml acetone plus butylated hydroxytoluene (BHT) as antioxidant and an internal standard, e.g., ethyl arachidate. Cell debris is then pelleted by centrifugation and the supernatant (extractant) is taken for analysis by GC. For accurate quantitation of the molecules, flame ionization detection (FID) was used as opposed to MS total ion count. The concentrations of the esters or fatty acids in the biological extracts were calculated using calibration relationships between GC-FID peak area and known concentrations of authentic standards. Knowing the volume of the extractant, the resulting concentrations of the products in the extractant, and the dry cell weight of the cell pellet extracted, the percentage of dry cell weight that comprised the esters or fatty acids can be determined.
The term “region” as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.
The term “domain” as used herein refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also include distinct, non-contiguous regions of a biomolecule. Examples of protein domains include, but are not limited to, an Ig domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.
As used herein, the term “molecule” means any compound, including, but not limited to, a small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can be natural or synthetic.
“Carbon-based Products of Interest” include alcohols such as ethanol, propanol, isopropanol, butanol, fatty alcohols, fatty acid esters, wax esters; hydrocarbons and alkanes such as propane, octane, diesel, Jet Propellant 8 (JP8); polymers such as terephthalate, 1,3-propanediol, 1,4-butanediol, polyols, Polyhydroxyalkanoates (PHA), poly-beta-hydroxybutyrate (PHB), acrylate, adipic acid, ε-caprolactone, isoprene, caprolactam, rubber; commodity chemicals such as lactate, Docosahexaenoic acid (DHA), 3-hydroxypropionate, γ-valerolactone, lysine, serine, aspartate, aspartic acid, sorbitol, ascorbate, ascorbic acid, isopentenol, lanosterol, omega-3 DHA, lycopene, itaconate, 1,3-butadiene, ethylene, propylene, succinate, citrate, citric acid, glutamate, malate, 3-hydroxypropionic acid (HPA), lactic acid, THF, gamma butyrolactone, pyrrolidones, hydroxybutyrate, glutamic acid, levulinic acid, acrylic acid, malonic acid; specialty chemicals such as carotenoids, isoprenoids, itaconic acid; pharmaceuticals and pharmaceutical intermediates such as 7-aminodeacetoxycephalosporanic acid (7-ADCA)/cephalosporin, erythromycin, polyketides, statins, paclitaxel, docetaxel, terpenes, peptides, steroids, omega fatty acids and other such suitable products of interest. Such products are useful in the context of biofuels, industrial and specialty chemicals, as intermediates used to make additional products, such as nutritional supplements, neutraceuticals, polymers, paraffin replacements, personal care products and pharmaceuticals.
Biofuel: A biofuel refers to any fuel that derives from a biological source. Biofuel can refer to one or more hydrocarbons, one or more alcohols, one or more fatty esters or a mixture thereof.
The term “hydrocarbon” generally refers to a chemical compound that consists of the elements carbon (C), hydrogen (H) and optionally oxygen (O). There are essentially three types of hydrocarbons, e.g., aromatic hydrocarbons, saturated hydrocarbons and unsaturated hydrocarbons such as alkenes, alkynes, and dienes. The term also includes fuels, biofuels, plastics, waxes, solvents and oils. Hydrocarbons encompass biofuels, as well as plastics, waxes, solvents and oils. A “fatty acid” is a carboxylic acid with a long unbranched aliphatic tail (chain), which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of four to 28 carbons.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this present invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.
Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Esters are chemical compounds with the basic formula:
where R and R′ denote any alkyl or aryl group. In one embodiment, the invention provides one or more isolated or recombinant nucleic acids encoding one or more genes which, when recombinantly expressed in a photosynthetic microorganism, catalyze the synthesis of esters by the microorganism. The first gene is a thioesterase, which catalyzes the synthesis of fatty acids from an acyl-Acyl Carrier Protein (“acyl-ACP”) molecule. The second gene is an acyl-CoA synthetase, which synthesizes fatty acyl-CoA from a fatty acid. The third gene is a wax synthase, which synthesizes esters from a fatty acyl-CoA molecule and an alcohol (e.g., methanol, ethanol, proponal, butanol, etc.). In certain related embodiments, additional genes expressing a recombinant resistance nodulation cell division type (“RND-type”) transporter such as TolC/AcrAB are also recombinantly expressed to facilitate the transport of ethyl esters outside of the engineered photosynthetic cell and into the culture medium.
Accordingly, the present invention provides isolated nucleic acid molecules for genes encoding thioesterase, acyl-CoA synthetases and wax synthase enzymes, and variants thereof. An exemplary full-length expression optimized nucleic acid sequence for a gene encoding a thioesterase is presented as SEQ ID NO: 4. The corresponding amino acid sequences is presented as SEQ ID NO: 1. Additional genes encoding thioesterases are presented in Table 3A. An exemplary full-length expression-optimized nucleic acid sequence for a gene encoding an acyl-CoA synthetase is presented as SEQ ID NO: 5, and the corresponding amino acid sequence is presented as SEQ ID NOs: 2. Additional genes encoding acyl-CoA synthetases are presented in Table 3B. An exemplary full-length expression-optimized nucleic acid sequence for a gene encoding an acyl-CoA synthetase is presented as SEQ ID NO: 6, and the corresponding amino acid sequence is presented as SEQ ID NOs: 3. Additional genes encoding acyl-CoA synthetases are presented in Table 3C.
One skilled in the art will recognize that the redundancy of the genetic code will allow many other nucleic acid sequences to encode the identical enzymes. The sequences of the nucleic acids disclosed herein can be optimized as needed to yield the desired expression levels in a particular photosynthetic microorganism. Such a nucleic acid sequence can have 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9% or even higher identity to the native gene sequence.
In another embodiment, the nucleic acid molecule of the present invention encodes a polypeptide having the amino acid sequence of SEQ ID NO:1, 2, 3, 7, 8, or 9. Preferably, the nucleic acid molecule of the present invention encodes a polypeptide sequence of at least 50%, 60, 70%, 80%, 85%, 90% or 95% identity to SEQ ID NO:1, 2, 3, 7, 8 or 9 and the identity can even more preferably be 96%, 97%, 98%, 99%, 99.9% or even higher.
The present invention also provides nucleic acid molecules that hybridize under stringent conditions to the above-described nucleic acid molecules. As defined above, and as is well known in the art, stringent hybridizations are performed at about 25° C. below the thermal melting point (Tm) for the specific DNA hybrid under a particular set of conditions, where the Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. Stringent washing is performed at temperatures about 5° C. lower than the Tm for the specific DNA hybrid under a particular set of conditions.
Nucleic acid molecules comprising a fragment of any one of the above-described nucleic acid sequences are also provided. These fragments preferably contain at least 20 contiguous nucleotides. More preferably the fragments of the nucleic acid sequences contain at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous nucleotides.
The nucleic acid sequence fragments of the present invention display utility in a variety of systems and methods. For example, the fragments may be used as probes in various hybridization techniques. Depending on the method, the target nucleic acid sequences may be either DNA or RNA. The target nucleic acid sequences may be fractionated (e.g., by gel electrophoresis) prior to the hybridization, or the hybridization may be performed on samples in situ. One of skill in the art will appreciate that nucleic acid probes of known sequence find utility in determining chromosomal structure (e.g., by Southern blotting) and in measuring gene expression (e.g., by Northern blotting). In such experiments, the sequence fragments are preferably detectably labeled, so that their specific hydridization to target sequences can be detected and optionally quantified. One of skill in the art will appreciate that the nucleic acid fragments of the present invention may be used in a wide variety of blotting techniques not specifically described herein.
It should also be appreciated that the nucleic acid sequence fragments disclosed herein also find utility as probes when immobilized on microarrays. Methods for creating microarrays by deposition and fixation of nucleic acids onto support substrates are well known in the art. Reviewed in DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosures of which are incorporated herein by reference in their entireties. Analysis of, for example, gene expression using microarrays comprising nucleic acid sequence fragments, such as the nucleic acid sequence fragments disclosed herein, is a well-established utility for sequence fragments in the field of cell and molecular biology. Other uses for sequence fragments immobilized on microarrays are described in Gerhold et al., Trends Biochem. Sci. 24:168-173 (1999) and Zweiger, Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosure of each of which is incorporated herein by reference in its entirety.
As is well known in the art, enzyme activities can be measured in various ways. For example, the pyrophosphorolysis of OMP may be followed spectroscopically (Grubmeyer et al., (1993) J. Biol. Chem. 268:20299-20304). Alternatively, the activity of the enzyme can be followed using chromatographic techniques, such as by high performance liquid chromatography (Chung and Sloan, (1986) J. Chromatogr. 371:71-81). As another alternative the activity can be indirectly measured by determining the levels of product made from the enzyme activity. These levels can be measured with techniques including aqueous chloroform/methanol extraction as known and described in the art (Cf M. Kates (1986) Techniques of Lipidology; Isolation, analysis and identification of Lipids. Elsevier Science Publishers, New York (ISBN: 0444807322)). More modern techniques include using gas chromatography linked to mass spectrometry (Niessen, W. M. A. (2001). Current practice of gas chromatography—mass spectrometry. New York, N.Y.: Marcel Dekker. (ISBN: 0824704738)). Additional modern techniques for identification of recombinant protein activity and products including liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography (HPLC), capillary electrophoresis, Matrix-Assisted Laser Desorption Ionization time of flight-mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), near-infrared (NIR) spectroscopy, viscometry (Knothe, G (1997) Am. Chem. Soc. Symp. Series, 666: 172-208), titration for determining free fatty acids (Komers (1997) Fett/Lipid, 99(2): 52-54), enzymatic methods (Bailer (1991) Fresenius J. Anal. Chem. 340(3): 186), physical property-based methods, wet chemical methods, etc. can be used to analyze the levels and the identity of the product produced by the organisms of the present invention. Other methods and techniques may also be suitable for the measurement of enzyme activity, as would be known by one of skill in the art.
Also provided are vectors, including expression vectors, which comprise the above nucleic acid molecules of the present invention, as described further herein. In a first embodiment, the vectors include the isolated nucleic acid molecules described above. In an alternative embodiment, the vectors of the present invention include the above-described nucleic acid molecules operably linked to one or more expression control sequences. The vectors of the instant invention may thus be used to express a thioesterase, an acyl-CoA synthease, and/or a wax synthase, contributing to the synthesis of esters by the cell.
In a related embodiment, vectors may include nucleic acid molecules encoding an RND-type transporter such as TolC/AcrAB to facilitate the extracellular transport of esters. Exemplary vectors of the invention include any of the vectors expressing a thioesterase, an acyl-CoA synthease, wax synthase, and/or TolC/AcrAB transporter disclosed here, e.g., pJB532, pJB634, pJB578 and pJB1074. The invention also provides other vectors such as pJB161 which are capable of receiving nucleic acid sequences of the invention. Vectors such as pJB161 comprise sequences which are homologous with sequences that are present in plasmids which are endogenous to certain photosynthetic microorganisms (e.g., plasmids pAQ7 or pAQ1 of certain Synechococcus species). Recombination between pJB161 and the endogenous plasmids in vivo yield engineered microbes expressing the genes of interest from their endogenous plasmids. Alternatively, vectors can be engineered to recombine with the host cell chromosome, or the vector can be engineered to replicate and express genes of interest independent of the host cell chromosome or any of the host cell's endogenous plasmids.
Vectors useful for expression of nucleic acids in prokaryotes are well known in the art.
According to another aspect of the present invention, isolated polypeptides (including muteins, allelic variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules of the present invention are provided. In one embodiment, the isolated polypeptide comprises the polypeptide sequence corresponding to SEQ ID NO:1, 2, 3, 7, 8, or 9. In an alternative embodiment of the present invention, the isolated polypeptide comprises a polypeptide sequence at least 85% identical to SEQ ID NO:1, 2, 3, 7, 8, or 9. Preferably the isolated polypeptide of the present invention has at least 50%, 60, 70%, 80%, 85%, 90%, 95%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or even higher identity to SEQ ID NO:1, 2, 3, 7, 8 or 9.
According to other embodiments of the present invention, isolated polypeptides comprising a fragment of the above-described polypeptide sequences are provided. These fragments preferably include at least 20 contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous amino acids.
The polypeptides of the present invention also include fusions between the above-described polypeptide sequences and heterologous polypeptides. The heterologous sequences can, for example, include sequences designed to facilitate purification, e.g. histidine tags, and/or visualization of recombinantly-expressed proteins. Other non-limiting examples of protein fusions include those that permit display of the encoded protein on the surface of a phage or a cell, fusions to intrinsically fluorescent proteins, such as green fluorescent protein (GFP), and fusions to the IgG Fc region.
In another aspect of the present invention, host cells transformed with the nucleic acid molecules or vectors of the present invention, and descendants thereof, are provided. In some embodiments of the present invention, these cells carry the nucleic acid sequences of the present invention on vectors, which may but need not be freely replicating vectors. In other embodiments of the present invention, the nucleic acids have been integrated into the genome of the host cells and/or into an endogenous plasmid of the host cells.
In a preferred embodiment, the host cell comprises one or more recombinant thioesterase-, acyl-CoA synthase-, wax synthase-, or TolC/AcrAB-encoding nucleic acids which express thioesterase-, acyl-CoA synthase, wax synthase or TolC/AcrAB respectively in the host cell.
In an alternative embodiment, the host cells of the present invention can be mutated by recombination with a disruption, deletion or mutation of the isolated nucleic acid of the present invention so that the activity of a native thioesterase, acyl-CoA synthase, wax synthase, and/or TolC/AcrAB protein in the host cell is reduced or eliminated compared to a host cell lacking the mutation.
Microorganism: Includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms “microbial cells” and “microbes” are used interchangeably with the term microorganism.
A variety of host organisms can be transformed to produce a product of interest. Photoautotrophic organisms include eukaryotic plants and algae, as well as prokaryotic cyanobacteria, green-sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteria.
Extremophiles are also contemplated as suitable organisms. Such organisms withstand various environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension, and chemicals. They include hyperthermophiles, which grow at or above 80° C. such as Pyrolobus fumarii; thermophiles, which grow between 60-80° C. such as Synechococcus lividis; mesophiles, which grow between 15-60° C. and psychrophiles, which grow at or below 15° C. such as Psychrobacter and some insects. Radiation tolerant organisms include Deinococcus radiodurans. Pressure-tolerant organisms include piezophiles, which tolerate pressure of 130 MPa. Weight-tolerant organisms include barophiles. Hypergravity (e.g., >1 g) hypogravity (e.g., <1 g) tolerant organisms are also contemplated. Vacuum tolerant organisms include tardigrades, insects, microbes and seeds. Dessicant tolerant and anhydrobiotic organisms include xerophiles such as Artemia salina; nematodes, microbes, fungi and lichens. Salt-tolerant organisms include halophiles (e.g., 2-5 M NaCl) Halobacteriacea and Dunaliella salina. pH-tolerant organisms include alkaliphiles such as Natronobacterium, Bacillus firmus OF4, Spirulina spp. (e.g., pH>9) and acidophiles such as Cyanidium caldarium, Ferroplasma sp. (e.g., low pH). Anaerobes, which cannot tolerate O2 such as Methanococcus jannaschii; microaerophils, which tolerate some O2 such as Clostridium and aerobes, which require O2 are also contemplated. Gas-tolerant organisms, which tolerate pure CO2 include Cyanidium caldarium and metal tolerant organisms include metalotolerants such as Ferroplasma acidarmanus (e.g., Cu, As, Cd, Zn), Ralstonia sp. CH34 (e.g., Zn, Co, Cd, Hg, Pb). Gross, Michael. Life on the Edge: Amazing Creatures Thriving in Extreme Environments. New YorK: Plenum (1998) and Seckbach, J. “Search for Life in the Universe with Terrestrial Microbes Which Thrive Under Extreme Conditions.” In Cristiano Batalli Cosmovici, Stuart Bowyer, and Dan Wertheimer, eds., Astronomical and Biochemical Origins and the Search for Life in the Universe, p. 511. Milan: Editrice Compositori (1997).
Plants include but are not limited to the following genera: Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia and Zea.
Algae and cyanobacteria include but are not limited to the following genera: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritractus, Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema, Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysostephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus, Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum, Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis, Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium, Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion, Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis, Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema, Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria, Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora, Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma, Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate, Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas, Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma, Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium, Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira, Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and Zygonium.
Additional cyanobacteria include members of the genus Chamaesiphon, Chroococcus, Cyanobacterium, Cyanobium, Cyanothece, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus, Prochloron, Synechococcus, Synechocystis, Cyanocystis, Dermocarpella, Stanieria, Xenococcus, Chroococcidiopsis, Myxosarcina, Arthrospira, Borzia, Crinalium, Geitlerinemia, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Oscillatoria, Planktothrix, Prochiorothrix, Pseudanabaena, Spirulina, Starria, Symploca, Trichodesmium, Tychonema, Anabaena, Anabaenopsis, Aphanizomenon, Cyanospira, Cylindrospermopsis, Cylindrospermum, Nodularia, Nostoc, Scylonema, Calothrix, Rivularia, Tolypothrix, Chlorogloeopsis, Fischerella, Geitieria, Iyengariella, Nostochopsis, Stigonema and Thermosynechococcus.
Green non-sulfur bacteria include but are not limited to the following genera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Thermomicrobium.
Green sulfur bacteria include but are not limited to the following genera:
Chlorobium, Clathrochloris, and Prosthecochloris.
Purple sulfur bacteria include but are not limited to the following genera: Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis,
Purple non-sulfur bacteria include but are not limited to the following genera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio, and Roseospira.
Aerobic chemolithotrophic bacteria include but are not limited to nitrifying bacteria such as Nitrobacteraceae sp., Nitrobacter sp., Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomonas sp., Nitrosococcus sp., Nitrosospira sp., Nitrosolobus sp., Nitrosovibrio sp.; colorless sulfur bacteria such as, Thiovulum sp., Thiobacillus sp., Thiomicrospira sp., Thiosphaera sp., Thermothrix sp.; obligately chemolithotrophic hydrogen bacteria such as Hydrogenobacter sp., iron and manganese-oxidizing and/or depositing bacteria such as Siderococcus sp., and magnetotactic bacteria such as Aquaspirillum sp.
Archaeobacteria include but are not limited to methanogenic archaeobacteria such as Methanobacterium sp., Methanobrevibacter sp., Methanothermus sp., Methanococcus sp., Methanomicrobium sp., Methanospirillum sp., Methanogenium sp., Methanosarcina sp., Methanolobus sp., Methanothrix sp., Methanococcoides sp., Methanoplanus sp.; extremely thermophilic S-Metabolizers such as Thermoproteus sp., Pyrodictium sp., Sulfolobus sp., Acidianus sp. and other microorganisms such as, Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp., Ralstonia sp., Rhodococcus sp., Corynebacteria sp., Brevibacteria sp., Mycobacteria sp., and oleaginous yeast.
Preferred organisms for the manufacture of esters according to the methods disclosed herein include: Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, and Zea mays (plants); Botryococcus braunii, Chlamydomonas reinhardtii and Dunaliela salina (algae); Synechococcus sp PCC 7002, Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803, Thermosynechococcus elongatus BP-1 (cyanobacteria); Chlorobium tepidum (green sulfur bacteria), Chloroflexus auranticus (green non-sulfur bacteria); Chromatium tepidum and Chromatium vinosum (purple sulfur bacteria); Rhodospirillum rubrum, Rhodobacter capsulatus, and Rhodopseudomonas palusris (purple non-sulfur bacteria).
Yet other suitable organisms include synthetic cells or cells produced by synthetic genomes as described in Venter et al. US Pat. Pub. No. 2007/0264688, and cell-like systems or synthetic cells as described in Glass et al. US Pat. Pub. No. 2007/0269862.
Still, other suitable organisms include microorganisms that can be engineered to fix carbon dioxide, such as Escherichia coli, Acetobacter aceti, Bacillus subtilis, yeast and fungi such as Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis.
The capability to use carbon dioxide as the sole source of cell carbon (autotrophy) is found in almost all major groups of prokaryotes. The CO2 fixation pathways differ between groups, and there is no clear distribution pattern of the four presently-known autotrophic pathways. See, e.g., Fuchs, G. 1989. Alternative pathways of autotrophic CO2 fixation, p. 365-382, in H. G. Schlegel, and B. Bowien (ed.), Autotrophic bacteria. Springer-Verlag, Berlin, Germany. The reductive pentose phosphate cycle (Calvin-Bassham-Benson cycle) represents the CO2 fixation pathway in almost all aerobic autotrophic bacteria, for example, the cyanobacteria.
For producing esters via the recombinant expression of thioesterase, acyl-CoA synthetase and/or wax synthase enzymes, an engineered cyanobacteria, e.g., a Synechococcus or Thermosynechococcus species, is especially preferred. Other preferred organisms include Synechocystis, Klebsiella oxytoca, Escherichia coli or Saccharomyces cerevisiae. Other prokaryotic, archaea and eukaryotic host cells are also encompassed within the scope of the present invention. Engineered ester-producing organisms expressing thioesterase, acyl-CoA synthetase and/or wax synthase enzymes can be further engineered to express recombinant TolC/AcrAB to enhance the extracellular transport of esters.
In various embodiments of the invention, desired esters or a mixture thereof can be produced. For example, by including a particular alcohol or mixture of alcohols in the culture media, methyl esters, ethyl esters, propyl esters, butyl esters, and esters of higher chain length alcohols (or mixtures thereof, depending on the substrate alcohols available to the photosynthetic microbe) can be synthesized. The carbon chain lengths of the esters can vary from C10 to C20, e.g., using ethanol as a substrate, diverse esters including, e.g., ethyl myristate, ethyl palmitate, ethyl oleate, and/or ethyl stearate and/or mixtures thereof can be produced by a single engineered photosynthetic microorganism of the invention. Accordingly, the invention provides methods and compositions for the production of various chain lengths of esters, each of which is suitable for use as a fuel or any other chemical use.
In preferred aspects, the methods provide culturing host cells for direct product secretion for easy recovery without the need to extract biomass. These carbon-based products of interest are secreted directly into the medium. Since the invention enables production of various defined chain length of hydrocarbons and alcohols, the secreted products are easily recovered or separated. The products of the invention, therefore, can be used directly or used with minimal processing.
One skilled in the art will recognize that a variety of media and culture conditions can be used in conjunction with the methods and engineered cyanobacteria disclosed herein for the bioproduction of fatty acid esters (see, e.g., Rogers and Gallon, Biochemistry of the Algae and Cyanobacteria, Clarendon Press Oxford (1988); Burlwe, Algal Culture: From Laboratory to Pilot Plant, Carnegie Institution of Washington Publication 600 Washington, D.C., (1961); and Round, F. E. The Biology of the Algae. St Martin's Press, New York, 1965; Golden S S et al. (1987) Methods Enzymol 153:215-231; Golden and Sherman, J. Bacteriology 158:36 (1984), each of which is incorporated herein by reference). Exemplary culture conditions and media are also described in, e.g., WO/2010/068288, filed May 21, 2009, published Jun. 17, 2010, and incorporated by reference herein. Typical culture conditions for the methods of the present invention include the use of JB 2.1 culture media or A+ media. A recipe for one liter of JB 2.1 appears in Table A, below.
As described in more detail in the Examples, below, in certain embodiments one or more alcohols (e.g., methanol, ethanol, propanol, butanol, etc.) may be added during culturing to produce the desired fatty acid ester(s) of interest (e.g., a fatty acid methyl ester, a fatty acid ethyl ester, etc., and mixtures thereof). For organisms that require or metabolize most efficiently in the presence of light and carbon dioxide, either carbon dioxide or bicarbonate can be used during culturing.
In various embodiments, compositions produced by the methods of the invention are used as fuels. Such fuels comply with ASTM standards, for instance, standard specifications for diesel fuel oils D 975-09b, and Jet A, Jet A-1 and Jet B as specified in ASTM Specification D. 1655-68. Fuel compositions may require blending of several products to produce a uniform product. The blending process is relatively straightforward, but the determination of the amount of each component to include in a blend is much more difficult. Fuel compositions may, therefore, include aromatic and/or branched hydrocarbons, for instance, 75% saturated and 25% aromatic, wherein some of the saturated hydrocarbons are branched and some are cyclic. Preferably, the methods of the invention produce an array of hydrocarbons, such as C13-C17 or C10-C15 to alter cloud point. Furthermore, the compositions may comprise fuel additives, which are used to enhance the performance of a fuel or engine. For example, fuel additives can be used to alter the freezing/gelling point, cloud point, lubricity, viscosity, oxidative stability, ignition quality, octane level, and flash point. Fuels compositions may also comprise, among others, antioxidants, static dissipater, corrosion inhibitor, icing inhibitor, biocide, metal deactivator and thermal stability improver.
In addition to many environmental advantages of the invention such as CO2 conversion and renewable source, other advantages of the fuel compositions disclosed herein include low sulfur content, low emissions, being free or substantially free of alcohol and having high cetane number.
Biologically-produced carbon-based products, e.g., ethanol, fatty acids, alkanes, isoprenoids, represent a new commodity for fuels, such as alcohols, diesel and gasoline. Such biofuels have not been produced using biomass but use CO2 as its carbon source. These new fuels may be distinguishable from fuels derived form petrochemical carbon on the basis of dual carbon-isotopic fingerprinting. Such products, derivatives, and mixtures thereof may be completely distinguished from their petrochemical derived counterparts on the basis of 14C (fM) and dual carbon-isotopic fingerprinting, indicating new compositions of matter.
There are three naturally occurring isotopes of carbon: 12C, 13C, and 14C. These isotopes occur in above-ground total carbon at fractions of 0.989, 0.011, and 10−12, respectively. The isotopes 12C and 13C are stable, while 14C decays naturally to 14N, a beta particle, and an anti-neutrino in a process with a half-life of 5730 years. The isotope 14C originates in the atmosphere, due primarily to neutron bombardment of 14N caused ultimately by cosmic radiation. Because of its relatively short half-life (in geologic terms), 14C occurs at extremely low levels in fossil carbon. Over the course of 1 million years without exposure to the atmosphere, just 1 part in 1050 will remain 14C.
The 13C:12C ratio varies slightly but measurably among natural carbon sources. Generally these differences are expressed as deviations from the 13C:12C ratio in a standard material. The international standard for carbon is Pee Dee Belemnite, a form of limestone found in South Carolina, with a 13C fraction of 0.0112372. For a carbon source a, the deviation of the 13C:12C ratio from that of Pee Dee Belemnite is expressed as: δa=(Ra/Rs)−1, where Ra=13C:12C ratio in the natural source, and Rs=13C:12C ratio in Pee Dee Belemnite, the standard. For convenience, δa is expressed in parts per thousand, or ‰. A negative value of δa shows a bias toward 12C over 13C as compared to Pee Dee Belemnite. Table 1 shows δa and 14C fraction for several natural sources of carbon.
Stable Isotopes in Ecology and Environmental Science,
Biological processes often discriminate among carbon isotopes. The natural abundance of 14C is very small, and hence discrimination for or against 14C is difficult to measure. Biological discrimination between 13C and 12C, however, is well-documented. For a biological product p, we can define similar quantities to those above: δp=(Rp/Rs)−1, where Rp=13C:12C ratio in the biological product, and Rs=13C:12C ratio in Pee Dee Belemnite, the standard. Table 2 shows measured deviations in the 13C:12C ratio for some biological products.
13C:12C variations in selected biological products
10-20
Stable Isotopes in Ecology and Environmental Science,
Stable Isotopes in Ecology and Environmental Science,
E. coli lipid from plant sugar
Table 2 introduces a new quantity, D. This is the discrimination by a biological process in its utilization of 12C vs. 13C. We define D as follows: D=(Rp/Ra)−1. This quantity is very similar to δa and δp, except we now compare the biological product directly to the carbon source rather than to a standard. Using D, we can combine the bias effects of a carbon source and a biological process to obtain the bias of the biological product as compared to the standard. Solving for δp, we obtain: δp=(D)(δa)+D+δa, and, because (D)(δa) is generally very small compared to the other terms, δp≈δa+D.
For a biological product having a production process with a known D, we may therefore estimate δp by summing δa and D. We assume that D operates irrespective of the carbon source. This has been done in Table 1 for cyanobacterial lipid and biomass produced from fossil carbon. As shown in the Table 1 and Table 2, above, cyanobacterial products made from fossil carbon (in the form of, for example, flue gas or other emissions) will have a higher δp than those of comparable biological products made from other sources, distinguishing them on the basis of composition of matter from these other biological products. In addition, any product derived solely from fossil carbon will have a negligible fraction of 14C, while products made from above-ground carbon will have a 14C fraction of approximately 10−12.
Accordingly, in certain aspects, the invention provides various carbon-based products of interest characterized as −δp(‰) of about 63.5 to about 66 and −D(‰) of about 37.5 to about 40.
The following examples are for illustrative purposes and are not intended to limit the scope of the present invention.
In one embodiment of the invention, a cyanobacterium strain is transformed or engineered to express one or more enzymes selected from the following list: a wax synthase (EC: 2.3.175), a thioesterase (EC: 3.1.2.-, 3.1.2.14), and an acyl-CoA synthase (EC: 6.2.1.3). For example, a typical embodiment utilizes a thioesterase gene from E. coli (tesA; SEQ ID NO:1), an acyl-CoA synthetase gene from E. coli (fadD; SEQ ID NO:2), and a wax synthase gene from A. baylyi (wax; SEQ ID NO:3). Thioesterase generates fatty acid from acyl-ACP. Acyl-CoA synthetase (also referred to as acyl-CoA ligase) generates fatty acyl-CoA from fatty acid. Wax synthase (EC 2.3.1.75) generates fatty acid esters using acyl-CoA and acyl alcohol as substrates (e.g., methanol, ethanol, butanol, etc).
Additional thioesterase, acyl-CoA synthetase and wax synthases genes that can be recombinantly expressed in cyanobacteria are set forth in Table 3A, Table 3B, and Table 3C, respectively.
E. coli
Cuphea
hookeriana
Umbellularia
california
Cinnamonum
camphorum
Arabidopsis
thaliana
E. coli
Geobacillus
thermodenitrificans
Acinetobacter baylyi
Mycobacterium
tuberculosis H37Rv
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Rattus
norvegicus (rat)
Fundibacter
jadensis
Acinetobacter sp.
H. sapiens
Fragaria xananassa
Malus xdomestica
Simmondsia
chinensis
Mus musculus
The engineered cyanobacterium expressing one or more of the thioesterase, acyl-CoA synthetase, and wax synthase genes set forth above is grown in suitable media, under appropriate conditions (e.g., temperature, shaking, light, etc.). After a certain optical density is reached, the cells are separated from the spent medium by centrifugation. The cell pellet is re-suspended and the cell suspension and the spent medium are then extracted with a suitable solvent, e.g., ethyl acetate. The resulting ethyl acetate phases from the cell suspension and the supernatant are subjected to GC-MS analysis. The fatty acid esters in the ethyl acetate phases can be quantified, e.g., using commercial palmitic acid ethyl ester as a reference standard.
Fatty acid esters can be made according to this method by adding an alcohol (e.g., methanol, propanol, isopropanol, butanol, etc.) to the fermentation media, whereby fatty acid esters of the added alcohols are produced by the engineered cyanobacterium. Alternatively, one or more alcohols can be synthesized by the engineered cyanobacterium, natively or recombinantly, and used as substrates for fatty acid ester synthesis by a recombinantly expressed wax synthase. As detailed in the Examples below, the engineered cyanobacterium can also be modified to recombinantly expresses a TolC/AcrAB transporter to facilitate secretion of the fatty acid esters into the culture medium.
Genes and Plasmids:
The pJB5 base vector was designed as an empty expression vector for recombination into Synechococcus sp. PCC 7002. Two regions of homology, the Upstream Homology Region (UHR) and the Downstream Homology Region (DHR), are designed to flank the construct of interest. These 500 bp regions of homology correspond to positions 3301-3800 and 3801-4300 (Genbank Accession NC—005025) for UHR and DHR respectively. The aadA promoter, gene sequence, and terminator were designed to confer spectinomycin and streptomycin resistance to the integrated construct. For expression, pJB5 was designed with the aphII kanamycin resistance cassette promoter and ribosome binding site (RBS). Downstream of this promoter and RBS, the restriction endonuclease recognition site for NdeI, EcoRI, SpeI and PacI were inserted. Following the EcoRI site, the natural terminator from the alcohol dehydrogenase gene from Zymomonas mobilis (adhII) terminator was included. Convenient XbaI restriction sites flank the UHR and the DHR allowing cleavage of the DNA intended for recombination from the rest of the vector.
The E. coli thioesterase tesA gene with the leader sequence removed (SEQ ID NO:4; Genbank # NC—000913; Chot and Cronan, 1993), the E. coli acyl-CoA synthetase fadD (SEQ ID NO:5; Genbank # NC—000913; Kameda and Nunn, 1981) and the wax synthase gene (wax) from Acinetobacter baylyi strain ADPI (SEQ ID NO:6; Genbank # AF529086.1; Stöveken et al. 2005) were purchased from DNA 2.0, following codon optimization, checking for secondary structure effects, and removal of any unwanted restriction sites (NdeI, XhoI, BamHI, NgoMIV, NcoI, SacI, BsrGI, AvrII, BmtI, MluI, EcoRI, SbfI, NotI, SpeI, XbaI, Pad, AscI, FseI). These genes were received on a pJ201 vector and assembled into a three-gene operon (tesA-fadD-wax, SEQ ID NO: 10) with flanking NdeI-EcoRI sites on the recombination vector pJB5 under the control of the PaphII kanamycin resistance cassette promoter. A second plasmid (pJB532; SEQ ID NO:11) was constructed which is identical to pJB494 except the PaphII promoter was replaced with SEQ ID NO:12, a Ptrc promoter and a lacIq repressor. As a control, a third plasmid (pJB413) was prepared with only tesA under the control of the PaphII promoter. These plasmid constructs were named pJB494, pJB532, and pJB413, respectively.
Strain Construction:
The constructs described above were integrated onto the plasmid pAQ1 in Synechococcus sp. PCC 7002 according to the following protocol. Synechococcus 7002 was grown for 48 h from colonies in an incubated shaker flask at 37° C. at 2% CO2 to an OD730 of 1 in A+ medium described in Frigaard et al., Methods Mol. Biol., 274:325-340 (2004). 450 μL of culture was added to a epi-tube with 50 μL of 5 μg of plasmid DNA digested with XbaI ((New England Biolabs; Ipswitch, Mass.)) that was not purified following restriction digest. Cells were incubated in the dark for four hours at 37° C. The entire volume of cells was plated on A+ medium plates with 1.5% agarose and grown at 37° C. in a lighted incubator (40-60 μE/m2/s PAR, measured with a LI-250A light meter (LI-COR)) for about 24 hours. 25 μg/mL of spectinomycin was underlayed on the plates. Resistant colonies were visible in 7-10 days after further incubation, and recombinant strains were confirmed by PCR using internal and external primers to check insertion and confirm location of the genes on pAQ1 in the strains (Table 4).
Ethyl Ester Production Culturing Conditions:
One colony of each of the four strains listed in Table 4 was inoculated into 10 ml of A+ media containing 50 μg/ml spectinomycin and 1% ethanol (v/v). These cultures were incubated for about 4 days in a bubble tube at 37° C. sparged at approximately 1-2 bubbles of 1% CO2/air every 2 seconds in light (40-50 μE/m2/s PAR, measured with a LI-250A light meter (LI-COR)). The cultures were then diluted so that the following day they would have OD730 of 2-6. The cells were washed with 2×10 ml JB 2.1/spec200, and inoculated into duplicate 28 ml cultures in JB 2.1/spec200+1% ethanol (v/v) media to an OD730=0.07. IPTG was added to the JCC803 cultures to a final concentration of 0.5 mM. These cultures were incubated in a shaking incubator at 150 rpm at 37° C. under 2% CO2/air and continuous light (70-130 μE m2/s PAR, measured with a LI-250A light meter (LI-COR)) for ten days. Water loss through evaporation was replaced with the addition of sterile Milli-Q water. 0.5% (v/v) ethanol was added to the cultures to replace loss due to evaporation every 48 hours. At 68 and 236 hours, 5 ml and 3 ml of culture were removed from each flask for ethyl ester analysis, respectively. The OD730 values reached by the cultures are given in Table 5.
The culture aliquots were pelleted using a Sorvall RC6 Plus superspeed centrifuge (Thermo Electron Corp) and a F13S-14X50CY rotor (5000 rpm for 10 min). The spent media supernatant was removed and the cells were resuspended in 1 ml of Milli-Q water. The cells were pelleted again using a benchtop centrifuge, the supernatant discarded and the cell pellet was stored at −80° C. until analyzed for the presence of ethyl esters.
Detection and Quantification of Ethyl Esters in Strains:
Cell pellets were thawed and 1 ml aliquots of acetone (Acros Organics 326570010) containing 100 mg/L butylated hydroxytoluene (Sigma-Aldrich B1378) and 50 mg/L ethyl valerate (Fluka 30784) were added. The cell pellets were mixed with the acetone using a Pasteur pipettes and vortexed twice for 10 seconds (total extraction time of 1-2 min). The suspensions were centrifuged for 5 min to pellet debris, and the supernatants were removed with Pasteur pipettes and subjected to analysis with a gas chromatograph using flame ionization detection (GC/FID).
An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used to detect the ethyl esters. One μL of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was 50° C., hold one minute; 10°/min increase to 280° C.; hold ten minutes. The GC/MS interface was 290° C., and the MS range monitored was 25 to 600 amu. Ethyl myristate [C14:0; retention time (rt): 17.8 min], ethyl palmitate (C16:0; rt: 19.8 min) and ethyl stearate (C18:0; rt: 21.6 min) were identified based on comparison to a standard mix of C4-C24 even carbon saturated fatty acid ethyl esters (Supelco 49454-U). Ethyl oleate (C18:1; rt: 21.4 min) was identified by comparison with an ethyl oleate standard (Sigma Aldrich 268011). These identifications were confirmed by GC/MS (see following Methyl Ester Production description for details). Calibration curves were constructed for these ethyl esters using the commercially available standards, and the concentrations of ethyl esters present in the extracts were determined and normalized to the concentration of ethyl valerate (internal standard).
Four different ethyl esters were found in the extracts of JCC723 and JCC803 (Table 6 and Table 7). In general, JCC803 produced 2-10× the amount of each ethyl ester than JCC723, but ethyl myristate (C14:0) was only produced in low quantities of 1 mg/L or less for all these cultures. Both JCC723 and JCC803 produced ethyl esters with the relative amounts C16:0>C18:0>C18:1 (cis-9)>C14:0. No ethyl esters were found in the extracts of JCC879 or JCC750, indicating that the strain cannot make ethyl esters naturally and that expression of only the tesA gene is not sufficient to confer production of ethyl esters.
Methyl Ester Production Culturing Conditions:
One colony of JCC803 (Table 1) was inoculated into 10 mL of A+ media containing 50 μg/ml spectinomycin and 1% ethanol (v/v). This culture was incubated for 3 days in a bubble tube at 37° C. sparged at approximately 1-2 bubbles of 1% CO2/air every 2 seconds in light (40-50 μE/m2/s PAR, measured with a LI-250A light meter (LI-COR)). The culture was innoculated into two flasks to a final volume of 20.5 ml and OD730=0.08 in A+ media containing 200 μg/ml spectinomycin and 0.5 mM IPTG with either 0.5% methanol or 0.5% ethanol (v/v). These cultures were incubated in a shaking incubator at 150 rpm at 37° C. under 2% CO2/air and continuous light (70-130 μE m2/s PAR, measured with a LI-250A light meter (LI-COR)) for three days. Water loss through evaporation was replaced with the addition of sterile Milli-Q water. Samples of 5 ml of these cultures (OD730=5-6) were analyzed for the presence of ethyl or methyl esters.
Detection of Methyl Esters and Comparison with Ethyl Ester Production in the Same Strain:
Cell pellets were thawed and 1 ml aliquots of acetone (Acros Organics 326570010) containing 100 mg/L butylated hydroxytoluene (Sigma-Aldrich B1378) and 50 mg/L ethyl valerate (Fluka 30784) were added. The cell pellets were mixed with the acetone using a Pasteur pipette and vortexed twice for 10 seconds (total extraction time of 1-2 min). The suspensions were centrifuged for 5 min to pellet debris, and the supernatants were removed with Pasteur pipettes and subjected to analysis with a gas chromatograph using mass spectral detection (GC/MS).
An Agilent 7890A GC/5975C EI-MS equipped with a 7683 series autosampler was used to measure the ethyl esters. One μL of each sample was injected into the GC inlet using pulsed splitless injection (pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was 50° C., hold one minute; 10°/min increase to 280° C.; hold ten minutes. The GC/MS interface was 290° C., and the MS range monitored was 25 to 600 amu. Compounds indicated by peaks present in total ion chromatograms were identified by matching experimentally determined mass spectra associated with the peaks with mass spectral matches found by searching in a NIST 08 MS database.
The culture of JCC803 incubated with ethanol contained ethyl palmitate [C16:0; retention time (rt): 18.5 min], ethyl heptadecanoate (C17rt: 19.4 min), ethyl oleate (C18:1; rt: 20.1 min) and ethyl stearate (C18:0; rt: 20.3 min) (
No ethyl esters were detected in the strain incubated with methanol. Instead, methyl palmitate (C16:0; retention time (“rt”): 17.8 min), methyl heptadecanoate (C17:0; rt: 18.8 min) and methyl stearate (C18:0) were found (
The data presented herein shows that JCC803 and other cyanobacterial strains engineered with tesA-fadD-wax genes can utilize methanol, ethanol, butanol, and other alcohols, including exogenously added alcohols, to produce a variety of fatty acid esters. In certain embodiments, multiple types of exogenous or endogenous alcohols (e.g., methanol and ethanol; butanol or ethanol; methanol and butanol; etc.) could be added to the culture medium and utilized as substrates.
In order to compare the yields of fatty-acid esters produced by recombinant strains expressing tesA-fadD or fadD-wax (i.e., two of the three genes in the tesA-fadD-wax synthetic operon), fadD-wax and tesA-fadD and were assembled as two-gene operons and inserted into pJB5 to yield pJB634 and pJB578, respectively. These recombination plasmids were transformed into Synechococcus sp. PCC 7002 as described in Example 1, above to generate the strains listed in Table 8. Table 8 also lists JCC723, described above.
Synechococcus sp. PCC 7002
One 30-ml culture of each strain listed in Table 1 was prepared in JB 2.1 medium containing 200 mg/L spectinomycin and 1% ethanol (vol/vol) at an OD730=0.1 in 125 ml flasks equipped with foam plugs (inocula were from five ml A+ cultures containing 200 mg/L spectinomyin started from colonies incubated for 3 days in a Multitron II Infors shaking photoincubator under continuous light of ˜100 μE m−2s−1 photosynthetically active radiation (PAR) at 37° C. at 150 rpm in 2% CO2-enriched air). The cultures were incubated for seven days in the Infors incubators under continuous light of ˜100 μE m−2s−1 photosynthetically active radiation (PAR) at 37° C. at 150 rpm in 2% CO2-enriched air. Fifty percent of the starting volume of ethanol was added approximately at day 5 based on experimentally determined stripping rates of ethanol under these conditions. Water loss was compensated by adding back milli-Q water (based on weight loss of flasks). Optical density measurements at 730 nm (OD730) were taken (Table 8), and esters were extracted from cell pellets using the acetone procedure detailed in Example 2, above. Ethyl arachidate (Sigma A9010) at 100 mg/L was used as an internal standard instead of ethyl valerate. The dry cell weights (DCWs) were estimated based on the OD measurement using an experimentally determined average of 300 mg L−1 OD730−1.
The acetone extracts were analyzed by GC/FID (for instrument conditions, see Example 2). In order to quantify the various esters, response factors (RF) were estimated from RFs measured for authentic ethyl ester standards and these RFs were used to determine the titres in the acetone extracts. The % DCW of the fatty-acid esters and the sum of the esters as % DCW is given in Table 8. Expression of fadD-wax was sufficient to allow production of fatty-acid ethyl esters (FAEEs), while expression of tesA-fadD did not result in any FAEEs (
Seven 30-ml cultures of JCC803 (prepared from a single JCC803 culture that was diluted into 250 ml of JB 2.1 media containing 200 mg/L spectinomycin at an OD730=0.1) in 125-ml flasks were used to evaluate the ability of JCC803 to esterify different alcohols with fatty acids. Seven different alcohols were added at concentrations previously determined to allow growth of JCC803 (Table 9). The cultures were incubated for seven days in a Multitron II Infors shaking photoincubator under continuous light of ˜100 μE m−2s−1 photosynthetically active radiation (PAR) at 37° C. at 150 rpm in 2% CO2-enriched air. Water loss was compensated by adding back milli-Q water (based on weight loss of flasks). Optical density measurements at 730 nm (OD730) were taken (Table 3), and esters were extracted from cell pellets using the acetone procedure detailed in Example 2, above. Ethyl arachidate (Sigma A9010) at 100 mg/L was used as an internal standard instead of ethyl valerate. The dry cell weights (DCWs) were also determined for each culture so that the % DCW of the esters could be reported.
The acetone extracts were analyzed by GC/MS and GC/FID, as described above. The compounds indicated by peaks present in the total ion chromatograms were identified by matching the mass spectra associated with the peaks with mass spectral matches found by searching the NIST 08 MS database or by interpretation of the mass spectra when a respective mass spectrum of an authentic standard was not available in the database. In all cases, the corresponding alcohol esters of fatty acids were produced by JCC803 (
In general, the provision of longer-chain alcohols increased the yields of fatty-acid esters. The addition of butanol resulted in the highest yields of fatty-acid esters. Because butanol can be made biosynthetically (Nielsen et al. 2009, and references therein), exogenous butanol biosynthetic pathways could be expressed by one skilled in the art to generate a photosynthetic strain which can produce butyl esters without the addition of butanol. The use of butanol and butanol-producing pathways in other microbes containing the tesA-fadD-wax pathway would also be expected to increase yields of fatty-acid esters.
Six 30-ml cultures of JCC803 (prepared from a single JCC803 culture that was diluted into 200 ml of JB 2.1 media/spec200 at an OD730=0.1) in 125 ml flasks were used to evaluate the ability of JCC803 cultures to produce butyl esters when containing different concentrations of butanol. Six different concentrations were tested (Table 12). The cultures were incubated for 21 days in a Multitron II Infors shaking photoincubator under continuous light at ˜100 μE m−2s−1 PAR at 37° C. at 150 rpm in 2% CO2-enriched air. Fifty percent of the starting volume of butanol was added approximately every 3.5 days based on experimentally determined stripping rates of butanol under these conditions. Water loss was compensated by adding back milli-Q water (based on weight loss of flasks). OD730s were taken and esters were extracted from cell pellets using the acetone procedure detailed above. 100 mg/L ethyl arachidate (Sigma A9010) was used as an internal standard instead of ethyl valerate. The dry cell weights (DCWs) were also determined for each culture so that the % DCW of the esters could be reported.
An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used to quantify the butyl esters. One microliter of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min), which was at a temperature of 280° C. The column was an HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm), and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was: 50° C., hold one minute; 10°/min increase to 280° C.; hold ten minutes. Butyl myristate, butyl palmitate, butyl margarate, butyl oleate and butyl stearate were quantified by determining appropriate response factors for the number of carbons present in the butyl esters from commercially available fatty-acid ethyl esters (“FAEEs”) and fatty acid butyl esters (“FABEs”). The calibration curves were prepared for ethyl laurate (Sigma 61630), ethyl myristate (Sigma E39600), ethyl palmitate (Sigma P9009), ethyl oleate (Sigma 268011), ethyl stearate (Fluka 85690), butyl laurate (Sigma W220604) and butyl stearate (Sigma S5001). The concentrations of the butyl esters present in the extracts were determined and normalized to the concentration of ethyl arachidate (internal standard).
The yields of the JCC803 cultures as given by the % DCW of the fatty acid butyl esters is given in Table 12. The highest yield of 14.7% resulted from the culture incubated with 0.05% butanol (vol/vol) although the 0.075% butanol-containing culture was approximately the same.
Plasmids.
Escherichia coli exports alkanes and other hydrophobic molecules out of the cell via the TolC-AcrAB transporter complex (Tsukagoshi and Aono, 2000; Chollet et al. 2004). PCR primer sets were designed to amplify tolC (Genbank # NC—000913.2, locus b3035) and acrA-acrB as an operon (Genbank # NC—000913.2, loci b0463, b0462) from E. coli MG1655 (ATCC #700926). The tolC and acrAB genes were amplified from MG1655 genomic DNA using the Phusion High-Fidelity PCR kit F-553 from New England BioLabs (Ipswich, Mass.) following the manufacturer's instructions. Buffer GC and 3% dimethyl sulfoxide (DMSO) were used for the PCR reactions. The amplicons were assembled into a three-gene, two-promoter construct (“transporter insert”; PpsaA-tolC-Ptsr2142-acrAB) and placed in multiple cloning site of recombination vector pJB161 (SEQ ID #15) to yield pJB1074. pJB161 (and pJB161-derived plasmids, including pJB1074) contain an upstream homology region (UHR) and a downstream homology region (DHR) that allows recombination into the pAQ7 plasmid of Synechococcus sp. PCC7002 at the lactate dehydrogenase locus (for pAQ7 plasmid sequence, see Genbank # CP000957). The homology regions flank a multiple cloning site (mcs), the natural terminator from the alcohol dehydrogenase gene from Zymomonas mobilis (adhII) and a kanamycin cassette which provides resistance in both E. coli and Synechococcus sp. PCC 7002. The transporter insert with flanking homology regions is provided as SEQ ID 16.
Strain Construction.
As described above, JCC803 is a strain of Synechococcus sp. PCC 7002 that has been engineered to produce esters of fatty acids (such as those found in biodiesel) when incubated in the presence of alcohols. The strain contains a thioesterase (tesA), an acyl-CoA synthetase (fadD) and a wax synthase (wxs) inserted into plasmid pAQ1 by homologous recombination.
The genes present in pJB161 and pJB1074 were integrated into the plasmid pAQ7 in Synechococcus sp. PCC 7002 (specifically, strain JCC803) using the following procedure. A 5 ml culture of JCC803 in A+ medium containing 200 mg/L spectinomycin was incubated in an Infors shaking incubator at 150 rpm at 37° C. under 2% CO2/air and continuous light (70-130 μE m−2 s−1 PAR, measured with a LI-250A light meter (LI-COR)) until it reached an OD730 of 1.14. For each plasmid, 500 μl of culture and 5 μg of plasmid DNA were added into a microcentrifuge tube. The tubes were then incubated at 37° C. in the dark rotating on a Rotamix RKSVD (ATR, Inc.) on a setting of approximately 20. After 4 hours for pJB161 or 7 hours for pJB1074, the cells were pelleted using a microcentrifuge. All but ˜100 μl of the supernatants were removed and the cell pellets were resuspended using the remaining supernatant and plated on A+ agar plates. The plates were incubated overnight in a Percival lighted incubator under constant illumination (40-60 μE m−2 s−1 PAR, measured with a LI-250A light meter (LI-COR)) at 37° C. for about 24 hours. On the following day, spectinomycin and kanamycin solution was added underneath the agar of the plates to estimated concentration of 25 mg/L spectinomycin and 50 mg/L kanamycin (assuming 40 ml A+ agar in the plate). These plates were placed back into the incubator until tiny colonies became visible. The plates were moved to another Percival incubator under the same conditions except that 1% CO2 was maintained in the air (allows for faster growth). Approximately 110 colonies formed for recombinant strains resulting from the pJB1074 transformation and 2800 colonies resulting from the pJB160 transformation. A colony from the pJB161 transformation plate was designated JCC1132.
Thirty colonies were picked from the tolC-acrAB transformation plate and streaked onto both an A+ plate with 100 mg/L spectinomycin and 0.05 mg/L erythromycin and an A+ plate with 100 mg/L spectinomycin and 0.1 mg/L erythromycin. Erythromycin is a substrate for the TolC-AcrAB transporter (Chollet et al. 2004) and served to verify function of the transporter in naturally erythromycin-sensitive Synechococcus sp. PCC 7002. The plates were incubated in Percival lighted incubator at 37° C. under constant illumination (40-60 μE m−2 s−1 PAR, measured with a LI-250A light meter (LI-COR)) at 37° C. After two days, slight growth was visible on both plates. Eight days after streaking, variable growth and survival was evident on most of the streaks on the 0.05 mg/L erythromycin plate. On the 0.1 mg/L erythromycin plate, all of the streaks except for two had become nonviable. The same source colonies that produced the two viable streaks on 0.1 mg/L erythromycin produced streaks that were healthy on the 0.05 mg/L erythromycin plate. One of these strains on the 0.1 mg/L erythromycin plate was designated JCC1585 (see Table 13 for a list of strains).
Erythromycin Tolerance in Liquid Culture.
To verify the improved tolerance of JCC1585 to erythromycin compared to JCC1132, a 5 ml A+ culture containing 200 mg/L spectinomycin and 0.5 mg/L erythromycin (JCC1585) or containing 200 mg/L spectinomycin and 50 mg/L kanamycin (JCC1132) were used to inoculate 30 ml of JB 2.1 containing 200 mg/L spectinomycin and 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L erythromycin in 125 ml culture flasks at an OD730 of 0.1. These cultures were incubated in an Infors shaking incubator at 150 rpm at 37° C. under 2% CO2/air and continuous light (70-130 μE m−2 s−1 PAR, measured with a LI-250A light meter (LI-COR)). Timepoints were taken at 5 and 10 days of growth, during which water loss was replaced through addition of milli-Q water. Table 14 shows OD730 values of JCC1132 and JCC1585 cultures at day 5 and 10 with different concentrations of erythromycin present in the medium. The JCC1585 cultures were tolerant of erythromycin concentrations of up to 1 mg/L (highest concentration tested) after 10 days while the JCC1132 cultures had bleached under all concentrations of erythromycin tested.
To verify the improved tolerance of JCC1585 to erythromycin compared to JCC1132, a 5 ml A+ culture containing 200 mg/L spectinomycin and 0.5 mg/L erythromycin (JCC1585) or containing 200 mg/L spectinomycin and 50 mg/L kanamycin (JCC1132) were used to inoculate 30 ml of JB 2.1 media containing 200 mg/L spectinomycin and 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L erythromycin in 125 ml culture flasks at an OD730 of 0.1. These cultures were incubated in an Infors shaking incubator at 150 rpm at 37° C. under 2% CO2/air and continuous light (70-130 μE m2/s PAR, measured with a LI-250A light meter (LI-COR)). Timepoints were taken at 5 and 10 days of growth, during which water loss was replaced through addition of milli-Q water. The JCC1585 cultures were tolerant of erythromycin concentrations of up to 1 mg/L (highest concentration tested) after 10 days while the JCC1132 cultures had bleached under all concentrations of erythromycin tested (Table 14).
Culture Conditions.
To test for secretion of butyl esters, 5 ml A+ cultures with 200 mg/L spectinomycin and 50 mg/L kanamycin were inoculated from colonies for JCC1132 and JCC1585. These cultures were used to inoculate duplicate 30 ml cultures in JB2.1 medium containing 200 mg/L spectinomycin and 50 mg/L kanamycin. At the beginning of the experiment, 15 μl butanol (Sigma 34867) was added to each flask so that fatty acid butyl esters (FABEs) would be produced by the cultures. These cultures were incubated in an Infors shaking incubator at 150 rpm at 37° C. under 2% CO2/air and continuous light (70-130 μE m−2 s−1 PAR, measured with a LI-250A light meter (LI-COR)) for three days. At day 4 of the experiment, 7.5 μl butanol was added to the cultures to compensate for the experimentally determined stripping rate of butanol under these conditions. Water loss through evaporation was replaced with the addition of sterile Milli-Q water at day 7 and OD730 readings were taken for each culture.
Detection of Butyl Esters.
An aliquot of 250 μl was removed from each culture and centrifuged at 1500 rpm in Microcentrifuge 5424 (Eppendorf) for ˜2 min. The supernatants were removed and the pellets were suspended in 500 μl milli-Q H2O. The samples were centrifuged and the supernatants discarded. An additional centrifugation step for 4 min was performed, and any remaining supernatant was removed. The weight of the tube and the cell pellet were measured. One milliliter of acetone (Acros Organics 326570010) containing 100 mg/L butylated hydroxytoluene (BHT, Sigma-Aldrich B1378) and 100 mg/L ethyl arachidate (Sigma A9010) were added to each pellet, and the mixture was pipetted up and down until none of the pellet remained on the wall of the tube. Each tube was then vortexed for 15 s, and the weight of the tube, acetone solution, and cells was taken. The tubes were then spun down and 500 μl of supernatant was submitted for GC analysis. From these samples, the percent dry cell weights of fatty acid butyl esters in the cell pellets were determined.
In order to quantify FABE's in the medium, 300 μL of a 20% (v/v) Span80 (Fluka 85548) solution was added to each flask and mixed by swirling for 30 seconds. These mixtures were then poured into 50 mL Falcon tubes. Five mL of isooctane containing 0.01% BHT and 0.005% ethyl arachidate was added to the flasks and swirled for several seconds. The solutions were then poured into the appropriate 50 mL Falcon tubes containing the culture from the flasks. The tube was then shaken for 10 seconds and centrifuged using a Sorvall RC6 Plus superspeed centrifuge (Thermo Electron Corp) and a F13S-14X50CY rotor (6000 rpm for 20 min). One milliliter of the organic phase (upper phase) was removed and submitted for GC analysis.
The butyl esters produced by JCC803 and JCC803-derived strains were identified by GC/MS employing an Agilent 7890A GC/5975C ELMS equipped with a 7683 series autosampler. One microliter of each sample was injected into the GC inlet using a pulsed splitless injection (pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was 50° C., hold one minute; 10°/min increase to 280° C.; hold ten minutes. The GC/MS interface was 290° C., and the MS range monitored was 25 to 600 amu. Butyl myristate [retention time (rt): 19.72 min], butyl palmitate (rt: 21.58 min) butyl heptadecanoate (rt: 22.40 min), butyl oleate (rt: 23.04 min) and butyl stearate (rt: 23.24 min) were identified by matching experimentally determined mass spectra associated with the peaks with mass spectral matches found by searching in a NIST 08 MS database.
An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used to quantify the butyl esters. One microliter of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min), which was at a temperature of 280° C. The column was an HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm), and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was 50° C., hold one minute; 10°/min increase to 280° C.; hold ten minutes. Butyl myristate (rt: 19.68 min], butyl palmitate (rt: 21.48 min), butyl heptadecanoate (rt: 22.32 min), butyl oleate (rt: 22.95 min) and butyl stearate (rt: 23.14 min) were quantified by determining appropriate response factors for the number of carbons present in the butyl esters from commercially-available fatty acid ethyl esters (FAEEs) and FABEs. The calibration curves were prepared for ethyl laurate (Sigma 61630), ethyl myristate (Sigma E39600), ethyl palmitate (Sigma P9009), ethyl oleate (Sigma 268011), ethyl stearate (Fluka 85690), butyl laurate (Sigma W220604) and butyl stearate (Sigma S5001). The concentrations of the butyl esters present in the extracts were determined and normalized to the concentration of ethyl arachidate (internal standard).
Peaks with areas greater than 0.05 could be integrated by the Chemstation™ software (Agilent®), and the concentrations of the butyl esters in both media and supernatant were determined from these values. The dry cell weight (DCW) of these strains was based on a measurement of OD730 and calculated based on the observed average DCW/OD relationship of 0.29 g L−1 OD−1. In the case of the JCC1585 culture supernatant, small peaks for butyl myristate (flask 1 area: 1.26, flask 2: 2.23) and butyl palmitate (flask 1 area: 5.16, flask 2: 5.62) were observed while no peak with an area greater than 0.05 at these retention times was found in the media extraction of the JCC1132 cultures. The OD730 percent dry cell weights of the FABEs in the cell pellets and the media are given in Table 15. The total % DCW of FABE's found in the cell pellets is indicated, as is the % DCW of butyl myristate and butyl palmitate found in the pellets and the media.
Table 15 shows that the recombinant expression of to/C in an engineered cyanobacterium provides for the secretion of a detectable fraction of esters (in this case, butyl esters) synthesized by the engineered cell. The amount of secretion achieved can be modulated by increasing concentrations of erythromycin or other transporter substrates, and/or through optimization of expression levels (promoter strength and codon optimization strategies) and/or specifically targeting a cyanobacterial membrane by employing appropriate cyanobacterial N-terminal leader sequences.
Strain Construction.
Thermosynechoccocus elongatus BP-1 long-chain-fatty-acid CoA ligase gene (aas, GenBank accession number NP—682091.1) was replaced with a thermostable kanamycin resistance marker (kan_HTK, GenBank accession number AB121443.1) as follows:
Regions of homology flanking the BP-1 aas gene (Accession Number: NP—682091.1) were amplified directly from BP-1 genomic DNA using the primers in Table 16. PCR amplifications were performed with Phusion High Fidelity PCR Master Mix (New England BioLabs) and standard amplification conditions.
The amplified upstream homologous region (UHR) was cloned into the UHR of a pJB5 expression vector containing kan_HTK by digesting the insert and vector individually with SbfI and NotI restriction endonucleases (New England BioLabs) following well known laboratory techniques. Digestions were isolated on 1% TAE agarose gel, purified using a Gel Extraction Kit (Qiagen), and ligated with T4 DNA Ligase (New England BioLabs) incubated at room temperature for 1 hour. The ligated product was transformed into NEB 5-alpha chemically competent E. coli cells (New England BioLabs) using standard techniques and confirmed by PCR. The downstream homologous region (DHR) was cloned into the resulting plasmid following a similar protocol using AscI and FseI restriction endonucleases (New England BioLabs). The final plasmid (pJB1349) was purified using QIAprep Spin Miniprep kit (Qiagen) and the construct was confirmed by digestion with HindIII, AseI, and PstI restriction endonucleases (New England BioLabs).
BP-1 was grown in 5 ml B-HEPES liquid media in a glass test tube (45° C., 120 rpm, 2% CO2) to OD7301.28. A 1 ml aliquot of culture was transferred to a fresh tube and combined with 1 ug of purified pJB1349. The culture was incubated in the dark (45° C., 120 rpm, 2% CO2) for 4 hours. 4 ml of fresh B-HEPES liquid media were added and the culture was incubated with light (45° C., 120 rpm, 2% CO2) overnight. 500 μl of the resulting culture were plated in 3 ml of B-HEPES soft agar on B-HEPES plates containing 60 μg/ml kanamycin and placed in an illuminated incubator (45° C., ambient CO2) until colonies appeared (1 week), then moved into a 2% CO2 illuminated incubator for an additional week.
Four randomly selected colonies (samples A-D) were independently grown in 5 ml B-HEPES liquid media with 60 μg/ml kanamycin in glass test tubes (45° C., 120 rpm, 2% CO2) for one week. Replacement of aas gene was confirmed by PCR of whole cell genomic DNA by a culture PCR protocol as follows. Briefly, 100 μl of each culture was resuspended in 50 μl lysis buffer (96.8% diH2O, 1% Triton X-100, 2% 1M Tris pH 8.5, 0.2% 1M EDTA). 10 μl of each suspension were heated 10 min at 98° C. to lyse cells. 1 μl of lysate was used in 15 μl standard PCR reactions using Quick-Load Taq 2× Master Mix (New England BioLabs). The PCR product showed correct bands for an unsegregated knockout.
All cultures were maintained in fresh B-HEPES liquid media with 60 μg/ml kanamycin for an additional week. The PCR reaction described above was repeated, again showing correct bands for an unsegregated knockout. Cultures were maintained in liquid culture, and one representative culture was saved as JCC1862.
Detection and Quantification of Free Fatty Acids in Strains.
Each of the four independently inoculated cultures described above (samples A-D), as well as BP-1, was analyzed for secretion of free fatty acids. OD730 was measured, and the volume in each culture tube was recorded. Fresh B-HEPES liquid media was added to each tube to bring the total volume to 5 ml and free fatty acids were extracted as follows:
Samples were acidified with 50 μl 1N HCl. 500 μl of 250 g/L methyl-β-cyclodextrin solution was added and samples were transferred to 15-ml conical tubes after pulse-vortexing. 1 ml of 50 mg/L butylated hydroxytoluene in isooctane was added to each tube. Samples were vortexed 20 s, then centrifuged 5 min at 6000 RCF to fractionate. 500 μl of the isooctane layer were placed into a new tube and submitted for GC analysis.
Concentrations of octanoic acid, decanoic acid, lauric acid, myristic acid, palmitoleic acid, palmitic acid, oleic acid, stearic acid, and 1-nonadecene extractants were quantitated by gas chromatography/flame ionization detection (GC/FID). Unknown peak areas in biological samples were converted to concentrations via linear calibration relationships determined between known authentic standard concentrations and their corresponding GC-FID peak areas. Standards were obtained from Sigma. GC-FID conditions were as follows. An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used. 1 μl of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 ml/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 ml/min. The GC oven temperature program was 50° C., hold one minute; 10° C./min increase to 280° C.; hold ten minutes.
GC results showed that the unsegregated aas knockout increased fatty acid production relative to BP-1 (Table 17), with myristic and oleic acid making up the majority of the increase (Table 18).
Transformation of BP-1.
As disclosed in PCT/US2010/042667, filed Jul. 20, 2010, Thermosynechococcus elongatus BP-1 is transformed with integration or expression plasmids using the following protocol. 400 ml Thermosynechococcus elongatus BP-1 in B-HEPES medium is grown in a 2.8 l Fernbach flask to an OD730 of 1.0 in an Infors Multritron II shaking photoincubator (55° C.; 3.5% CO2; 150 rpm). For each transformation, 50 ml cell culture is pelleted by centrifugation for 20 min (22° C.; 6000 rpm). After removing the supernatant, the cell pellet is resuspended in 500 μl B-HEPES and transferred to a 15 ml Falcon tube. To each 500 μl BP-1 cell suspension (OD730 of ˜100), 25 μg undigested plasmid (or no DNA) is added. The cell-DNA suspension is incubated in a New Brunswick shaking incubator (45° C.; 250 rpm) in low light (˜3 μmol photons m−2 s1). Following this incubation, the cell-DNA suspension is made up to 1 ml by addition of B-HEPES, mixed by gentle vortexing with 2.5 ml of molten B-HEPES 0.82% top agar solution equilibrated at 55° C., and spread out on the surface of a B-HEPES 1.5% agar plate (50 ml volume). Plates are left to sit at room temperature for 10 min to allow solidification of the top agar, after which time plates are placed in an inverted position in a Percival photoincubator and left to incubate for 24 hr (45° C.; 1% CO2; 95% relative humidity) in low light (7-12 μmol photons m−2 s1). After 24 hr, the plates are underlaid with 300 μl of 10 mg/ml kanamycin so as to obtain a final kanamycin concentration of 60 μg/ml following complete diffusion in the agar. Underlaid plates are placed back in the Percival incubator and left to incubate (45° C.; 1% CO2; 95% relative humidity; 7-12 μmol photons m−2 s1) for twelve days.
Increased Fatty Acids in BP-1.
Thermosynechococcus elongatus BP-1 (Δaas) is first constructed as described in the above Example. BP-1(Δaas) is shown to have elevated levels of both intracellular and extracellular levels of free fatty acids relative to wild-type because mechanistic analysis suggests that cells lacking an acyl-ACP synthetase have the inability to recycle exogenous or extracellular fatty acids; the extracellular fatty acid chains are diverted away from transport into the inner cellular membrane while other transport systems are thought to continue to export fatty acids. Therefore, to up-regulate fatty acid production, BP-1(Δaas) is transformed with a plasmid (e.g., pJB1349) carrying a thioesterase gene (see Table 3A). Increased cellular level of fatty acid production may be attributed to the combination of the aas deletion decreasing extracellular import of fatty acids and the addition of the thioesterase gene and/or thioesterase gene homologues.
Fatty Acid Esters.
The thioesterase gene with or without the leader sequence removed (Genbank # NC 000913, ref: Chot and Cronan, 1993), the E. coli acyl-CoA synthetase fadD (Genbank # NC 000913, ref: Kameda and Nunn, 1981) and the wax synthase (wxs) from Acinetobacter baylyi strain ADPI (Genbank # AF529086.1, ref: Stóveken et al. 2005) genes are designed for codon optimization, checking for secondary structure effects, and removal of any unwanted restriction sites (NdeI, XhoI, BamHI, NgoMIV, NcoI, SacI, BsrGI, AvrII, BmtI, MiuI, EcoRI, SbfI, NotI, SpeI, XbaI, Pad, AscI, FseI). These genes are engineered into plasmid or integration vectors (e.g., pJB1349) and assembled into a two gene operon (fadD-wxs) or a three gene operon (tesA-fadD-wxs) with flanking sites on the integration vector corresponding to integration sites for transformation into Thermosynechococcus elongatus BP-1. Integration sites include TS1, TS2, TS3 and TS4. A preferred integration site is the site of the aas gene. Host cells are cultured in the presence of small amounts of ethanol (1-10%) in the growth media under an appropriate promoter such as Pnir for the production of fatty acid esters.
In another embodiment, Thermosynechococcus elongatus BP-1 host cell with a two gene operon (fadD-wxs) or a three gene operon (tesA-fadD-wxs) is engineered to have ethanol producing genes (PCT/US2009/035937, filed Mar. 3, 2009; PCT/US2009/055949, filed Sep. 3, 2009; PCT/US2009/057694, filed Sep. 21, 2009) conferring the ability to produce fatty acid esters. In one plasmid construct, genes for ethanol production, including pyruvate decarboxylase from Zymomonas mobilis (pdcZm) and alcohol dehydrogenase from Moorella sp. HUC22-1 (adhAM), are engineered into a plasmid and transformed into BP-1. In an alternate plasmid construct, the pyruvate decarboxylase from Zymobacter palmae (pdcZp) and alcohol dehydrogenase from Moorella sp. HUC22-1 (adhAM), are engineered into a plasmid and transformed into BP-1. These genes are engineered into plasmid or integration vectors (e.g., pJB1349) with flanking sites on the integration vector corresponding to integration sites for transformation into Thermosynechococcus elongatus BP-1. Integration sites include TS1, TS2, TS3 and TS4. A preferred integration site is the site of the aas gene. In one configuration, expression of pdcZm and adhAM are driven by λ phage cI (“PcI”) and pEM7 and in another expression strain driven by PcI and PtRNAGlu. In one embodiment, a single promoter is used to control the expression of both genes. In another embodiment each gene expression is controlled by separate promoters with PaphII or Pcpcb controlling one and PcI controlling the other.
Strain Construction.
DNA sequences for thioesterase genes tesA, fatB, fatB1, and fatB2 were obtained from Genbank and were purchased from DNA 2.0 following codon optimization, checking for secondary structure effects, and removal of any unwanted restriction sites. Thioesterase gene fatB_mat is a modified form of fatB with its leader sequence removed.
Escherichia coli
Umbellularia californica
Cinnamomum camphora
Cuphea hookeriana
The thioesterase genes were cloned into a pJB5 expression vector containing upstream and downstream regions of homology to aquI (SYNPCC7002_A1189), pAQ3, and pAQ4 by digesting the inserts and vectors individually with AscI and NotI restriction endonucleases (New England BioLabs) following known laboratory techniques. Digestions were isolated on 1% TAE agarose gel, purified using a Gel Extraction Kit (Qiagen), and ligated with T4 DNA Ligase (New England BioLabs) incubated at room temperature for one hour. The ligated product was transformed into NEB 5-alpha chemically competent E. coli cells (New England BioLabs) using standard techniques. Purified plasmid was extracted using the QIAprep Spin Miniprep kit (Qiagen) and constructs were confirmed by PCR.
Synechococcus sp. PCC 7002 (Δaas) was grown in 5 ml A+ liquid media with 25 μg/ml gentamicin in a glass test tube (37° C., 120 rpm, 2% CO2) to OD730 of 0.98-1.1. 500 μl of culture was combined with 1 μg purified plasmid in 1.5 ml microcentrifuge tubes and incubated in darkness 3-4 hours. Samples were then plated on A+ agar plates with 3 or 6 mM urea and incubated overnight 37° C. in the light. Selective antibiotics were introduced to the plates by placing stock solution spectinomycin under the agar at a final concentration of 10 μg/mL, and incubating to allow diffusion of the antibiotic. Plates were incubated at 37° C. with light until plates cleared and individual colonies formed. Plates were then moved to an illuminated incubator at 2% CO2. Cultures were maintained on liquid or agar A+ media containing 3-6 mM urea with 25 μg/ml gentamicin, 100-200 μg/ml spectomycin, to promote plasmid segregation.
Thioesterase integration and attenuation was confirmed by PCR of whole-cell genomic DNA by a “culture PCR” protocol. Briefly, 100 μl of each culture was resuspended in 50 μl water or lysis buffer (96.8% diH2O, 1% Triton X-100, 2% 1M tris pH 8.5, 0.2% 1M EDTA). 10 μl of each suspension were heated 10 min at 98° C. to lyse cells. 1 μl of lysate was used in 10 μl standard PCR reactions using Quick-Load Taq 2× Master Mix (New England BioLabs) or Platinum PCR Supermix HiFi (Invitrogen). PCR products showed correct bands for segregated aquI, pAQ4 and unsegregated (pAQ3) integrants.
Detection and Quantification of Free Fatty Acids in Strains.
Individual colonies were grown in A+ liquid media with 3 mM urea, 50 μg/ml gentamicin, 200 μg/ml spectomycin in glass test tubes (see Table 20). Cultures were maintained in liquid culture to promote segregation (37° C., 120 rpm, 2% CO2). Liquid cultures were diluted to OD730=0.2 in 5 ml A+ liquid media with 3 mM urea and no antibiotics in glass test tubes and incubated for seven days (37° C., 120 rpm, 2% CO2). After one week, OD730 was recorded and free fatty acids were extracted as follows:
Samples were acidified with 50 μl 1N HCl. 500 μl of 250 g/L methyl-β-cyclodextrin solution was added, and samples were transferred to 15-ml conical tubes after pulse-vortexing. 1 ml of 50 mg/L butylated hydroxytoluene in isooctane was added to each tube. Samples were vortexed 20 s and immediately centrifuged 5 min at 6000 RCF to fractionate. 500 μl of the isooctane layer were sub-sampled into a new tube and submitted for GC analysis.
Concentrations of octanoic acid, decanoic acid, lauric acid, myristic acid, palmitoleic acid, palmitic acid, oleic acid, stearic acid, and 1-nonadecene extractants were quantitated by gas chromatography/flame ionization detection (GC/FID). Unknown peak areas in biological samples were converted to concentrations via linear calibration relationships determined between known authentic standard concentrations and their corresponding GC-FID peak areas. Standards were obtained from Sigma. GC-FID conditions were as follows. An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used. 1 μl of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 ml/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 ml/min. The GC oven temperature program was 50° C., hold one minute; 10° C./min increase to 280° C.; hold ten minutes.
GC results showed increased fatty acid secretion in the thioesterase strains relative to Synechococcus sp. PCC 7002 JCC138 (Table 20). The specific enrichment profile of each culture was thioesterase dependent (Table 21).
Individual colonies of JCC1704, JCC1705, and JCC1706 were grown for three days in A+ liquid media with 3 mM urea, 25 μg/ml gentamicin, 100 μg/ml spectomycin in glass test tubes (37° C., 120 rpm, 2% CO2). Cultures were diluted to OD730=0.2 in 5 ml A+ liquid media with 3 mM urea and no antibiotics in glass test tubes and incubated at 37° C., 120 rpm, 2% CO2. After 11 days, OD730 was recorded and free fatty acids were extracted as follows:
Samples were acidified with 50 μl 1N HCl. 500 μl of 250 g/L methyl-β-cyclodextrin solution was added and samples were transferred to 15-ml conical tubes after pulse-vortexing. 1 ml of 50 mg/L butylated hydroxytoluene in isooctane was added to each tube. Samples were vortexed 20 s and immediately centrifuged 5 min at 6000 RCF to fractionate. 500 μl of the isooctane layer were sub-sampled into a new tube and submitted for GC analysis.
Concentrations of octanoic acid, decanoic acid, lauric acid, myristic acid, palmitoleic acid, palmitic acid, oleic acid, stearic acid, and 1-nonadecene extractants were quantitated by gas chromatograph/flange ionization detection (GC/FID), Unknown peak areas in biological samples were converted to concentrations via linear calibration relationships determined between known authentic standard concentrations and their corresponding GC-FID peak areas. Standards were obtained. from Sigma. GC-FID conditions were as follows. An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used 1 μl of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 ml/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 ml/min. The GC oven temperature program was 50° C., hold one minute; 10° C./min increase to 280° C.; hold ten minutes.
GC results showed increased fatty acid secretion relative to JCC138 but to a lesser degree than tesA or fatB_mat (Table 22). The specific enrichment profile of each culture was thioesterase dependent (Table 23).
Construction of the Promoter-uidA Expression Plasmid.
The E. coli uidA gene (Genbank AAB30197) was synthesized by DNA 2.0 (Menlo Park, Calif.), and was subcloned into pJB5. The DNA sequences of the ammonia-repressible nitrate reductase promoters P(nirA) (SEQ ID NO:17), P(nir07) (SEQ ID NO:18), and P(nir09) (SEQ ID NO:19) were obtained from Genbank. The nickel-inducible P(nrsB) promoter (SEQ ID NO:20), nrsS and nrsR were amplified from Synechocystis sp. PCC 6803. The promoters were cloned between NotI and NdeI sites immediately upstream of uidA, which is flanked by NdeI and EcoRI.
In addition, plasmids containing two 750-bp regions of homology designed to remove the native aquI (A1189) or the ldh (G0164) gene from Synechococcus sp. PCC 7002 were obtained by contract synthesis from DNA 2.0 (Menlo Park, Calif.). Using these vectors, 4 constructs were engineered and tested for GUS activity. Final transformation constructs are listed in Table 24. All restriction and ligation enzymes were obtained from New England Biolabs (Ipswich, Mass.). Ligated constructs were transformed into NEB 5-α competent E. coli (High Efficiency) (New England Biolabs: Ipswich, Mass.).
Plasmid Transformation into JCC138.
The constructs as described above were integrated onto either the genome or pAQ7 of JCC138, both of which are maintained at approximately 7 copies per cell. The following protocol was used for integrating the DNA cassettes. JCC138 was grown in an incubated shaker flask at 37° C. at 1% CO2 to an OD730 of 0.8 in A+ medium. 500 μl of culture was added to a microcentrifuge tube with 1 μg of DNA. DNA was prepared using a Qiagen Qiaprep Spin Miniprep Kit (Valencia, Calif.) for each construct. Cells were incubated in the dark for one hour at 37° C. The entire volume of cells was plated on A+ plates with 1.5% agar supplemented with 3 mM urea when necessary and grown at 37° C. in an illuminated incubator (40-60 μE/m2/s PAR, measured with a LI-250A light meter (LI-COR)) for approximately 24 hours. 25 μg/mL of spectinomycin or 50 μg/mL of kanamycin was introduced to the plates by placing the stock solution of antibiotic under the agar, and allowing it to diffuse up through the agar. After further incubation, resistant colonies became visible in 6 days. One colony from each plate was restreaked onto A+ plates with 1.5% agar supplemented with 6 mM urea when necessary and 200 μg/mL spectinomycin or 50 μg/mL of kanamycin.
Measurement of GUS Activity.
The GUS (beta-glucuronidase) reporter system was used to test the inducibility or repressibility of several promoters. This system measures the activity of beta-glucuronidase, an enzyme from E. coli that transforms colorless or non-fluorescent substrates into colored or fluorescent products. In this case, MUG (4-methylumbelliferyl β-D-glucuronide) is the substrate, and is hydrolyzed by beta-glucuronidase to produce the florescent product MU (4-methylumbelliferone), which is subsequently detected and quantified with a fluorescent spectrophotometer.
Strains containing uidA constructs under urea repression were incubated to OD730 between 1.8 and 4. These cells were subcultured to OD730 0.2 in 5 mL A+ media supplemented with 0, 3, 6, or 12 mM urea plus either 100 μg/mL spectinomycin or 50 μg/ml kanamycin and incubated for 24 hours. JCC138 was cultured in 5 mL A+ media for 24 hours. The strain containing gus under nickel-inducible expression was cultured for 3 days, then subcultured to OD730 0.2 in 5 mL A+ supplemented with 0, 2, 4, or 8 M NiSO4. These cells were incubated for 6 hours. To harvest cells, cultures were spun for 5 minute at 6000 rpm. Pellets were resuspended in 1 mL 1×GUS extraction buffer (1 mM EDTA, 5.6 mM 2-mercaptoethanol, 0.1 M sodium phosphate, pH 7) and lysed with microtip sonication pulsing 0.5 seconds on and 0.5 seconds off for 2 min. Total protein was analyzed with Bio-Rad (Hercules, Calif.) Quick Start Bradford assay, and extracts were subsequently analyzed for GUS activity using a Sigma (St Louis, Mo.) 0-Glucuronidase Fluorescent Activity Detection Kit. Relative activities of the 4 promoters are found in Table 25.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. All publications, patents and other references mentioned herein are hereby incorporated by reference in their entirety.
E. coli TesA amino acid sequence
E. coli FadD amino acid sequence
A. baylyi ADP1 wax synthase amino acids sequence
E. coli tesA optimized nucleic acid sequence
E. coli fadD optimized nucleic acid sequence
A. baylyi wsadpl optimized nucleic acid sequence
E. coli TolC amino acid sequence
E. coli AcrA amino acid sequence
E. coli AcrB amino acid sequence
AAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTCATATGG
CGGATACTCTGCTGATTCTGGGTGATTCTCTGTCTGCAGGCTACCGTATGTCCGCCTCCGCGGC
CTGGCCAGCTCTGCTGAATGATAAGTGGCAGTCTAAGACGTCCGTTGTGAACGCATCCATCTCT
GGCGACACGAGCCAGCAGGGCCTGGCCCGTCTGCCTGCACTGCTGAAACAGCACCAACCGCGC
TGGGTCCTGGTGGAGCTGGGCGGTAACGACGGTCTGCGCGGCTTCCAGCCGCAGCAGACCGAA
CAGACTCTGCGTCAGATTCTGCAGGACGTGAAAGCTGCTAACGCGGAACCGCTGCTGATGCAGA
TTCGTCTGCCAGCGAACTATGGCCGCCGTTACAACGAAGCGTTCTCTGCAATCTACCCAAAACT
GGCGAAAGAGTTTGACGTCCCGCTGCTGCCGTTCTTCATGGAGGAAGTATACCTGAAACCGCAG
TGGATGCAAGATGACGGCATCCACCCGAACCGTGATGCGCAGCCGTTCATCGCTGACTGGATGG
CGAAGCAACTGCAGCCGCTGGTAAACCACGATTCCTAATTAAAGATCTGTAGTAGGATCCATGTAG
TAACCCAGATCGTTACCAGTCCCTGGTTGACATGTTCGAACAGTCCGTGGCTCGCTACGCCGAT
CAGCCTGCTTTCGTCAACATGGGTGAGGTAATGACCTTTCGCAAACTGGAGGAGCGTTCCCGTG
CTTTCGCGGCATACCTGCAGCAGGGTCTGGGCCTGAAGAAAGGCGACCGCGTGGCCCTGATGAT
GCCGAACCTGCTGCAATATCCTGTGGCGCTGTTCGGTATCCTGCGTGCTGGTATGATCGTTGTC
AATGTTAACCCTCTGTATACCCCTCGTGAACTGGAGCACCAGCTGAATGACTCTGGTGCGTCTG
CTATCGTTATCGTTTCCAATTTCGCACATACGCTGGAGAAAGTGGTTGATAAAACCGCAGTGCAG
CATGTCATTCTGACTCGCATGGGTGACCAGCTGTCCACCGCTAAAGGTACTGTAGTCAACTTCGT
TGTGAAATACATTAAGCGCCTGGTTCCGAAATACCACCTGCCAGATGCAATTAGCTTTCGCTCTG
CACTGCATAACGGTTACCGTATGCAGTACGTAAAACCAGAGCTGGTGCCGGAAGACCTGGCCTT
TCTGCAGTATACCGGCGGCACCACCGGCGTGGCAAAGGGCGCGATGCTGACCCATCGTAACATG
CTGGCGAACCTGGAGCAGGTTAACGCAACGTACGGCCCGCTGCTGCACCCGGGTAAAGAACTG
GTAGTTACGGCACTGCCTCTGTATCACATCTTTGCACTGACGATCAACTGTCTGCTGTTCATTGA
ACTGGGTGGTCAGAACCTGCTGATCACCAACCCGCGTGACATTCCGGGCCTGGTAAAAGAGCTG
GCTAAGTACCCGTTCACCGCCATTACTGGCGTAAACACTCTGTTTAACGCGCTGCTGAACAACAA
AGAGTTTCAGCAGCTGGACTTCTCTAGCCTGCACCTGAGCGCTGGCGGTGGCATGCCGGTTCAG
CAGGTTGTGGCAGAGCGTTGGGTGAAACTGACCGGCCAGTATCTGCTGGAGGGTTATGGTCTGA
CCGAGTGTGCACCGCTGGTCAGCGTTAACCCGTATGATATTGATTACCACTCTGGTTCTATTGGT
CTGCCGGTTCCGTCCACGGAAGCCAAACTGGTGGACGATGACGACAACGAAGTACCTCCGGGCC
AGCCGGGTGAGCTGTGTGTCAAGGGTCCGCAGGTTATGCTGGGCTACTGGCAGCGCCCGGACG
CCACCGACGAAATCATTAAAAACGGTTGGCTGCATACCGGTGATATCGCTGTAATGGACGAAGA
AGGTTTCCTGCGTATCGTGGACCGTAAGAAAGATATGATTCTGGTGAGCGGTTTCAACGTGTAC
CCGAACGAAATTGAGGACGTAGTTATGCAACACCCTGGCGTGCAGGAGGTGGCAGCCGTGGGC
GTGCCGTCCGGTTCTTCTGGTGAGGCTGTGAAAATCTTTGTCGTTAAAAAGGACCCGTCCCTGA
CCGAAGAATCTCTGGTGACGTTTTGCCGCCGTCAACTGACTGGCTACAAAGTGCCGAAACTGGT
CGAGTTCCGCGATGAGCTGCCAAAATCTAACGTGGGTAAGATCCTGCGCCGCGAGCTGCGTGAC
GAGGCACGTGGCAAAGTTGACAATAAAGCATAACCGCGTAGGAGGACAGCTATGCGCCCACTTCA
TCCGATCGATTTCATTTTCCTGTCCCTGGAGAAACGCCAGCAGCCGATGCACGTAGGTGGTCTG
TTCCTGTTCCAGATCCCGGATAACGCTCCGGACACCTTTATTCAGGACCTGGTGAACGATATCCG
TATCTCCAAGTCTATTCCGGTTCCGCCGTTCAACAACAAGCTGAACGGTCTGTTCTGGGACGAA
GACGAGGAGTTCGATCTGGATCACCATTTCCGTCATATTGCGCTGCCGCACCCGGGTCGCATCC
GTGAGCTGCTGATTTACATCTCTCAGGAACACAGCACTCTCCTCGATCGCGCTAAACCTCTGTGG
ACTTGCAACATCATTGAAGGTATCGAGGGTAACCGTTTCGCCATGTACTTCAAGATTCATCATGC
GATGGTGGATGGTGTGGCGGGTATGCGTCTGATTGAGAAAAGCCTGTCCCATGATGTTACTGAA
AAGAGCATCGTACCGCCGTGGTGCGTTGAGGGCAAACGTGCTAAACGCCTGCGTGAACCGAAG
ACCGGCAAAATTAAGAAAATCATGTCTGGTATTAAATCTCAGCTCCAGGCCACCCCGACCGTTAT
TCAAGAACTGTCTCAGACGGTCTTCAAAGACATCGGCCGTAATCCGGACCACGTTTCCTCTTTCC
AGGCGCCGTGCTCCATCCTCAACCAGCGTGTGTCTTCTTCTCGTCGTTTCGCAGCACAGAGCTTT
GACCTGGACCGTTTCCGCAACATCGCCAAATCTCTGAACGTGACCATTAACGACGTTGTCCTGG
CTGTGTGTAGCGGTGCTCTGCGCGCTTATCTGATGTCTCATAACTCTCTGCCATCCAAACCGCTG
ATCGCTATGGTCCCAGCAAGCATCCGCAACGATGATTCTGATGTGTCCAACCGTATTACTATGAT
TCTGGCCAACCTCGCTACTCACAAAGACGACCCTCTGCAGCGTCTGGAAATCATCCGCCGCTCC
GTCCAGAACTCTAAACAGCGTTTTAAACGCATGACTTCCGACCAGATTCTGAACTATTCTGCGGT
TGTATACGGCCCGGCTGGTCTGAACATTATCAGCGGTATGATGCCGAAACGTCAGGCTTTTAAC
CTGGTAATCAGCAACGTTCCTGGCCCGCGTGAGCCGCTGTACTGGAACGGCGCAAAACTGGACG
CACTGTACCCGGCTTCCATCGTTCTGGATGGCCAGGCTCTGAACATCACTATGACCTCTTACCTG
GACAAACTGGAAGTAGGTCTGATCGCGTGTCGCAATGCACTGCCGCGCATGCAGAACCTGCTGA
CCCACCTGGAGGAGGAAATCCAGCTGTTTGAGGGCGTTATCGCCAAACAGGAAGATATCAAAAC
GGCGAACTAACCATGGTTGAATTC
CAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTG
AGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC
CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTGACGGCGGGATATAAC
ATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCG
GACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGT
GGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGT
CGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCA
GACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACC
CAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGT
TGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTC
CACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGCTGCG
CGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACC
ACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGC
GTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTT
GTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTT
TCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGC
ATACTCTGCGACATCGTATAACGTTACTGGTTTCATATTCACCACCCTGAATTGACTCTCTTC
CGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCACCATTCGATGGTGTCAACGTAAATGC
ATGCCGCTTCGCCTTCCAATTGGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCA
TCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGC
GCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGA
AATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACA
GGGTGATTCTCTGTCTGCAGGCTACCGTATGTCCGCCTCCGCGGCCTGGCCAGCTCTG
CTGAATGATAAGTGGCAGTCTAAGACGTCCGTTGTGAACGCATCCATCTCTGGCGACA
CGAGCCAGCAGGGCCTGGCCCGTCTGCCTGCACTGCTGAAACAGCACCAACCGCGCTG
GGTCCTGGTGGAGCTGGGCGGTAACGACGGTCTGCGCGGCTTCCAGCCGCAGCAGAC
CGAACAGACTCTGCGTCAGATTCTGCAGGACGTGAAAGCTGCTAACGCGGAACCGCTG
CTGATGCAGATTCGTCTGCCAGCGAACTATGGCCGCCGTTACAACGAAGCGTTCTCTG
CAATCTACCCAAAACTGGCGAAAGAGTTTGACGTCCCGCTGCTGCCGTTCTTCATGGA
GGAAGTATACCTGAAACCGCAGTGGATGCAAGATGACGGCATCCACCCGAACCGTGAT
GCGCAGCCGTTCATCGCTGACTGGATGGCGAAGCAACTGCAGCCGCTGGTAAACCACG
ATTCCTAA
TTAAAGATCTGTAGTAGGATCCATGTAGGGTGAGGTTATAGCTATGAAGAAAG
TTTGGCTGAACCGTTATCCGGCAGATGTACCGACTGAAATTAACCCAGATCGTTACCAG
TCCCTGGTTGACATGTTCGAACAGTCCGTGGCTCGCTACGCCGATCAGCCTGCTTTCGT
CAACATGGGTGAGGTAATGACCTTTCGCAAACTGGAGGAGCGTTCCCGTGCTTTCGCG
GCATACCTGCAGCAGGGTCTGGGCCTGAAGAAAGGCGACCGCGTGGCCCTGATGATG
CCGAACCTGCTGCAATATCCTGTGGCGCTGTTCGGTATCCTGCGTGCTGGTATGATCG
TTGTCAATGTTAACCCTCTGTATACCCCTCGTGAACTGGAGCACCAGCTGAATGACTCT
GGTGCGTCTGCTATCGTTATCGTTTCCAATTTCGCACATACGCTGGAGAAAGTGGTTGA
TAAAACCGCAGTGCAGCATGTCATTCTGACTCGCATGGGTGACCAGCTGTCCACCGCT
AAAGGTACTGTAGTCAACTTCGTTGTGAAATACATTAAGCGCCTGGTTCCGAAATACCA
CCTGCCAGATGCAATTAGCTTTCGCTCTGCACTGCATAACGGTTACCGTATGCAGTACG
TAAAACCAGAGCTGGTGCCGGAAGACCTGGCCTTTCTGCAGTATACCGGCGGCACCAC
CGGCGTGGCAAAGGGCGCGATGCTGACCCATCGTAACATGCTGGCGAACCTGGAGCA
GGTTAACGCAACGTACGGCCCGCTGCTGCACCCGGGTAAAGAACTGGTAGTTACGGCA
CTGCCTCTGTATCACATCTTTGCACTGACGATCAACTGTCTGCTGTTCATTGAACTGGG
TGGTCAGAACCTGCTGATCACCAACCCGCGTGACATTCCGGGCCTGGTAAAAGAGCTG
GCTAAGTACCCGTTCACCGCCATTACTGGCGTAAACACTCTGTTTAACGCGCTGCTGAA
CAACAAAGAGTTTCAGCAGCTGGACTTCTCTAGCCTGCACCTGAGCGCTGGCGGTGGC
ATGCCGGTTCAGCAGGTTGTGGCAGAGCGTTGGGTGAAACTGACCGGCCAGTATCTGC
TGGAGGGTTATGGTCTGACCGAGTGTGCACCGCTGGTCAGCGTTAACCCGTATGATAT
TGATTACCACTCTGGTTCTATTGGTCTGCCGGTTCCGTCCACGGAAGCCAAACTGGTG
GACGATGACGACAACGAAGTACCTCCGGGCCAGCCGGGTGAGCTGTGTGTCAAGGGT
CCGCAGGTTATGCTGGGCTACTGGCAGCGCCCGGACGCCACCGACGAAATCATTAAAA
ACGGTTGGCTGCATACCGGTGATATCGCTGTAATGGACGAAGAAGGTTTCCTGCGTAT
CGTGGACCGTAAGAAAGATATGATTCTGGTGAGCGGTTTCAACGTGTACCCGAACGAA
ATTGAGGACGTAGTTATGCAACACCCTGGCGTGCAGGAGGTGGCAGCCGTGGGCGTG
CCGTCCGGTTCTTCTGGTGAGGCTGTGAAAATCTTTGTCGTTAAAAAGGACCCGTCCCT
GACCGAAGAATCTCTGGTGACGTTTTGCCGCCGTCAACTGACTGGCTACAAAGTGCCG
AAACTGGTCGAGTTCCGCGATGAGCTGCCAAAATCTAACGTGGGTAAGATCCTGCGCC
GCGAGCTGCGTGACGAGGCACGTGGCAAAGTTGACAATAAAGCATAA
CCGCGTAGGAG
AGCAGCCGATGCACGTAGGTGGTCTGTTCCTGTTCCAGATCCCGGATAACGCTCCGGA
CACCTTTATTCAGGACCTGGTGAACGATATCCGTATCTCCAAGTCTATTCCGGTTCCGC
CGTTCAACAACAAGCTGAACGGTCTGTTCTGGGACGAAGACGAGGAGTTCGATCTGGA
TCACCATTTCCGTCATATTGCGCTGCCGCACCCGGGTCGCATCCGTGAGCTGCTGATTT
ACATCTCTCAGGAACACAGCACTCTCCTCGATCGCGCTAAACCTCTGTGGACTTGCAAC
ATCATTGAAGGTATCGAGGGTAACCGTTTCGCCATGTACTTCAAGATTCATCATGCGAT
GGTGGATGGTGTGGCGGGTATGCGTCTGATTGAGAAAAGCCTGTCCCATGATGTTACT
GAAAAGAGCATCGTACCGCCGTGGTGCGTTGAGGGCAAACGTGCTAAACGCCTGCGTG
AACCGAAGACCGGCAAAATTAAGAAAATCATGTCTGGTATTAAATCTCAGCTCCAGGC
CACCCCGACCGTTATTCAAGAACTGTCTCAGACGGTCTTCAAAGACATCGGCCGTAATC
CGGACCACGTTTCCTCTTTCCAGGCGCCGTGCTCCATCCTCAACCAGCGTGTGTCTTCT
TCTCGTCGTTTCGCAGCACAGAGCTTTGACCTGGACCGTTTCCGCAACATCGCCAAATC
TCTGAACGTGACCATTAACGACGTTGTCCTGGCTGTGTGTAGCGGTGCTCTGCGCGCT
TATCTGATGTCTCATAACTCTCTGCCATCCAAACCGCTGATCGCTATGGTCCCAGCAAG
CATCCGCAACGATGATTCTGATGTGTCCAACCGTATTACTATGATTCTGGCCAACCTCG
CTACTCACAAAGACGACCCTCTGCAGCGTCTGGAAATCATCCGCCGCTCCGTCCAGAA
CTCTAAACAGCGTTTTAAACGCATGACTTCCGACCAGATTCTGAACTATTCTGCGGTTG
TATACGGCCCGGCTGGTCTGAACATTATCAGCGGTATGATGCCGAAACGTCAGGCTTT
TAACCTGGTAATCAGCAACGTTCCTGGCCCGCGTGAGCCGCTGTACTGGAACGGCGCA
AAACTGGACGCACTGTACCCGGCTTCCATCGTTCTGGATGGCCAGGCTCTGAACATCA
CTATGACCTCTTACCTGGACAAACTGGAAGTAGGTCTGATCGCGTGTCGCAATGCACT
GCCGCGCATGCAGAACCTGCTGACCCACCTGGAGGAGGAAATCCAGCTGTTTGAGGGC
GTTATCGCCAAACAGGAAGATATCAAAACGGCGAACTAA
CCATGGTTGAATTCGGTTTTC
GCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGGGGTACAGTCTATGCCTCGGGCAT
CCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACG
CAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGTGATCGCCGAAGTATC
GACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTAC
ATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGG
TGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTT
CCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTC
CGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATA
GCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTG
AGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAAT
GTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGT
CGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGAC
AGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCC
ACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTAACAATTCGTTCAAGCCGAC
GCCGCTTCGCGGCGCGGCTTAACTCAAGCGTTAGATGCACTAAGCACATAATTGCTCACAGCCAAA
CTATCAGGTCAAGTCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATTGGGAGATA
TATCATGA
GGCGCGCCacgagtgcggggaaatttcgggggcgatcgcccctatatcgcaaaaaggagttaccccatcagagctatagtcg
AGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGG
GGTCATAGATCTGTAGTAGGATCCATGTAGGGTGAGGTTATAGCTATGAAGAAAGTTTGGC
TGAACCGTTATCCGGCAGATGTACCGACTGAAATTAACCCAGATCGTTACCAGTCCCTG
GTTGACATGTTCGAACAGTCCGTGGCTCGCTACGCCGATCAGCCTGCTTTCGTCAACAT
GGGTGAGGTAATGACCTTTCGCAAACTGGAGGAGCGTTCCCGTGCTTTCGCGGCATAC
CTGCAGCAGGGTCTGGGCCTGAAGAAAGGCGACCGCGTGGCCCTGATGATGCCGAAC
CTGCTGCAATATCCTGTGGCGCTGTTCGGTATCCTGCGTGCTGGTATGATCGTTGTCAA
TGTTAACCCTCTGTATACCCCTCGTGAACTGGAGCACCAGCTGAATGACTCTGGTGCGT
CTGCTATCGTTATCGTTTCCAATTTCGCACATACGCTGGAGAAAGTGGTTGATAAAACC
GCAGTGCAGCATGTCATTCTGACTCGCATGGGTGACCAGCTGTCCACCGCTAAAGGTA
CTGTAGTCAACTTCGTTGTGAAATACATTAAGCGCCTGGTTCCGAAATACCACCTGCCA
GATGCAATTAGCTTTCGCTCTGCACTGCATAACGGTTACCGTATGCAGTACGTAAAACC
AGAGCTGGTGCCGGAAGACCTGGCCTTTCTGCAGTATACCGGCGGCACCACCGGCGTG
GCAAAGGGCGCGATGCTGACCCATCGTAACATGCTGGCGAACCTGGAGCAGGTTAACG
CAACGTACGGCCCGCTGCTGCACCCGGGTAAAGAACTGGTAGTTACGGCACTGCCTCT
GTATCACATCTTTGCACTGACGATCAACTGTCTGCTGTTCATTGAACTGGGTGGTCAGA
ACCTGCTGATCACCAACCCGCGTGACATTCCGGGCCTGGTAAAAGAGCTGGCTAAGTA
CCCGTTCACCGCCATTACTGGCGTAAACACTCTGTTTAACGCGCTGCTGAACAACAAAG
AGTTTCAGCAGCTGGACTTCTCTAGCCTGCACCTGAGCGCTGGCGGTGGCATGCCGGT
TCAGCAGGTTGTGGCAGAGCGTTGGGTGAAACTGACCGGCCAGTATCTGCTGGAGGGT
TATGGTCTGACCGAGTGTGCACCGCTGGTCAGCGTTAACCCGTATGATATTGATTACCA
CTCTGGTTCTATTGGTCTGCCGGTTCCGTCCACGGAAGCCAAACTGGTGGACGATGAC
GACAACGAAGTACCTCCGGGCCAGCCGGGTGAGCTGTGTGTCAAGGGTCCGCAGGTTA
TGCTGGGCTACTGGCAGCGCCCGGACGCCACCGACGAAATCATTAAAAACGGTTGGCT
GCATACCGGTGATATCGCTGTAATGGACGAAGAAGGTTTCCTGCGTATCGTGGACCGT
AAGAAAGATATGATTCTGGTGAGCGGTTTCAACGTGTACCCGAACGAAATTGAGGACG
TAGTTATGCAACACCCTGGCGTGCAGGAGGTGGCAGCCGTGGGCGTGCCGTCCGGTTC
TTCTGGTGAGGCTGTGAAAATCTTTGTCGTTAAAAAGGACCCGTCCCTGACCGAAGAA
TCTCTGGTGACGTTTTGCCGCCGTCAACTGACTGGCTACAAAGTGCCGAAACTGGTCG
AGTTCCGCGATGAGCTGCCAAAATCTAACGTGGGTAAGATCCTGCGCCGCGAGCTGCG
TGACGAGGCACGTGGCAAAGTTGACAATAAAGCATAA
CTCGACGCGTAGGAGGACAGCT
ATGCGCCCACTTCATCCGATCGATTTCATTTTCCTGTCCCTGGAGAAACGCCAGCAGCC
GATGCACGTAGGTGGTCTGTTCCTGTTCCAGATCCCGGATAACGCTCCGGACACCTTT
ATTCAGGACCTGGTGAACGATATCCGTATCTCCAAGTCTATTCCGGTTCCGCCGTTCAA
CAACAAGCTGAACGGTCTGTTCTGGGACGAAGACGAGGAGTTCGATCTGGATCACCAT
TTCCGTCATATTGCGCTGCCGCACCCGGGTCGCATCCGTGAGCTGCTGATTTACATCTC
TCAGGAACACAGCACTCTCCTCGATCGCGCTAAACCTCTGTGGACTTGCAACATCATTG
AAGGTATCGAGGGTAACCGTTTCGCCATGTACTTCAAGATTCATCATGCGATGGTGGA
TGGTGTGGCGGGTATGCGTCTGATTGAGAAAAGCCTGTCCCATGATGTTACTGAAAAG
AGCATCGTACCGCCGTGGTGCGTTGAGGGCAAACGTGCTAAACGCCTGCGTGAACCGA
AGACCGGCAAAATTAAGAAAATCATGTCTGGTATTAAATCTCAGCTCCAGGCCACCCC
GACCGTTATTCAAGAACTGTCTCAGACGGTCTTCAAAGACATCGGCCGTAATCCGGAC
CACGTTTCCTCTTTCCAGGCGCCGTGCTCCATCCTCAACCAGCGTGTGTCTTCTTCTCG
TCGTTTCGCAGCACAGAGCTTTGACCTGGACCGTTTCCGCAACATCGCCAAATCTCTGA
ACGTGACCATTAACGACGTTGTCCTGGCTGTGTGTAGCGGTGCTCTGCGCGCTTATCT
GATGTCTCATAACTCTCTGCCATCCAAACCGCTGATCGCTATGGTCCCAGCAAGCATCC
GCAACGATGATTCTGATGTGTCCAACCGTATTACTATGATTCTGGCCAACCTCGCTACT
CACAAAGACGACCCTCTGCAGCGTCTGGAAATCATCCGCCGCTCCGTCCAGAACTCTA
AACAGCGTTTTAAACGCATGACTTCCGACCAGATTCTGAACTATTCTGCGGTTGTATAC
GGCCCGGCTGGTCTGAACATTATCAGCGGTATGATGCCGAAACGTCAGGCTTTTAACC
TGGTAATCAGCAACGTTCCTGGCCCGCGTGAGCCGCTGTACTGGAACGGCGCAAAACT
GGACGCACTGTACCCGGCTTCCATCGTTCTGGATGGCCAGGCTCTGAACATCACTATG
ACCTCTTACCTGGACAAACTGGAAGTAGGTCTGATCGCGTGTCGCAATGCACTGCCGC
GCATGCAGAACCTGCTGACCCACCTGGAGGAGGAAATCCAGCTGTTTGAGGGCGTTAT
CGCCAAACAGGAAGATATCAAAACGGCGAACTAA
CCATGGTTGAATTCGGTTTTCCGTCC
TGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCA
GCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGG
GCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAAC
TATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACG
GCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTA
AGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGA
GAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGT
TATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTC
GAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCC
TTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTA
AATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCT
TACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCG
ACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTAT
CTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTG
AAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTAACAATTCGTTCAAGCCGACGCCGCTTC
GCGGCGCGGCTTAACTCAAGCGTTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGG
TCAAGTCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATTGGGAGATATATCATGA
AGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGG
GGTCATATGGCGGATACTCTGCTGATTCTGGGTGATTCTCTGTCTGCAGGCTACCGTAT
GTCCGCCTCCGCGGCCTGGCCAGCTCTGCTGAATGATAAGTGGCAGTCTAAGACGTCC
GTTGTGAACGCATCCATCTCTGGCGACACGAGCCAGCAGGGCCTGGCCCGTCTGCCTG
CACTGCTGAAACAGCACCAACCGCGCTGGGTCCTGGTGGAGCTGGGCGGTAACGACG
GTCTGCGCGGCTTCCAGCCGCAGCAGACCGAACAGACTCTGCGTCAGATTCTGCAGGA
CGTGAAAGCTGCTAACGCGGAACCGCTGCTGATGCAGATTCGTCTGCCAGCGAACTAT
GGCCGCCGTTACAACGAAGCGTTCTCTGCAATCTACCCAAAACTGGCGAAAGAGTTTG
ACGTCCCGCTGCTGCCGTTCTTCATGGAGGAAGTATACCTGAAACCGCAGTGGATGCA
AGATGACGGCATCCACCCGAACCGTGATGCGCAGCCGTTCATCGCTGACTGGATGGCG
AAGCAACTGCAGCCGCTGGTAAACCACGATTCCTAA
TTAAAGATCTGTAGTAGGATCCAT
CGACTGAAATTAACCCAGATCGTTACCAGTCCCTGGTTGACATGTTCGAACAGTCCGTG
GCTCGCTACGCCGATCAGCCTGCTTTCGTCAACATGGGTGAGGTAATGACCTTTCGCA
AACTGGAGGAGCGTTCCCGTGCTTTCGCGGCATACCTGCAGCAGGGTCTGGGCCTGAA
GAAAGGCGACCGCGTGGCCCTGATGATGCCGAACCTGCTGCAATATCCTGTGGCGCTG
TTCGGTATCCTGCGTGCTGGTATGATCGTTGTCAATGTTAACCCTCTGTATACCCCTCG
TGAACTGGAGCACCAGCTGAATGACTCTGGTGCGTCTGCTATCGTTATCGTTTCCAATT
TCGCACATACGCTGGAGAAAGTGGTTGATAAAACCGCAGTGCAGCATGTCATTCTGAC
TCGCATGGGTGACCAGCTGTCCACCGCTAAAGGTACTGTAGTCAACTTCGTTGTGAAA
TACATTAAGCGCCTGGTTCCGAAATACCACCTGCCAGATGCAATTAGCTTTCGCTCTGC
ACTGCATAACGGTTACCGTATGCAGTACGTAAAACCAGAGCTGGTGCCGGAAGACCTG
GCCTTTCTGCAGTATACCGGCGGCACCACCGGCGTGGCAAAGGGCGCGATGCTGACCC
ATCGTAACATGCTGGCGAACCTGGAGCAGGTTAACGCAACGTACGGCCCGCTGCTGCA
CCCGGGTAAAGAACTGGTAGTTACGGCACTGCCTCTGTATCACATCTTTGCACTGACG
ATCAACTGTCTGCTGTTCATTGAACTGGGTGGTCAGAACCTGCTGATCACCAACCCGC
GTGACATTCCGGGCCTGGTAAAAGAGCTGGCTAAGTACCCGTTCACCGCCATTACTGG
CGTAAACACTCTGTTTAACGCGCTGCTGAACAACAAAGAGTTTCAGCAGCTGGACTTCT
CTAGCCTGCACCTGAGCGCTGGCGGTGGCATGCCGGTTCAGCAGGTTGTGGCAGAGC
GTTGGGTGAAACTGACCGGCCAGTATCTGCTGGAGGGTTATGGTCTGACCGAGTGTGC
ACCGCTGGTCAGCGTTAACCCGTATGATATTGATTACCACTCTGGTTCTATTGGTCTGC
CGGTTCCGTCCACGGAAGCCAAACTGGTGGACGATGACGACAACGAAGTACCTCCGGG
CCAGCCGGGTGAGCTGTGTGTCAAGGGTCCGCAGGTTATGCTGGGCTACTGGCAGCGC
CCGGACGCCACCGACGAAATCATTAAAAACGGTTGGCTGCATACCGGTGATATCGCTG
TAATGGACGAAGAAGGTTTCCTGCGTATCGTGGACCGTAAGAAAGATATGATTCTGGT
GAGCGGTTTCAACGTGTACCCGAACGAAATTGAGGACGTAGTTATGCAACACCCTGGC
GTGCAGGAGGTGGCAGCCGTGGGCGTGCCGTCCGGTTCTTCTGGTGAGGCTGTGAAA
ATCTTTGTCGTTAAAAAGGACCCGTCCCTGACCGAAGAATCTCTGGTGACGTTTTGCCG
CCGTCAACTGACTGGCTACAAAGTGCCGAAACTGGTCGAGTTCCGCGATGAGCTGCCA
AAATCTAACGTGGGTAAGATCCTGCGCCGCGAGCTGCGTGACGAGGCACGTGGCAAAG
TTGACAATAAAGCATAA
CAATTCGGTTTTCCGTCCTGTCTTGATTTTCAAGCAAACAATGCC
AGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTG
TTTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGAT
GTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACA
TCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAA
GCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGC
TTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGA
AGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATT
TGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCT
GGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAAC
TCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTC
GCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCG
CAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGC
CCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGC
CTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCG
GCAAATAATGTCTAACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCGTTA
GATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAGTCTGCTTTTATTATTTTTAAGC
GTGCATAATAAGCCCTACACAAATTGGGAGATATATCATGA
GGCGCGCCacgagtgcggggaaatttcgggggc
TCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTAC
ATAAACAGTAATACAAGGGGTCATATGTAACAGGAATTCGGTTTTCCGTCCTGTCTTGATT
TTCAAGCAAACAATGCCTCCGATTTCTAATCGGAGGCATTTGTTTTTGTTTATTGCAAA
AACAAAAAATATTGTTACAAATTTTTACAGGCTATTAAGCCTACCGTCATAAATAATTT
GCCATTTACTAGTTTTTAATTAAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCC
GCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGATTGAACAA
GATGGCCTGCATGCTGGTTCTCCGGCTGCTTGGGTGGAACGCCTGTTTGGTTACGACTGGGCTCA
GCTGACTATTGGCTGTAGCGATGCAGCGGTTTTCCGTCTGTCTGCACAGGGTCGTCCGGTTCTGTT
TGTGAAAACCGACCTGTCCGGCGCACTGAACGAACTGCAGGACGAAGCGGCCCGTCTGTCCTGG
CTCGCGACGACTGGTGTTCCGTGCGCGGCAGTTCTGGACGTAGTTACTGAAGCCGGTCGCGATTG
GCTGCTGCTGGGTGAAGTTCCGGGTCAGGATCTGCTGAGCAGCCACCTCGCTCCGGCAGAAAAAG
TTTCCATCATGGCGGACGCGATGCGCCGTCTGCACACCCTGGACCCGGCAACTTGCCCGTTTGAC
CATCAGGCTAAACACCGTATTGAACGTGCACGCACTCGTATGGAAGCGGGTCTGGTTGATCAGGA
CGACCTGGATGAAGAGCACCAGGGCCTCGCACCGGCGGAACTGTTTGCACGTCTGAAAGCCCGC
ATGCCGGACGGCGAAGACCTGGTGGTAACGCATGGCGACGCTTGTCTGCCAAACATTATGGTGGA
AAACGGCCGCTTCTCTGGTTTTATTGACTGTGGCCGTCTGGGTGTAGCTGATCGCTATCAGGATAT
CGCCCTCGCTACCCGCGATATTGCAGAAGAACTGGGTGGTGAATGGGCTGACCGTTTCCTGGTGC
TGTACGGTATCGCAGCGCCGGATTCTCAGCGCATTGCCTTCTACCGTCTGCTGGATGAGTTCTTCT
AA
GGCGCGCCgaaactgcgccaagaatagctcacttcaaatcagtcacggttttgtttagggcttgtctggcgattttggtgacatagacagtcaca
CCCCCACAATCATGTCAAGAATTCAAGCATCTTAAATAATGTTAATTATCGGCAAAGTCTGT
GCTCCCCTTCTATAATGCTGAATTGAGCATTCGCCTCCTGAACGGTCTTTATTCTTCCATTGT
GGGTCTTTAGATTCACGATTCTTCACAATCATTGATCTAAAGATCTTTCTAGATTCTCGAGGC
gcacgccttagtaacccggaattgcgtaagtctgccgccgatcgtgatgctgcctttgaaaaaattaatgaagcgcgcagtccattactgccaca
gctaggtttaggtgcagattacacctatagcaacggctaccgcgacgcgaacggcatcaactctaacgcgaccagtgcgtccttgcagttaact
caatccatttttgatatgtcgaaatggcgtgcgttaacgctgcaggaaaaagcagcagggattcaggacgtcacgtatcagaccgatcagcaaa
ccttgatcctcaacaccgcgaccgcttatttcaacgtgttgaatgctattgacgttctttcctatacacaggcacaaaaagaagcgatctaccgtc
aattagatcaaaccacccaacgttttaacgtgggcctggtagcgatcaccgacgtgcagaacgcccgcgcacagtacgataccgtgctggcga
acgaagtgaccgcacgtaataaccttgataacgcggtagagcagctgcgccagatcaccggtaactactatccggaactggctgcgctgaatg
tcgaaaactttaaaaccgacaaaccacagccggttaacgcgctgctgaaagaagccgaaaaacgcaacctgtcgctgttacaggcacgcttga
gccaggacctggcgcgcgagcaaattcgccaggcgcaggatggtcacttaccgactctggatttaacggcttctaccgggatttctgacacctct
tatagcggttcgaaaacccgtggtgccgctggtacccagtatgacgatagcaatatgggccagaacaaagttggcctgagcttctcgctgccga
tttatcagggcggaatggttaactcgcaggtgaaacaggcacagtacaactttgtcggtgccagcgagcaactggaaagtgcccatcgtagcgt
cgtgcagaccgtgcgttcctccttcaacaacattaatgcatctatcagtagcattaacgcctacaaacaagccgtagtttccgctcaaagctcatt
agacgcgatggaagcgggctactcggtcggtacgcgtaccattgttgatgtgttggatgcgaccaccacgttgtacaacgccaagcaagagctg
gcgaatgcgcgttataactacctgattaatcagctgaatattaagtcagctctgggtacgttgaacgagcaggatctgctggcactgaacaatgc
gctgagcaaaccggtttccactaatccggaaaacgttgcaccgcaaacgccggaacagaatgctattgctgatggttatgcgcctgatagcccg
gcaccagtcgttcagcaaacatccgcacgcactaccaccagtaacggtcataaccctttccgtaactga
GGATCCAAGGTGGCTA
CTTCAACGATAGCTTAAACTTCGCTGCTCCAGCGAGGGGATTTCACTGGTTTGAATGCTTCA
ATGCTTGCCAAAAGAGTGCTACTGGAACTTACAAGAGTGACCCTGCGTCAGGGGAGCTAGC
ACTCAAAAAAGACTCCTCCAATTCCGTCCatgaacaaaaacagagggtttacgcctctggcggtcgttctgatgctctca
ggcagcttagccctaacaggatgtgacgacaaacaggcccaacaaggtggccagcagatgcccgccgttggcgtagtaacagtcaaaactga
acctctgcagatcacaaccgagcttccgggtcgcaccagtgcctaccggatcgcagaagttcgtcctcaagttagcgggattatcctgaagcgta
atttcaaagaaggtagcgacatcgaagcaggtgtctctctctatcagattgatcctgcgacctatcaggcgacatacgacagtgcgaaaggtga
tctggcgaaagcccaggctgcagccaatatcgcgcaattgacggtgaatcgttatcagaaactgctcggtactcagtacatcagtaagcaagag
tacgatcaggctctggctgatgcgcaacaggcgaatgctgcggtaactgcggcgaaagctgccgttgaaactgcgcggatcaatctggcttaca
ccaaagtcacctctccgattagcggtcgcattggtaagtcgaacgtgacggaaggcgcattggtacagaacggtcaggcgactgcgctggcaa
ccgtgcagcaacttgatccgatctacgttgatgtgacccagtccagcaacgacttcctgcgcctgaaacaggaactggcgaatggcacgctgaa
acaagagaacggcaaagccaaagtgtcactgatcaccagtgacggcattaagttcccgcaggacggtacgctggaattctctgacgttaccgtt
gatcagaccactgggtctatcaccctacgcgctatcttcccgaacccggatcacactctgctgccgggtatgttcgtgcgcgcacgtctggaaga
agggcttaatccaaacgctattttagtcccgcaacagggcgtaacccgtacgccgcgtggcgatgccaccgtactggtagttggcgcggatgac
aaagtggaaacccgtccgatcgttgcaagccaggctattggcgataagtggctggtgacagaaggtctgaaagcaggcgatcgcgtagtaata
agtgggctgcagaaagtgcgtcctggtgtccaggtaaaagcacaagaagttaccgctgataataaccagcaagccgcaagcggtgctcagcct
gaacagtccaagtcttaa
cttaaacaggagccgttaagacatgcctaatttctttatcgatcgcccgatttttgcgtgggtgatcgccattatcatcat
gttggcaggggggctggcgatcctcaaactgccggtggcgcaatatcctacgattgcaccgccggcagtaacgatctccgcctcctaccccggc
gctgatgcgaaaacagtgcaggacacggtgacacaggttatcgaacagaatatgaacggtatcgataacctgatgtacatgtcctctaacagt
gactccacgggtaccgtgcagatcaccctgacctttgagtctggtactgatgcggatatcgcgcaggttcaggtgcagaacaaactgcagctgg
cgatgccgttgctgccgcaagaagttcagcagcaaggggtgagcgttgagaaatcatccagcagcttcctgatggttgtcggcgttatcaacac
cgatggcaccatgacgcaggaggatatctccgactacgtggcggcgaatatgaaagatgccatcagccgtacgtcgggcgtgggtgatgttca
gttgttcggttcacagtacgcgatgcgtatctggatgaacccgaatgagctgaacaaattccagctaacgccggttgatgtcattaccgccatca
aagcgcagaacgcccaggttgcggcgggtcagctcggtggtacgccgccggtgaaaggccaacagcttaacgcctctattattgctcagacgc
gtctgacctctactgaagagttcggcaaaatcctgctgaaagtgaatcaggatggttcccgcgtgctgctgcgtgacgtcgcgaagattgagctg
ggtggtgagaactacgacatcatcgcagagtttaacggccaaccggcttccggtctggggatcaagctggcgaccggtgcaaacgcgctggat
accgctgcggcaatccgtgctgaactggcgaagatggaaccgttcttcccgtcgggtctgaaaattgtttacccatacgacaccacgccgttcgt
gaaaatctctattcacgaagtggttaaaacgctggtcgaagcgatcatcctcgtgttcctggttatgtatctgttcctgcagaacttccgcgcgacg
ttgattccgaccattgccgtaccggtggtattgctcgggacctttgccgtccttgccgcctttggcttctcgataaacacgctaacaatgttcgggat
ggtgctcgccatcggcctgttggtggatgacgccatcgttgtggtagaaaacgttgagcgtgttatggcggaagaaggtttgccgccaaaagaa
gctacccgtaagtcgatggggcagattcagggcgctctggtcggtatcgcgatggtactgtcggcggtattcgtaccgatggccttctttggcggt
tctactggtgctatctatcgtcagttctctattaccattgtttcagcaatggcgctgtcggtactggtggcgttgatcctgactccagctctttgtgcca
ccatgctgaaaccgattgccaaaggcgatcacggggaaggtaaaaaaggcttcttcggctggtttaaccgcatgttcgagaagagcacgcacc
actacaccgacagcgtaggcggtattctgcgcagtacggggcgttacctggtgctgtatctgatcatcgtggtcggcatggcctatctgttcgtgc
gtctgccaagctccttcttgccagatgaggaccagggcgtgtttatgaccatggttcagctgccagcaggtgcaacgcaggaacgtacacagaa
agtgctcaatgaggtaacgcattactatctgaccaaagaaaagaacaacgttgagtcggtgttcgccgttaacggcttcggctttgcgggacgtg
gtcagaataccggtattgcgttcgtttccttgaaggactgggccgatcgtccgggcgaagaaaacaaagttgaagcgattaccatgcgtgcaac
acgcgctttctcgcaaatcaaagatgcgatggttttcgcctttaacctgcccgcaatcgtggaactgggtactgcaaccggctttgactttgagctg
attgaccaggctggccttggtcacgaaaaactgactcaggcgcgtaaccagttgcttgcagaagcagcgaagcaccctgatatgttgaccagc
gtacgtccaaacggtctggaagataccccgcagtttaagattgatatcgaccaggaaaaagcgcaggcgctgggtgtttctatcaacgacatta
acaccactctgggcgctgcatggggcggcagctatgtgaacgactttatcgaccgcggtcgtgtgaagaaagtttatgtcatgtcagaagcgaa
ataccgtatgctgccggatgatatcggcgactggtatgttcgtgctgctgatggtcagatggtgccattctcggcgttctcctcttctcgttgggagt
acggttcgccgcgtctggaacgttacaacggcctgccatccatggaaatcttaggccaggcggcaccgggtaaaagtaccggtgaagcaatgg
agctgatggaacaactggcgagcaaactgcctaccggtgttggctatgactggacggggatgtcctatcaggaacgtctctccggcaaccagg
caccttcactgtacgcgatttcgttgattgtcgtgttcctgtgtctggcggcgctgtacgagagctggtcgattccgttctccgttatgctggtcgttc
cgctgggggttatcggtgcgttgctggctgccaccttccgtggcctgaccaatgacgtttacttccaggtaggcctgctcacaaccattgggttgtc
ggcgaagaacgcgatccttatcgtcgaattcgccaaagacttgatggataaagaaggtaaaggtctgattgaagcgacgcttgatgcggtgcg
gatgcgtttacgtccgatcctgatgacctcgctggcgtttatcctcggcgttatgccgctggttatcagtactggtgctggttccggcgcgcagaac
gcagtaggtaccggtgtaatgggcgggatggtgaccgcaacggtactggcaatcttcttcgttccggtattctttgtggtggttcgccgccgcttta
gccgcaagaatgaagatatcgagcacagccatactgtcgatcatcattga
GAGCTCttGAATTCGGTTTTCCGTCCTGTCT
CATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGATTGAACAAGAT
GGCCTGCATGCTGGTTCTCCGGCTGCTTGGGTGGAACGCCTGTTTGGTTACGACTGGGCTCAGCT
GACTATTGGCTGTAGCGATGCAGCGGTTTTCCGTCTGTCTGCACAGGGTCGTCCGGTTCTGTTTGT
GAAAACCGACCTGTCCGGCGCACTGAACGAACTGCAGGACGAAGCGGCCCGTCTGTCCTGGCTC
GCGACGACTGGTGTTCCGTGCGCGGCAGTTCTGGACGTAGTTACTGAAGCCGGTCGCGATTGGCT
GCTGCTGGGTGAAGTTCCGGGTCAGGATCTGCTGAGCAGCCACCTCGCTCCGGCAGAAAAAGTTT
CCATCATGGCGGACGCGATGCGCCGTCTGCACACCCTGGACCCGGCAACTTGCCCGTTTGACCAT
CAGGCTAAACACCGTATTGAACGTGCACGCACTCGTATGGAAGCGGGTCTGGTTGATCAGGACGA
CCTGGATGAAGAGCACCAGGGCCTCGCACCGGCGGAACTGTTTGCACGTCTGAAAGCCCGCATG
CCGGACGGCGAAGACCTGGTGGTAACGCATGGCGACGCTTGTCTGCCAAACATTATGGTGGAAAA
CGGCCGCTTCTCTGGTTTTATTGACTGTGGCCGTCTGGGTGTAGCTGATCGCTATCAGGATATCGC
CCTCGCTACCCGCGATATTGCAGAAGAACTGGGTGGTGAATGGGCTGACCGTTTCCTGGTGCTGT
ACGGTATCGCAGCGCCGGATTCTCAGCGCATTGCCTTCTACCGTCTGCTGGATGAGTTCTTCTAA
G
Synechococcus sp. PCC 7002 rbcL RBS
This application is a divisional of U.S. patent application Ser. No. 13/765,211, filed Feb. 12, 2013, which is a divisional of U.S. patent application Ser. No. 13/243,165, filed Sep. 23, 2011, which is a continuation of U.S. patent application Ser. No. 12/876,056, filed Sep. 3, 2010, which is a continuation-in-part of international application PCT/US/2009/035937, filed Mar. 3, 2009, which claims the benefit of earlier filed U.S. Provisional Patent Application No. 61/121,532, filed Dec. 10, 2008, U.S. Provisional Patent Application No. 61/033,411 filed Mar. 3, 2008, and U.S. Provisional Application No. 61/033,402, filed Mar. 3, 2008; this application also claims priority to U.S. Provisional Application 61/353,145, filed Jun. 9, 2010. The disclosures of each of these applications are incorporated hereinby reference, in their entirety, for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 61121532 | Dec 2008 | US | |
| 61033411 | Mar 2008 | US | |
| 61033402 | Mar 2008 | US | |
| 61353145 | Jun 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13765211 | Feb 2013 | US |
| Child | 14559168 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13243165 | Sep 2011 | US |
| Child | 13765211 | US | |
| Parent | 12876056 | Sep 2010 | US |
| Child | 13243165 | US | |
| Parent | PCT/US2009/035937 | Mar 2009 | US |
| Child | 12876056 | US |
