WO2012135766A1 - Procédé pour retirer des marqueurs génétiques - Google Patents

Procédé pour retirer des marqueurs génétiques Download PDF

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WO2012135766A1
WO2012135766A1 PCT/US2012/031696 US2012031696W WO2012135766A1 WO 2012135766 A1 WO2012135766 A1 WO 2012135766A1 US 2012031696 W US2012031696 W US 2012031696W WO 2012135766 A1 WO2012135766 A1 WO 2012135766A1
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host cell
protein
recombinase
recombinase protein
gene
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PCT/US2012/031696
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English (en)
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Yu Xu
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Joule Unlimited Technologies, Inc.
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Publication of WO2012135766A1 publication Critical patent/WO2012135766A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays

Definitions

  • the disclosure relates to methods for removing genetic markers from photosynthetic microorganisms .
  • Genetic marker genes such as antibiotic resistance genes transformed into microorganisms can also form the basis for concerns over lateral transfer of the marker gene to other wild-type organisms.
  • Engineered bacteria having an antibiotic resistance gene that are released into natural habitats could conceivably transfer the antibiotic resistance gene to other wild-type bacteria (although there is no known occurrence for this happening in cyanobacteria, this does happen frequently in pathogens, for example Enterococcus, Bacillus, etc.). Conferring such antibiotic resitance to wild type populations could, in turn, result in the transfer of the marker to other wild type bacteria.
  • the present disclosure embodies a method for screening a host cell comprising culturing an engineered unicellular non-diazotrophic cyanobacterial host cell comprising a gene encoding a selectable genetic marker, the genetic marker further comprising flanking nucleotide sequences, and contacting the host cell with an exogenous nucleic acid sequence encoding a recombinase protein, wherein the recombinase protein excises the selectable genetic marker.
  • the method further comprises isolating the host cell.
  • the host cell is light dependent or fixes carbon.
  • the host cell comprises a recombinant nucleic acid sequence encoding a carbon-based product.
  • the host cell releases, permeates, or exports the carbon-based product of interest.
  • the carbon-based product is selected from alkanes, alkenes, aliphatic and aromatic alkane and alkene mixtures, alcohols, alkanals and alkenols, alkanoic and alkenoic acids, hydroxy alkanoic acids, keto acids, alkyl alkanoates, ethers, amino acids, lactams, organic polymers, isoprenoids and pharmaceuticals/multifunctional group molecule.
  • the host cell is thermophilic, halophilic or both.
  • the host cell is selected from Chamaesiphon sp., Chlorococcus sp., Gloeothece sp., Gloeobacter sp., Prochlorococcus sp., Acaryochloris sp., Xenococcus sp., Dactylococcopsis sp.,
  • the selectable genetic marker can be an antibiotic resistance gene. In another embodiment, the selectable genetic marker is an auxotrophic selectable marker.
  • the exogenous nucleic acid encoding a recombinase protein is of EC 2.7.7.-.
  • the reocmbinase protein is selected from the tyrosine recombinase family.
  • the recombinase protein is a site-specific recombinase protein.
  • the recombinase protein is selected from coliphage HK022 Int and coliphage ⁇ Int.
  • the recombinase protein in the host cell does not require exogenous co-factors.
  • flanking nucleotide sequences comprise attR and attL. In a further embodiment, the flanking nucleotide sequences are altered to confer a decreased binding efficiency for the recombinase protein. In another embodiment, the nucleotide sequence expressing the recombinase protein comprises SEQ ID NO:3.
  • the selectable genetic marker and exogenous nucleic acid sequence are segregated from the host cell in less than about 2, less than about 3, less than about 4, less than about 5, less than about 6, less than about 7, less than about 8, less than about 9 or less than about 10 sequential platings of said host cell.
  • a unicellular, non- diazotrophic cyanobateria host cell comprising a nucleotide sequence encoding an exogenous recombinase protein having activity of EC 2.7.7.-.
  • the expression of the exogenous recombinase protein is operably linked to an inducible promoter.
  • the recombinase protein is selected from the Tyrosine recombinase family. In one aspect, the recombinase protein is a site-specific recombinase protein. In another aspect, the recombinase protein is selected from coliphage HK022 Int and coliphage ⁇ Int. In still another aspect, the recombinase protein does not require exogenous co-factors.
  • the host cell is selected from Chamaesiphon sp., Chlorococcus sp., Gloeothece sp., Gloeobacter sp., Prochlorococcus sp., Acaryochloris sp., Xenococcus sp., Dactylococcopsis sp., Prochloron sp., Chroogloeocystis sp., Coelosphaerium sp., Cyanodictyon sp., Geminocystis sp., Johannesbaptistia sp., Limnococcus sp., Radiocystis sp., Rhabdoderma sp., Rubidibacter sp., Snowella sp., Sphaerocavum sp., Synechococcus sp., Synechocystis spp., Cyanobacterium s
  • the nucleic acid encoding a recombinase protein comprises SEQ ID NO:3.
  • the inducible promoter is a nickel-inducible promoter nrS B, a copper-inducible promoter pe tE, a nitrogen regulated promoter nir , an IPTG inducible promoter, a promoter of genes involved in iron chelating and uptake, a promoter of genes involved in dark anaerobic fermentation, or a promoter of genes involved in C0 2 - concentrating and C0 2 -uptake mechanisms.
  • an improved recombinase comprising altered substrate affinity, improved thermostability, enzyme activity, improved stability in halophilic conditions, improved stability at varying pH, and optimized codon usage for improved expression in a host cell.
  • a method for deleting a gene comprising: adding an integrative vector comprising a selection marker and flanking regions comprising regions homologous to said gene into a culture of host cells comprising said gene; selecting for host cells expressing said integrative vector; inserting an integrase into said host cells to remove said selection marker from said integrative vector; and selecting for host cells no longer expressing said selection marker.
  • said deleted gene is the nifJ gene.
  • the deleted gene is the IdhA gene.
  • FIG. 1 depicts the results of a DNA gel for a PCR analysis of colonies transformed with pEx_P cpcB _int_aadA and grown in the presence of spectinomycin.
  • FIG. 2 depicts the results of a DNA gel for a PCR analysis performed on transformant colonies #4 and #9 grown on solid A + medium without any antibiotics.
  • FIG. 3 depicts the results of a SDS-PAGE gel for a Western Blot immunological analyses showing expression of recombinase Int in transformed host cells.
  • accession numbers throughout this description are derived from the NCBI database (National Center for Biotechnology Information) maintained by the National Institute of Health, U.S.A. The accession numbers are as provided in the database on November 1 st , 2010.
  • Amino acid Triplets of nucleotides, referred to as codons, in DNA molecules which code for amino acid in a peptide.
  • codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
  • 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 ⁇ -, a- disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present disclosure.
  • unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamate, C- ⁇ , ⁇ , ⁇ -trimethyllysine, C -Nacetyllysine, O-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5 -hydroxy lysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • 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.
  • 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.
  • fragments include 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).
  • antibodies can be produced by any known technique, including, but not limited to, harvest from cell culture of native B lymphocytes, harvest from culture of hybridomas, recombinant expression systems and phage display.
  • Attenuate 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.
  • Attenuation as applied to a nucleotide sequence encoding a gene or gene expression control sequence also refers to attenuation of the protein, and attenuation of a protein also refers to attenuation of the corresponding gene encoding the protein and/or the gene expression control sequence.
  • the sensitivity of a particular enzyme to feedback inhibition or inhibition caused by a composition that is not a product or a reactant is lessened such that the enzyme activity is not impacted by the presence of a compound.
  • an enzyme that has been altered to be less active can be referred to as attenuated.
  • Auxotrophs refers to organisms that do not have the ability to synthesize one or more particular compounds that are required for growth, and/or metabolic sustainability sufficient for the organism to maintain a living state or otherwise maintain viability, and is otherwise unable to synthesize or provide to itself intra-cellularly because of natural or genetic engineering means.
  • Biofuel A biofuel refers to any fuel that is derived from a biological source. Biofuel refers to one or more hydrocarbons, one or more alcohols, one or more fatty esters or a mixture thereof.
  • Carbon-based product of interest refers to, without limitation or implication that the scope of the claims are limited to the examples set forth herein, desirable end-products or metabolites produced by a biosynthetic pathway of an isolated host cell.
  • the end products or metabolites include, but are not limited to, alkanes (propane, octane), alkenes (ethylene, 1,3-butadiene, propylene, olefins, alkenes, isoprene, lycopene, terpenes) aliphatic and aromatic alkane and alkene mixtures (diesel, jet propellant 8 (JP8)), alkanols and alkenols (ethanol, propanol,
  • alkanoic and alkenoic acids acrylate, acrylic acid, adipic acid, itaconic acid, itaconate, docosahexaenoic acid, (DHA), omega-3 DHA, malonic acid, succinate, omega fatty acids
  • hydroxy alkanoic acids citrate, citric acid, malate, lactate, lactic acid, 3- hydroxypropionate, 3-hydroxypropionic acid (HP A), hydroxybutyrate), keto acid (levulinic acid, pyruvi acid), alkyl alkanoates (fatty acid esters, wax esters, ⁇ -caprolactone, gamma butyrolactone, ⁇ -valerolactone), ethers (THF), amino acids (glutamate, lysine, serine
  • Degenerate variant A degenerate variant of a referenced nucleic acid sequence, as used herein, 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.
  • degenerate oligonucleotide or “degenerate primer” is used to signify an oligonucleotide capable of hybridizing with target nucleotide sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.
  • Deletion The removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, where 3 ' and 5 ' ends of the nucleotide sequence may be removed, or the carboxy (C) and amino (N) terminal ends of the protein sequence removed and the nucleotide ends and/or amino/carboxy ends are subsequently re-ligated.
  • a deletion can also refer to the removal of an N- or C- terminal segment, or a 3' or 5' terminal end of a nucleotide sequence, wherein the translated or transcribed products are shorter in sequence length than the starting sequence.
  • Detectable Capable of having an existence or presence ascertained using various analytical methods as described throughout the description or otherwise known to a person skilled in the art.
  • DNA Deoxyribonucleic acid.
  • DNA is a long chain polymer which includes the genetic material of most living organisms (some viruses have genes including ribonucleic acid, RNA).
  • the repeating units in DNA polymers are four different nucleotides, each of which includes one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.
  • Domain refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be coextensive 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.
  • Down-regulation refers to when a gene is caused to be transcribed at a reduced rate compared to the endogenous gene transcription rate for that gene.
  • down-regulation additionally includes a reduced level of translation of the gene compared to the endogenous translation rate for that gene.
  • Methods of testing for down-regulation are well known to those in the art. For example, the transcribed RNA levels can be assessed using RT-PCR, and protein levels can be assessed using SDS-PAGE analysis.
  • Downstream when describing the location of a nucleic acid sequence, refers to 1) the nucleic acid sequence 3 ' to a nucleic acid sequence described, and/or 2) the translation, transcription, regulation or other related activity performed on a second nucleic acid sequence occurring after the translation, transcription, regulation or other related activity performed on a first nucleic acid sequence.
  • Endogenous refers to a nucleic acid sequence or peptide that is in the cell and was not introduced into the cell (or its progenitors) using recombinant engineering techniques. For example, a gene that was present in the cell when the cell was originally isolated from nature. A gene is still considered endogenous if the control sequences, such as a promoter or enhancer sequences that activate transcription or translation, have been altered through recombinant techniques.
  • Enzyme activity refers to an indicated enzyme (e.g., an "alcohol dehydrogenase activity") having measurable attributes in terms of, e.g., substrate specific activity, pH and temperature optima, and other standard measures of enzyme activity as the activity encoded by a reference enzyme (e.g. , alcohol dehydrogenase). Furthermore, the enzyme is at least 60% identical at a nucleic or amino acid level to the sequence of the reference enzyme as measured by a BLAST search.
  • Enzyme Classification Numbers The EC numbers provided throughout this description are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Kyoto. The EC numbers are as provided in the database on February 1, 2008.
  • Excise As used herein, the term “excise” (or “excises” and “excision”) with reference to a nucleic acid sequence, refers to the removal of a polynucleotide sequence from a host cell's plasmid or genome from an expressed recombinase protein.
  • the excised polynucleotide sequence can be a complete promoter nucleotide sequence or a partial sequence thereof, a complete protein encoding nucleotide sequence or partial sequence thereof, or a combination of a complete promoter nucleotide sequence and a complete protein encoding nucleotide sequence or partial sequences thereof.
  • excision results in the attenuation, disruption or complete absence of the trait conferred by the polynucleotide sequence (for example, as a nucleotide sequence recognized by a protein) or expression of the polynucleotide sequence.
  • exogenous when used with reference to a nucleic acid molecule and a particular cell or microorganism, refers to a nucleic acid sequence or peptide that was not present in the cell when the cell was originally isolated from nature.
  • a nucleic acid that originated in a different microorganism or synthesized de novo and was engineered into an alternate cell using recombinant DNA techniques or other methods for delivering said nucleic acid is exogenous.
  • Exogenous with reference to a compound or organic compound refers to an extracellular compound or organic compound required for the growth, propagation, sustenance, viability or activity of any metabolic activity, without specific reference to any one metabolic activity.
  • the exogenous compound or organic compound includes those that are subsequently converted by the microorganism to metabolites and/or intermediates necessary or useful for cellular function.
  • Expression The process by which nucleic acid encoded information of a gene is converted into the structures and functions of a cell, such as a protein, transfer RNA, or ribosomal RNA. Expressed genes include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated into protein (for example, transfer and ribosomal RNAs).
  • Expression control sequence refers to nucleic acid 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 mR A; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence.
  • 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.
  • Flanking As used herein, the term “flanking nucleotide sequences" (or
  • “flanking” as used to describe an upstream and/or downstream nucleotide sequence describes either the 5' or 3' targeted nucleotide sequences capable of being recognized by a recombinase protein, can be located upstream or downstream of a selectable marker gene, adjacent to a selectable marker gene, or embedded within and/or co-transcribed with the nucleotide sequences encoding the selectable marker gene.
  • 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 disclosure have particular utility.
  • the heterologous polypeptide included within the fusion protein of the present disclosure 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.
  • GFP green fluorescent protein
  • a genetic element refers to any functional, regulatory or structural nucleic acid or nucleic acid sequence such as, without limitation, ribonucleic acid and deoxyribonucleic acid (RNA, DNA), whether originating from exogenous or endogenous sources, derived synthetically or originating from any organism or virus, including, without limitation, cDNA, genomic DNA, mRNA, RNAi, snRNA, siRNA, miRNA, ta-siRNA, tRNA, double stranded and/or single stranded, co-suppression molecules, ribozyme molecules or related nucleic acid constructs.
  • RNA deoxyribonucleic acid
  • Hydrocarbon The term 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.
  • Isolated An "isolated" nucleic acid or polynucleotide ⁇ e.g., RNA, DNA or a mixed polymer) refers to 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.
  • the term embraces a nucleic acid or polynucleotide 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 "isolated polynucleotide” 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.
  • isolated or substantially pure also can be used in reference to recombinant or cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are
  • isolated does not necessarily require that the nucleic acid or polynucleotide so described has itself been physically removed from its native environment.
  • an endogenous nucleic acid sequence in the genome of an organism is deemed “isolated” herein if a heterologous sequence (i.e., a sequence that is not naturally adjacent to this endogenous nucleic acid sequence) is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this
  • endogenous nucleic acid sequence is altered.
  • a non-native promoter sequence can be substituted ⁇ e.g. by homologous recombination) for the native promoter of a gene in the genome of a human cell, such that this gene has an altered expression pattern.
  • This gene would now become “isolated” because it is separated from at least some of the sequences that naturally flank it.
  • a nucleic acid is also considered “isolated” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome.
  • an endogenous coding sequence is considered “isolated” if it contains an insertion, deletion or a point mutation introduced artificially, e.g. by human intervention.
  • an "isolated nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site, as well as a nucleic acid construct present as an episome. Moreover, an “isolated nucleic acid” can be substantially free of other cellular material or substantially free of culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.
  • 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).
  • 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.
  • isolated does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment.
  • Knock-out refers to a gene whose level of expression or activity has been reduced to zero.
  • a gene may be knocked-out with deletion of some or all of its coding sequence.
  • a gene may be knocked-out with an introduction of one or more nucleotides into its open-reading frame, which can result in translation of a non-sense or otherwise non-functional protein product.
  • 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
  • ligands which bind to labeled antiligands e.g., antibodies
  • labeled antiligands e.g., antibodies
  • fluorophores e.g., fluorophores
  • chemiluminescent agents e.g., enzymes
  • 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).
  • Mutation or 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 App lie.
  • 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 App lie.
  • 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)).
  • Nucleic acid molecule refers to both RNA and DNA molecules including, without limitation, cDNA, genomic DNA and mRNA, and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or
  • the nucleic acid molecule can be double-stranded or
  • nucleic acid molecule can be the sense strand or the antisense strand.
  • nucleic acid comprising SEQ. ID NO: l refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ. ID NO: l, or (ii) a sequence complimentary to SEQ. ID NO: l . The choice between the two is dictated by the context in which SEQ. ID NO: 1 is used.
  • nucleic acid sequences of the present disclosure 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 naturally occurring nucleotides with an analog, inter-nucleotide modifications such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendant moieties, (for example, polypeptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.).
  • uncharged linkages for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • charged linkages for example, phosphorothioates, phosphorodithioates, etc.
  • pendant moieties for example, polypeptides
  • intercalators for example, acridine
  • 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 a molecule.
  • Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as modifications found in "locked" nucleic acids.
  • Operably linked A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. Configurations of separate genes that are transcribed in tandem as a single messenger RNA are denoted as operons. Thus placing genes in close proximity, for example in a plasmid vector, under the transcriptional regulation of a single promoter, constitutes a synthetic operon.
  • Overexpression When a gene is caused to be transcribed at an elevated rate compared to the endogenous transcription rate for that gene.
  • overexpression additionally includes an elevated rate of translation of the gene compared to the endogenous translation rate for that gene.
  • Methods of testing for overexpression are well known in the art, for example transcribed RNA levels can be assessed using reverse transcriptase polymerase chain reaction (RT-PCR) and protein levels can be assessed using sodium dodecyl sulfate polyacrylamide gel elecrophoresis (SDS-PAGE) analysis.
  • a gene is considered to be overexpressed when it exhibits elevated activity compared to its endogenous activity, which may occur, for example, through reduction in concentration or activity of its inhibitor, or via expression of mutant version with elevated activity.
  • the host cell encodes an endogenous gene with a desired biochemical activity, it is useful to over-express an exogenous gene, which allows for more explicit regulatory control during growth and a means to potentially mitigate the effects of indigenous regulation, which is focused around the native genes explicitly.
  • Peptide 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.
  • Percent Sequence Identity As used herein, 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 nine nucleotides, usually about 20 nucleotides, more usually at least 24 nucleotides, typically at least about 28 nucleotides, more typically at least 32 nucleotides, and preferably at least about 36 or more nucleotides.
  • polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, WI.
  • FASTA provides alignments and percent sequence identity of the regions of the best overlap between query and search sequences. Pearson, Methods. Enzymology. 183:63-98 (1990) (and hereby incorporated by reference in its entirety).
  • percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters as provided in GCG Version 6.1 , herein incorporated by reference.
  • sequences can be compared using the computer program Basic Local Alignment Search Tool ("BLAST"; Altschul, et al, J. Mol. Biol.
  • Sequence homology for polypeptides 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.
  • GCG Genetics Computer Group
  • Bestfit programs
  • 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, blastx, tblastx or tblastn (Altschul et al., Nucleic Acids Res.
  • 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.
  • Polypeptide fragment refers to a polypeptide that has a deletion, e.g., an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide.
  • 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.
  • 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 naturallyoccurring protein. 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.
  • 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).
  • Protomer refers to a polymeric form of amino acids forming a subunit of a larger oligomeric protein structure.
  • Protomers of an oligomeric structure may be identical or non-identical.
  • Protomers can combine to form an oligomeric subunit, which can combine further with other identical or non-identical protomers to form a larger oligomeric protein.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified product preparation is one in which the product is more concentrated than the product is in its environment within a cell.
  • a purified wax is one that is substantially separated from cellular components (nucleic acids, lipids, carbohydrates, and other peptides) that can accompany it.
  • a purified wax preparation is one in which the wax is substantially free from contaminants, such as those that might be present following fermentation.
  • a recombinant nucleic acid molecule or protein is one that has a sequence that is not naturally occurring, has a sequence that is made by an artificial combination of two otherwise separated segments of sequence, or both. This artificial combination can be achieved, for example, by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules or proteins, such as genetic engineering techniques. Recombinant is also used to describe nucleic acid molecules that have been artificially manipulated, but contain the same regulatory sequences and coding regions that are found in the organism from which the nucleic acid was isolated.
  • recombinant host cell refers to a cell into which a recombinant vector has been introduced, e.g., a vector comprising acyl-CoA synthase. 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.
  • Release The movement of a compound from inside a cell (intracellular) to outside a cell (extracellular).
  • the movement can be active or passive.
  • When release is active it can be facilitated by one or more transporter peptides and in some examples it can consume energy.
  • release When release is passive, it can be through diffusion through the membrane facilitated or not by porters and can be facilitated by continually collecting the desired compound from the extracellular environment, thus promoting further diffusion. Release of a compound can also be accomplished by lysing a cell.
  • Segregation refers to the process of enriching a certain allelic locus (e.g. a disrupted or wild-type gene, a modified or intact non-coding genomic region) on microbial genome (including both chromosome and indigenous plasmids) until the allelic locus completely replaces other alleles, by imposing selection pressures such as antibiotic resistance, auxotrophy, etc.
  • Segregation process as a necessity to stabilize intended genetic traits, is usually applied to microorganisms that have multiple copies of genomic DNA (polyploids). For instance, many cyanobacteria are polypoids.
  • sequential plating(s) or sequential host cell plating(s) refers to the process of culturing a host cell in or on one sterile medium, then selecting an isolated colony from the culture medium to culture on a next sterile medium.
  • the culture medium can be, for example, an agar plate.
  • the culture medium may or may not have an integral selective agent, such as an antibiotic, that allows only the culturing of microorganisms that express an appropriate antibiotic resistance gene.
  • the process can be continued indefinitely to sequentially culture an inoculum from a preceding inoculated growth culture to a next sterile growth culture.
  • 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).
  • compositions that is a "substantially pure" compound is substantially free of one or more other compounds, i.e., the composition contains greater than 80 vol.%, greater than 90 vol.%, greater than 95 vol.%, greater than 96 vol.%, greater than 97 vol.%, greater than 98 vol.%, greater than 99 vol.%, greater than 99.5 vol.%), greater than 99.6 vol.%>, greater than 99.7 vol.%>, greater than 99.8 vol.%>, or greater than 99.9 vol.%> of the compound; or less than 20 vol.%>, less than 10 vol.%>, less than 5 vol.%), less than 3 vol.%>, less than 1 vol.%>, less than 0.5 vol.%>, less than 0.1 vol.%>, or less than 0.01 vol.% of the one or more other compounds, based on the total volume of the composition.
  • Suitable fermentation conditions generally refers to fermentation media and conditions adjustable with, pH, temperature, levels of aeration, etc., preferably optimum conditions that allow microorganisms to produce carbon-based products of interest.
  • the microorganism can be cultured for about 24 hours to one week after inoculation and a sample can be obtained and analyzed. The cells in the sample or the medium in which the cells are grown are tested for the presence of the desired product.
  • Up-regulation refers to when a gene is caused to be transcribed at an increased rate compared to the endogenous gene transcription rate for that gene.
  • up- regulation additionally includes an increased level of translation of the gene compared to the endogenous translation rate for that gene. Methods of testing for up-regulation are well known to those in the art. For example, the transcribed RNA levels can be assessed using RT-PCR, and protein levels can be assessed using SDS-PAGE analysis.
  • Vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated.
  • Other vectors include cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).
  • BACs bacterial artificial chromosomes
  • YACs yeast artificial chromosomes
  • viral vector Another type of 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).
  • integrative vectors can be integrated wholly or partially into the genome of a host cell upon introduction into the host cell via intended recombination, and are thereby replicated along with the host genome.
  • 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”).
  • a vector can also include one or more selectable marker genes and other genetic elements known in the art. Additionally, a vector can be all of integrative, recombinant and expression vectors.
  • wash out refers to the process of segregational loss of plasmids in host cells through the propagation of the host cells over successive generations. The process may require removing selection agent from the cell culture for which resulting selective pressure allows maintenance of the plasmid in the host cells. For example, in the absence of such a selective agent, a wild-type host cell plasmid may preferentially propagate through cell generations over a similar plasmid differing only in having an exogenous engineered gene. Thus, the engineered plasmid will be "washed out” of the cell line and be replaced by the wild type plasmid.
  • the Tyr recombinase family is based on the invariant sequences of His-X-X-Arg sequence and a Tyr residue, wherein the recombination reaction proceeds through a covalent intermediate in which the DNA 3 'phosphoryl group is esterified to the hydroxyl group of the tyrosyl residue.
  • Members of this family belong to enzyme classification 2.7.7.-. Many members function as an excisionase as well as an integrase in addition to contributing to the maintainence of plasmid copy number, eliminating dimers from replicated chromosomes, alteration of cell-surface components and regulating the life cycle of temperate phages.
  • a preferred embodiment of the present disclosure is the incorporation of a recombinase protein selected from the Tyr recombinase family.
  • the phage encoded integrase "Int" of the Tyr recombinase family catalyzes both excision and integration of phage DNA into the genome of the bacterial target cell. Both reactions are thought to require the presence of a host cell encoded accessory protein
  • HK022 heterologously expressed Int
  • HK022 has been demonstrated to actively integrate and excise so-called "atf sites both on plasmid and chromosome in Arabidopsis and human cells; Higher catalytic activities were observed with the provision of known accessory proteins (Kolot et al. 2003, Gottfried et al. 2005, Harel- Levi et al. 2008, Malchin et al. 2009).
  • This evidence indicates that plant and human cells may contain necessary, yet unidentified accessory proteins contributing to the functionality of recombinase proteins.
  • HK022 Int-att recombination system can be utilized in a broad spectrum of hosts with unacertainable co-factors that are known to be essential for Int function in wild type host cells. Therefore, in a preferred embodiment of the present disclosure, an exogenous recombinase protein does not require additional co-factors for its function in a host cell.
  • Coliphage HK022 (Refseq: NC 002166) encodes the recombinase HK022 integrase. Both integration into and excision from the host cell genome by the phage are mediated by HK022 integrase (Landy 1989). Integration is based on site-specific recombination between a host genome encoded receptor site attB and phage genome encoded docking site attP. After integration, the attB and attP sites are re-arranged to form attL and attR sites that flank the prophage genome. During phage genome excision, the attL and attR sites are recognized by Int.
  • the recombinase protein is a site-specific recombinase protein.
  • flanking exogenous attL and attR nucleotide sequences undergo a recombination event when contacted by the exogeous recombinase protein.
  • the exogenous recombinase protein is coliphage HK022 Int.
  • the E. coli attB consists of a 21 bp core sequence BOB', of which the 7 bp O sequence is identical in all four att sites and serves as the central recombination site during integration and excision. O is flanked by a 7 bp imperfect inverted repeat of B and B', which serve as weak binding sites for Int.
  • the phage attP site consists of a similar 21 bp core sequence COC which is flanked by longer arms of P and P' on each side (PCOC'P'). P and P' contain strong binding sites for Int, as well as binding sites for accessory factors that facilitate the Int-mediated site-specific integration and excision.
  • AttR and attL are arranged in the order of PCOB' and BOC'P', respectively (Dorgai et al. 1998).
  • the att-site specific integration/excision can also be carried out, less efficiently, by the integrase from Coliphage ⁇ , and vice versa (Yagil and Dolev, 1989).
  • the efficiency is mostly determined by the specific sequences of those extended cores (B, B', C and C) (Dorgai et al. 1998). Therefore, in a preferred embodiment of the present disclosure the flanking exogenous PCOB' and BOC'P 'nucleotide sequences undergo a recombination event when contacted by an exogenous recombinase protein.
  • coliphage ⁇ Int is the exogenous site-specific recombinase.
  • the present disclosure utilizes Int- family recombinases including, but not limited to those recombinases of enzyme classification (EC) 2.7.7.-, ⁇ InT (accession # P03700), HK022 (accession # PI 6407), 434 (accession # P27078), 21(P21) (accession # P27077), 186 (accession # P06723), P2 (accession # P36932), P4 (satellite phage) (accession # P08320), phi R73 (retrocj)) (accession # A42465), YjgC (P4-like) (accession # P39347), CP4-57 (P4- like) (accession # P32053), phi 80 (accession # P06155), SF6 (accession # P37317), YfdB (accession # P37326), P22 (accession # P04890), D
  • the recombinase protein chosen from the enzyme class 2.7.7.- is codon optimized for expression in the preferred host cell.
  • Selection markers are a gene whose product is required for survival during growth cycle of the host cell under selective pressure. Host cells lacking the selection marker, such as cells that have reverted back to the non-transformed or wild type state, are unable to survive. The use of selection markers is intended to ensure that only bacteria containing the expression systems and vectors survive, eliminating competition between the revertants and transformants. The most commonly used selection markers are antibiotic resistance genes. Host cells are grown in a medium supplemented with an antibiotic capable of being degraded by the selected antibiotic resistance gene product. Cells that do not contain the expression vector with the antibiotic resistance gene are killed by the antibiotic. Therefore, in an embodiment of the present disclosure, a selectable genetic marker is an antibiotic resistance gene.
  • selectable antibiotic markers for which resistance genes may be used in the host cells of the present disclosure include amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, geldanamycin, herbimycin, loracarbef, ertapenem, doripenim, imipenem/cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftobiprole, teicoplanin, vancomycin, telavan
  • Selection markers may include those that express a metabolite for which the host cell is auxotrophic.
  • Auxotrophy may be engineered into the cell, for example by knock out or attenuation of essential genes, or the wild-type host cell may be auxotrophic.
  • One or more than one metabolic activity may be selected for knock-out or replacement.
  • additional metabolic knockouts or replacements can be provided.
  • the auxotrophy-restoring selection markers can be of a biosynthetic-type (anabolic), of a utilization-type (catabolic), or may be chosen from both types.
  • one or more than one activity in a given biosynthetic pathway for the selected compound may be knocked-out; or more than one activity, each from different biosynthetic pathways, may be knocked-out.
  • the corresponding activity or activities are then provided by at least one recombinant vector which, upon transformation into the cell, restores prototrophy to the cell.
  • the selectable genetic marker is an auxotrophic selectable marker.
  • compounds and molecules whose biosynthesis or utilization can be targeted to produce auxotrophic host cells include: lipids, including, for example, fatty acids; mono- and disaccharides and substituted derivatives thereof, including, for example, glucose, fructose, sucrose, glucose-6-phosphate, and glucuronic acid, as well as Entner-Doudoroff, Pentose Phosphate, Calvin cycle and Kreb's cycle pathway intermediates and products; nucleosides, nucleotides, dinucleotides, including, for example, ATP, dNTP, FMN, FAD, NAD, NADP, nitrogenous bases, including, for example, pyridines, purines, pyrimidines, pterins, and hydro-, dehydro-, and/or substituted nitrogenous base derivatives, such as cofactors, for example, biotin, cobamamide, riboflavine, thiamine; organic acids, glycolysis, Kre
  • host cells are derived that have a recombinase gene stably transformed into the host cell genome wherein the gene encoding a recombinase protein is operably linked to an inducible promoter.
  • An inducible recombinase gene stably integrated into a host cell genome foregoes the need to transform a host cell with a transient Int expression vector subsequent to a first transformation of a desired gene and its selectable marker.
  • a selectable marker gene flanked by Int targeted nucleotide sequences is first transformed into a host cell and selected for, is then followed with a selection scheme, and finally the host cell is subjected to Int facilitated removal by culturing the selected-for colonies with an appropriate inducer wherein the inducer up- regulates expression of the recombinase.
  • the choice of a promoter used for recombinase expression is determined, in part, by its activity in a host cell and the desired level of gene product expression. High expression promoters exhibit higher levels of expression "leakage” when repressed, and low expression promoters exhibit lower levels of expression “leakage” when repressed. Because of the observation that successful transformation of a host cell is decreased in the presence of a recombinase protein, the desired expression levels must be appropriately engineered to account for recombinase activity due to expression leakage. Greater expression control over an exogenously regulated integrase (recombinase) expression system in a unicellular non- diazotrophic cyanobacteria host cell includes the appropriate choice of exogenous promoter.
  • Promoters contemplated for the present disclosure include, but are not limited to, nickel inducible promoters, copper-inducible promoters, nitrogen-source inducible promoters, IPTG inducible promoters, promoters of genes involved in iron chelating (siderophore), transport and utilization, genes involved in dark anaerobic fermentation, and promoters of genes involved in C0 2 -concentrating and C0 2 -uptake mechanisms in cyanobacteria. Because many promoters exhibit low levels of expression even under repressed conditions, additional promoter engineering is contemplated for greater control over the promoter expression activity.
  • Synechocystis sp. PCC 6803 composed by a two-component nickel signaling system (the nickel-sensing gene nrsS and the nickel-responding gene nrsR) and a nickel-inducible promoter P nrS B-
  • the promoter is turned on or off in response to nickel presence (above the induction threshold) or absence (below the induction threshold) via the NrsSR two- component signaling system. Therefore, one embodiment of the present disclosure uses a nickel-inducible promoter to control expression of a recombinase protein.
  • a copper-inducible promoter P petE from Synechocystis sp. PCC 6803 has been used to induce gene expression when the concentration of Cu 2+ was in a range from 6 to 400 nM (Gao, et ah, 2007, Gao and Xu, 2009).
  • Cyanobacterial promoters controlled by the global nitrogen regulator NtcA have also been used as nitrate-inducible promoters, which are strongly repressed in the presence of ammonia (or urea), and are significantly induced after switching from ammonia to nitrate as the sole nitrogen source (Qi et al. 2005). Therefore, in another embodiment of the present disclosure, a copper-inducible promoter is used to control expression of a recombinase protein.
  • Promoters of genes involved in iron chelating (siderophore), transport and utilization could be used as inducible promoters in response to iron depletion.
  • the isiAB gene operon in Synechocystis sp. PCC 6803, encoding iron-stress induced, photosystem I associated, chlorophyll a-binding protein and flavodoxin are significantly induced under iron- deplete conditions (Kunert et al. 2003). Therefore, in another embodiment of the present disclosure, a promoter responsive to cellular iron is used to control expression of a promoter responsive to cellular iron is used to control expression of a
  • genes involved in dark anaerobic fermentation could also potentially be used to construct inducible promoters that respond to light-dark transitions and/or low 0 2 levels.
  • Synechococcus sp. PCC 7002 are used to control expression of a recombinase protein.
  • Recent transcriptomic studies also revealed some cyanobacterial genes whose expressions are remarkably induced under micro-aerobic or semi-anaerobic conditions. For example, expression levels of a putative gene operon acsF2-ho2-hemN2 in Synechococcus sp. PCC 7002, as well as their homologs in Synechocystis sp.
  • PCC 6803 (sill 874-slll 876), encoding magnesium protoporphyrin IX monomethyl ester oxidative cyclases, heme oxygenases that synthesize biliverdin, and coproporphyrinogen III oxidases are significantly up-regulated under micro-aerobic phototrophic conditions (Minamizaki et al. 2008; Yilmaz et al. 2009; Goto et al. 2010, Ludwig and Bryant 2011). Therefore, in yet another embodiment of the present disclosure, promoters and their analogues used in the control of the putative gene operon acsF2-ho2-hemN2 in Synechococcus sp. PCC 7002 are used herein to control the expression of a recombombinase protein.
  • C0 2 level could be another regulatory factor as expression levels of some genes involved in C0 2 -concentrating and C0 2 -uptake mechanisms in cyanobacteria are remarkably up-regulated after switching from high C0 2 to ambient C0 2 level.
  • expression levels of a putative gene operon ndhF3-ndhD3-cupA in Synechococcus sp. PCC 7002, encoding two subunits of the inducible C02-concentrating complex and the C0 2 hydration protein, respectively are significantly induced after switching from high C0 2 to ambient C0 2 level (Woodger et al. 2007).
  • promoters and their analogues used in the control of the putative gene operon ndhF3-ndhD3- cupA in Synechococcus sp. PCC 7002 are used herein to control the expression of a recombombinase protein.
  • the core and distal attL and attR nucleotide recognition sequences are modified to alter the binding specificity of the integrase and potential accessory proteins, thus optimizing the excision rates for a desirable effect.
  • modifications provide an additional level of expression control over an exogenously regulated integrase (recombinase) expression system in a unicellular non-diazotrophic cyanobacteria host cell. For instance, Dorgai et al.
  • Another strategy for regulating promoter activity is to engineer recombinase protein variants designed to exhibit varying levels of activity for a specific set of targeted nucleotide flanking regions.
  • Dorgai et al. (1995) and Yagil et al. (1995) demonstrated that modified ⁇ integrases and HK022 integrases, through site-directed mutagenesis and/or peptide shuffling, can recognize both ⁇ and HK022 att sites in different efficiencies, and perform excision in different rates.
  • the present disclosure provides that the recombinase of the host cell is an improved recombinase.
  • the improved recombinase encompasses an engineered integrase having greater or lesser affinity towards a specific nucleotide target sequence to alter the recombinase binding activity yet still maintain its specificity for a particular targeted att sequence.
  • the improved recombinase comprises altered substrate affinity, enzyme activity, improved thermostability, improved stability in halophilic conditions, improved stability at varying pH, and optimized codon usage for improved expression in a host cell.
  • one method for the design of recombinase proteins of the present disclosure utilizes computational and b to inform atic analysis to design and select for advantageous changes in primary amino acid sequences encoding nucleotide
  • recombinase polypeptide sequences or related homologues in a complex with a DNA sequence substrate are obtained from the Protein Data Bank (PDB; HM Berman, et aL Nucleic Acids Research (2000) vol. 28:235- 242) for computational analysis on steady state and/or changes in Gibb's free energy relative to the wild type protem. Substitutions of one amino acid residue for another are accomplished in silica interactively as a means for identifying specific residue substitutions that optimize structural or catalytic contacts between the protein and substrate using standard software programs for viewing molecules as is well known to those skilled in the art.
  • PDB Protein Data Bank
  • silica structures are available through the RCSB Protein Data Bank, including phage lambda integrase 1NTDBD 1 -64 complexed with a P' att flanking nucleotide sequence (2WCC), a lambda integrase dimer bound to a COC core site (1Z19; 1Z1 B), a lambda integrase teramer bound to a Holliday Junction (1Z 1G), lambda integrase muiti er bound to double stranded DNA (1P7D), a lambda integrase catalytic core structure (1AE9; 20XO), and a lambda integrase amino terminal DNA binding domain (1KJK).
  • 2WCC P' att flanking nucleotide sequence
  • substitutions are rationally chosen based on substituted residue characteristics that optimize, for example. Van der Waal's interactions, hydr ⁇ ho bl city, hydrophilicity, steric non-interferences, pH-dependent electrostatics and related chemical interactions.
  • the overall energetic change of the substitution protein model when unbound and bound to i s substrate is calculated and assessed by one having skill i the art to be evaluated for the change in free energy for correlations to overall structural stability (e.g., Meiler, J. and D. Baker, Proteins (2006) 65:538-548).
  • a rational design change to the primary structure of the protein sequences of the present disclousre minimally alter the Gibb's free energy state of the unbound polypeptides and maintain a folded, functional and similar wild-type enzyme structure. More preferably a lower computational total free energy change of protein sequences of the present disclosure is achieved to indicate the potential for optimized enzyme structural stability.
  • the resultant mutant DNA sequences are genetically engineered into an appropriate vector to be expressed in a host ceil and analyzed to screen and select for the desired effect on whole cell production of a product or process of interest.
  • random mutagenesis of nucleotide sequences of the present disclosure is generated through error prone PCR. using techniques well known to one skilled in the art. Resultant nucleotide sequences are analyzed for structural and functional attributes through clonal screening assays and other methods as described herein.
  • a specifically desired protein mutant is generated by using site-directed mutagenesis.
  • site-directed mutagenesis For example, with overlap extension (An, el a!., Appi, Microbiol . Biotech. (2005) vol, 68(6):774-778) or mega-primer PCR (E. Burke and S. Barik, Methods ⁇ . Bio. (2003) vol 226:525-532) one can use nucleotide primers that have been altered at corresponding codon positions in the parent nucleotide to yield DNA progeny sequences containing the desired mutation. Alternatively, one can use cassette mutagenesis (Kegler- Ebo, et ah. Nucleic Acids Res. (1994) vol. 22(9): 1593-1599) as is commonly know by one skilled in the art.
  • Another embodiment of the present disclosure is to select for a polypeptide variant for expression in a recipient host ceil by comparing a first nucleic acid sequence encoding the polypeptide with the nucleic acid sequence of a second, related nucleic acid sequence encoding a polypeptide having more desirable qualities, and altering at least one codon of the first nucleic acid sequence to have identity with the corresponding codon of the second nucleic acid sequence, such that improved polypeptide activity, substrate specificity, substrate affinity, substrate catalytic conversion rate, improved thermostabi lity, activity at a different pH and/or optimized codon usage for expression and/or structure of the altered polypeptide is achieved in the host cell.
  • all amino acid residue variations are encoded at any desired, specified nucleotide codon positio using such methods as site saturation mutagenesis (Meyers, et al., Science ( 1985) Vol. 229:242-247; Derbyshire, et al, Gene (1986) Vol. 46:145-152; U.S. Patent 6,171,820).
  • Site saturation mutagenesis Melaton mutagenesis
  • Whole gene site saturation mutagenesis K. Kretz, et al., Meth. Enzym, (2004) Vol. 388:3-1 1 1
  • ail amino acid residue variations are encoded at every nucleotide codon position.
  • Both methods yield a population of protein variants differing from the parent polypeptide by one amino acid, with each amino acid substitution being correlated to structural/functional attributes at any position in the polypeptide.
  • Saturation mutagenesis uses PGR. and primers homologous to the parent sequence wherein one or more codon encoding nucleotide triplets is randomized. Randonuzation results in. the incorporation of codons corresponding to all amino acid replacements in the final, translated polypeptide.
  • Each PGR product is genetically engineered into an expression vector to be introduced into an expression host and screened for structural and functional attributes through clonal screening assays and other methods as described herein.
  • GSM correlated saturation mutagenesis
  • two or more amino acids at rationally designated positions are changed concomitantly to different amino acid residues to engineer unproved enzyme function and structure.
  • Correlated saturation mutagenesis allows for the identification of complimentary amino acid changes having positive, synergistic effects on. enzyme structure and function.
  • synergistic effects include, but are not limited to, significantly altered protien stability, substrate affinity or catalytic turnover rate, independently or concomitantly increasing advantageously the binding to a polynucleotide target sequence.
  • GSM is used at Int recombmase positions 1 -64 of the amino terminus which is involved in or located near the distal portions of the 3' and 5 ' flanking nucleotide regions known to be involved in polynucleotide sequence discrimination,
  • all three strategies can be combined to engineer the desired activity of the recombinase.
  • a tight-controlled inducible promoter could be used to repress the expression of an engineered int gene while using DNA segments flanked by engineered att sites. Due to the fact that int gene expression is tightly repressed, and the fact that remaining very low level of integrase has low excision activity, att- flanked DNA segments could be readily brought in. In the following removal step, integrase expression is significantly induced. Although the binding specificity for the engineered att sites is lower, and the exicision activity of the engineered integrase is truncated, high expression level of the integrase could still allow efficient removal of the targeted DNA segments.
  • Microorganisms include 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.
  • the engineered cell provided by the present disclosure may be derived from eukaryotic plants, industrially important organisms including, but not limited to, Xanthomonas spp., Escherichia coli, Corynebacterium spp., Lactobacillus spp., Aspergillus spp., Streptomyces spp., Acetobacter spp., Penicillin spp., Bacillus spp., Pseudomonad spp., Clostridium spp., Zymomonas spp., Salmonella spp., Serratia spp., Erwinia spp., Klebsiella spp., Shigella spp., Enteroccoccus spp., Alcaligenes spp., Paenibacillus spp., Arthrobacter spp., Brevibacterium spp., algae, cyanobacteria, green-sulfur
  • the cell is photoautotrophic in the presence of light and heterotrophic or mixotrophic in the absence of light. In other related aspects
  • the engineered cell is a plant cell selected from the group consisting of Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia and Zea.
  • the engineered cell of the present disclosure is an algae and/or cyanobacterial organism selected from the group consisting of 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,
  • Brachysira Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix, Campy lodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritr actus,
  • 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,
  • Compsogonopsis Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia,
  • 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,
  • 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,
  • 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,
  • 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,
  • Pseudoncobyrsa Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum,
  • 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, Molingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis,
  • the engineered cell provided by the present disclosure is derived from a Chloroflexus, Chloronema, Oscillochloris, Heliothrix,
  • Herpetosiphon, Roseiflexus, and Thermomicrobium cell a green sulfur bacteria selected from: Chlorobium, Clathrochloris, and Prosthecochloris; a purple sulfur bacteria is selected from: Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis; a purple non-sulfur bacteria is selected from: Phaeospirillum, Rhodobaca, Rhodobacter,
  • Rhodomicrobium Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio, and Roseospira
  • an aerobic chemolithotrophic bacteria selected from: nitrifying bacteria.
  • 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,
  • Hydrogenobacter sp. iron and manganese-oxidizing and/or depositing bacteria, Siderococcus sp., and magnetotactic bacteria, Aquaspirillum sp; an archaeobacteria selected from:
  • Methanothermus sp. Methanococcus sp., Methanomicrobium sp., Methanospirillum sp., Methanogenium sp., Methanosarcina sp., Methanolobus sp., Methanothrix sp., Methanococcoides sp., Methanoplanus sp.; extremely thermophilic sulfur-Metabolizers such as Thermoproteus sp., Pyrodictium sp., Sulfolobus sp., Acidianus sp., Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp., Ralstonia sp., Rhodococcus sp., Cory neb acteria sp., Brevibacteria sp., Mycobacteria sp., and oleaginous yeast.
  • the engineered cell provided by the present disclosure is derived from an extremophile that can withstand various environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension, and chemicals.
  • extremophiles which grow at or above 80°C such as Pyrolobus fumarii; thermophiles, which grow between 60-80°C such as Synechococcus lividis.
  • thermophilic, thermophile, hyperthermophile or hyperthermophilic generally refers to any microorganism adapted to have the ability to survive in environments of elevated or extreme temperatures.
  • 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 or barophiles which tolerate pressure of 130 MPa.
  • Hypergravity (e.g., >lg) hypogravity (e.g., ⁇ lg) 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.
  • halophiles or halophilic generally refers to any microorganism adapted to have the ability to survive in environments of elevated or extreme salinity.
  • 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).
  • microaerophils which tolerate some 0 2 such as Clostridium and aerobes, which require 0 2 are also contemplated.
  • Gas tolerant organisms which tolerate pure C0 2
  • 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) are also contemplated.
  • the host cell provided by the present disclosure is derived from 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 and 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).
  • Chlorobium tepidum green sulfur bacteria
  • Chloroflexus auranticus green non-sulfur bacteria
  • Chromatium tepidum and Chromatium vinosum purple sulfur bacteria
  • Rhodospirillum rubrum Rhodobacter capsulatus
  • Rhodopseudomonas palusris purple non-sulfur bacteria
  • the engineered cell provided by the present disclosure is a Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis cell.
  • the host cell is selected from Cyanobium sp., Dunaliella sp., Chlamydomonas sp., Spirulina sp., Cyanocethe sp., Chlorella sp., Botryococcus sp., Hamatococcus sp., Chamaesiphon sp., Chlorococcus sp., Gloeothece sp., Gloeobacter sp., Prochlorococcus sp., Acaryochloris sp., Xenococcus sp., Dactylococcopsis sp., Prochloron sp., Chroogloeocystis sp., Coelosphaerium sp.,
  • Cyanodictyon sp. Geminocystis sp., Johannesbaptistia sp., Limnococcus sp., Radiocystis sp., Rhabdoderma sp., Rubidibacter sp., Snowella sp., Sphaerocavum sp., Synechococcus sp., Synechocystis spp., Cyanobacterium sp., Cyanobium sp., Gleocapsa sp. and
  • the host cell provided by the present disclosure is capable of conducting or regulating at least one metabolic pathway selected from the group consisting of photosynthesis, sulfate reduction, methanogenesis, acetogenesis, reductive TCA cycle, Calvin cycle, 3-HPA cycle and 3HP/4HB cycle.
  • a common theme in selecting or engineering a suitable organism is autotrophic fixation of carbon, such as C0 2 to products. This would cover photosynthesis and
  • desired hydrocarbons and/or alcohols of certain chain length or a mixture thereof can be produced.
  • the host cell produces at least one of the following carbon-based products of interest: 1- dodecanol, 1- tetradecanol, 1-pentadecanol, ii-tridecane, n-ietradeeane, 15: 1 n-pentadeeane, n-pentadecane, 16: 1 /7-hexadecene, n-hexadecane, 1 7: 1 n-heptadecene, n-hepiadecane, 16: 1 n-hexadecen-ol, /7-hexadeean-l-ol and n-oetadeeen-i-ol, as shown in the Examples herein.
  • the carbon chain length ranges from C 10 to C 2 c- Accordingly, the present disclosure provides
  • a carbon-based product of interest is produced by a biosynthetic pathway of an isolated host cell.
  • the end products or metabolites include, but are not limited to, alkanes (propane, octane), alkenes (ethylene, 1,3 -butadiene, propylene, olefins, alkenes, isoprene, lycopene, terpenes) aliphatic and aromatic alkane and alkene mixtures (diesel, jet propellant 8 (JP8)), alkanols and alkenols (ethanol, propanol, isopropanol, butanol, fatty alcohols, 1,3-propanediol, 1 ,4-butanediol, polyols, sorbitol, isopentenol), alkanoic and alkenoic acids (acrylate, acrylic acid, adipic acid,
  • PHA polyhydroxyalkanoates
  • PHB poly-beta-hydroxybutyrate
  • PHB poly-beta-hydroxybutyrate
  • isoprenoids lanosterol, isoprenoids, carotenoids, steroids
  • pharmaceuticals/multi-functional group molecules ascorbate, ascorbic acid, paclitaxel, docetaxel, statins, erythromycin, polyketides, peptides, 7-aminodeacetoxycephalosporanic acid (7-ADCA)/cephalosporin
  • acetaldehyde acetaldehyde
  • the methods provide cuituring 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 present disclosure enables production of various defined chain length of hydrocarbons and alcohols, the secreted products are easily recovered or separated. The products of the present disclosure, therefore, can be used directly or used with minimal processing.
  • compositions produced by the methods of the disclosure are used as fuels.
  • Such fuels comply with ASTM standards, for instance, standard
  • 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 present disclosure produce an. array of hydrocarbons, such as C.3-C17 or C 10 -C15 to alter cloud point.
  • compositions may comprise fuel additives, which are used to enhance the performance of a fuel or engine.
  • 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.
  • Cyanobacteria are cultured in BG-11 medium (17.65 mM NaN0 3 , 0.18 mM K 2 HP04, 0.3 mM MgS0 4 , 0.25 mM CaCl 2 , 0.03 mM citric acid, 0.03 mM ferric ammonium citrate, 0.003 mM EDTA, 0.19 mM Na 2 C0 3 , 2.86 mg/L H 3 B0 3 , 1.81 mg/L MnCl 2 , 0.222 mg/L ZnS0 4 , 0.390 mg/L Na 2 Mo0 4 , 0.079 mg/L CuS0 4 , and 0.049 mg/L Co(N0 3 )2, pH 7.4)
  • Thermosynechococcus elongatus BP-1 (available from the Kazusa DNAResearch Institute, Japan) is propagated in BG11 medium supplemented with 20 mM TES-KOH (pH 8.2) as previously described [Iwai M, Katoh H, Katayama M, Ikeuchi M. "Improved genetic transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1.” Plant Cell Physiol (2004). 45(2): 171-175)]. Typically, cultures are maintained at 50°C and bubbled continuously with 5% C0 2 under a light intensity of 38 ⁇ photons m ⁇ 2 s "1 . T. elongatus BP-1 can be grown in A + medium also.
  • Chlamydomonas reinhardtii (available from the Chlamydomonas Center culture collection maintained by Duke University, Durham, North Carolina,) are grown in minimal salt medium consisting of 143 mg/L K 2 HP0 4 , 73 mg/L KH 2 P0 4 , 400 mg/L NH 4 N0 3 , 100 mg/L MgS0 4 -7H 2 0, 50 mg/L CaCl 2 -2 H 2 0, 1 mL/L trace elements stock, and 10 mL/L 2.0 M MOPS titrated with Tris base to pH 7.6 as described (Geraghty AM, Anderson JC, Spalding MH.
  • LEDs light emitting diodes
  • Carbon dioxide is supplied via inclusion of solid media supplements (i.e., sodium bicarbonate) or as a gas via its distribution into the growth incubator or media.
  • solid media supplements i.e., sodium bicarbonate
  • Most experiments are performed using concentrated carbon dioxide gas, at concentrations between 1 and 30%, which is directly bubbled into the growth media at velocities sufficient to provide mixing for the organisms.
  • concentrated carbon dioxide gas the gas originates in pure form from commercially-available cylinders, or preferentially from concentrated sources including off-gas or flue gas from coal plants, refineries, cement production facilities, natural gas facilities, breweries, and the like.
  • Synechococcus sp. PCC 7002 cells are transformed according to the optimized protocol previously described [Essich ES, Stevens Jr., E, Porter RD
  • Cells are grown in Medium A (18 g/L NaCl, 5 g/L MgS0 4 . 7 H 2 0, 30 mg/L Na 2 EDTA, 600 mg/L KC1, 370 mg/L CaCl 2 . 2 H 2 0, 1 g/L NaN0 3 , 50 mg/L KH 2 P0 4 , 1 g/L Trizma base pH 8.2, 4 ⁇ g/L Vitamin B i2 , 3.89 mg/L FeCl 3 . 6 H 2 0, 34.3 mg/L H 3 B0 3 , 4.3 mg/L MnCl 2 .
  • Medium A 18 g/L NaCl, 5 g/L MgS0 4 . 7 H 2 0, 30 mg/L Na 2 EDTA, 600 mg/L KC1, 370 mg/L CaCl 2 . 2 H 2 0, 1 g/L NaN0 3 , 50 mg/L KH 2 P0 4 , 1 g/L Trizma base pH 8.2, 4 ⁇ g/
  • Transformants are picked 3-4 days later. Selections are typically performed using 200 ⁇ g/ml kanamycin, 8 ⁇ g/ml chloramphenicol, 10 ⁇ g/ml spectinomycin on solid media, whereas 150 ⁇ g/ml kanamycin, 7 ⁇ g/ml chloramphenicol, and 5 ⁇ g/ml spectinomycin are employed in liquid media.
  • T. elongatus BP-1 cells are transformed according to the optimized protocol previously described ⁇ vide supra).
  • E. coli are transformed using standard techniques known to those skilled in the art, including heat shock of chemically competent cells and electroporation (Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif; Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y.; and Current Protocols in Molecular Biology, F. M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (through and including the 1997 Supplement)).
  • biosynthetic pathways as described herein are first tested and optimized using episomal plasmids described above.
  • Non- limiting optimizations include promoter swapping and tuning, ribosome binding site manipulation, alteration of gene order (e.g., gene ABC versus BAC, CBA, CAB, BCA), co-expression of molecular chaperones, random or targeted mutagenesis of gene sequences to increase or decrease activity, folding, or allosteric regulation, expression of gene sequences from alternate species, codon manipulation, addition or removal of intracellular targeting sequences such as signal sequences, and the like.
  • Each gene is optimized individually, or alternately, in parallel. Functional promoter and gene sequences are subsequently integrated into the E. coli chromosome to enable stable propagation in the absence of selective pressure (i.e., inclusion of antibiotics) using standard techniques known to those skilled in the art.
  • the model cyanobacterium used in this present disclosure is a unicellular coastal/marine cyanobacterium Synechococcus sp. PCC 7002 (JCC138). It has six
  • the small, high-copy and indispensible pAQl plasmid is an ideal platform to express HK022 Int, as an expression system pAQlEx based on pAQl has been developed (Scott et al. 2010, Xu et al. 2011), and also because that pAQlEx carrying Int expression can be easily washed out and replaced by the wild-type pAQl during final selection withdrawal.
  • the IdhA gene of JCC138 has been demonstrated to be easily and completely deleted (McNeely et al. 2010).
  • the in-house integrative vector pJB161 contains the upstream and downstream homologous regions of the JCC138 IdhA gene and an aphll gene conferring kanamycin resistance located between the two homologous regions.
  • the pJB161 (US
  • plasmid vector design for transforming an antibiotic marker into the host cell plasmid pAQ7 excluded an origin of replication in the attR-marker-attL construct. This exclusion will force the recombination reaction towards excision since the resulting circular DNA from the recombination reaction cannot replicate and subsequently is lost during propagation and/or "washing" of the resulting derived host cells. Excision of the marker by Int after a second transformation resulting in an expressed Int gene will result in a 21 -bp nucleotide "scar" corresponding to the attB sequence on the pAQ7 plasmid of the cured JCC138 strain. This attB scar is the recombinant truncation of the attR and attL targeted nucleotide sequences recognized by Int.
  • Antibiotic selection was done on solid A + medium containing gentamycin or spectinomycin having final concentrations of 50 ⁇ g/ml or 100 ⁇ g/ml, respectively.
  • the HK022 Int recombinase of the present disclosure uniquely recognizes conserved attL-attR sites that are flanking the selective markers (or any desired segmental targets), thus mediating the excision reaction for removal of the nucleotide sequence (encoding a genetic marker) between the attL and attR sequences.
  • the Int-targeted attR sequence is shown as SEQ ID NO: l .
  • the Int-targeted attL nucleotide sequence is shown as SEQ ID NO:2.
  • HK022 Int recombinase protein expression sequence is engineered into the pAQlEx plasmid, developed from the wild-type pAQl plasmid.
  • a plasmid origin of replication functional in JCC 138 is not included in the modified plasmid nucleotide sequence; an fi origin of replication sequence allows for propagation of the plasmid in an E. coli host cell.
  • the HK022 Int gene sequence has been codon optimized for expression in JCC 138.
  • the codon optimized protein coding Int recombinase nucleotide sequence is shown in SEQ ID NO:3.
  • HK022 integrase nucleotide encoding sequences were retrieved from the NCBI using search nucleotide search queries on the whole genome of HK022.
  • Core regions of the conserved nucleotide sequences comprising attR and attL were used as reference queries, including the conserved O, B, B', C and C sequences available from Dorgai, et al. (1998).
  • the P and P' regions were identified by tracing upstream approximately 150 bases and downstream approximately 90 bases.
  • the attR and attL sequences were made by combining the appropriate conserved P, P', B, B', C, C and O sequences.
  • Promoter selection to control Int expression has also been considered.
  • a strong constitutive promoter such as P cpC B, significantly accelerates the excision process when considering the multi-copy nature of the genome of JCC138. Strong Int expression also facilitated subsequent engineered pAQlEx wash-out process.
  • an inducible, moderate-strength promoter allowed for the excision process to be regulated as well as mitigate or minimize toxicity from Int expression to improve cell viability.
  • PCR primer sequences were generated to verify the integration or excision of desired polynucleotide sequences into or from the host cell genome.
  • F indicates forward or into the gene or genome from the 5' end
  • R indicates reverse or into the gene or genome from the 3' end.
  • SEQ ID NO:4 is the PCR primer for JCC138 IdhA gene upstream homology region (LH F).
  • SEQ ID NO: 5 is the PCR primer for JCC138 IdhA gene downstream homology region (LH R).
  • SEQ ID NO:6 is the PCR primer for JCC138 IdhA gene (L_F).
  • SEQ ID NO: 7 is the PCR primer for JCC138 IdhA gene (L_R).
  • SEQ ID NO:8 is the PCR primer used for the gentomycin gene region (G_F).
  • SEQ ID NO: 9 is the PCR primer used for the spectinomycin gene region (S_F).
  • SEQ ID NO: 10 is the PCR primer used for the upstream internal nucleotide spacer region between flanking recombinant homology regions located on pAQl(pAQl_F).
  • SEQ ID NO: 11 is the PCR primer used for the downstream internal nucleotide spacer region between flanking recombinant homology regions located on pAQl (pAQl R).
  • SEQ ID NO: 12 is the PCR primer used for the wild type pAQl plasmid downstream region (pEx_R).
  • tQgxdiSQ_CtermFLAG_BamHl and ii Pac ⁇ _attR_aacCl ⁇ gQnt)_attL_Asc ⁇ ” were synthesized by DNA 2.0.
  • the attR aacCl (gent) _attL fragment was subcloned into the integrative pJB161 vector by respective Pad and Ascl in the IdhA homology sites to form
  • JCC138 host cell cultures were grown to late exponential stage.
  • the plasmid DNA of AldhA::attR_aacCl_attL was mini-preped from E. coli NEB-5a (NEB) and transformation was performed by gently shaking 2 ⁇ g of the plasmid preparation with approximately 10 8 JCC138 cells under the standard conditions. After transformation, the cells were directly spread onto solid A + medium containing 50 ⁇ g/ml gentamycin and cultured for 5 days.
  • Engineered AldhA::attR_aacCl_attL strains were verified by PCR analysis using primers of LH F and LH R to confirm deletion of wild-type IdhA, and primers of G _F and LH R to confirm integration of AldhA: : attR_aacC 1 _attL construct.
  • Optimized HK022 coding sequences according to JCC138 codon usage were designed within the synthesized N ⁇ ieI_HK022mt_CtermFLAG_i?amHI fragment.
  • a FLAG tag was attached in-frame to the carboxyl terminus of the Int recombinase protein in order for expression levels to be determined.
  • JCC138 AldhA: :attR_aacCl _attL cultures were grown to late exponential stage.
  • the plasmid DNA of pAQlEx_P cpcB _int_aadA was mini-prepped from E. coli Epi400 (NEB) and transformed into JCC138: AldhA: :attR_aacCl _attL host cells by gently shaking 2 ⁇ g of plasmid DNA with approximate lylO 8
  • JCC ⁇ 38 AldhA::attR_ aacCl_attL ⁇ pAQ ⁇ Ex_P cpcB _int_aadA colonies were selected and re-streaked for further segregation onA+ media plates with spectinomycin, during which the Int recombinase att site-specific excision of the aacCI marker occurred. Marker removal was verified with PCR analysis. With reference to Fig. 1, PCR was done with two sets of primers, LH F/LH R (upper gel) and G F/LH R (lower gel).
  • AttR aacCl _attL nucleotide sequence encoding gentomycin resistance in transformants #1, #9 and #13 was completely removed from pAQ7 after only two rounds of plasmid segregation in 10 days, as indicated by the complete size shift from 1.7 kb to 600 bp in the upper gel and corresponding empty lanes in the lower gel.
  • Transformants shown but not numbered were in incomplete stages of aacCI removal due to either plasmid segregation or Int-mediated recombination.
  • Strain #4 is identified as an example of incomplete marker removal both partially due to plasmid segregation and from Int-mediated recombination.
  • Strain #4 later demonstrated replacement of pAQlEx (pEx_P cpcB _int_aadA) with wild type plasmid pAQl after being subjected to more rounds of washing out (see Fig. 3 and below).
  • genomic DNA of JCC138 was used as the PCR DNA control template for the control lane.
  • the constructed vector AldhA: :attR_aacCl _attL was used as the PCR DNA control template.
  • the cell pellet was resuspended with 100 ⁇ Laemmli sample buffer mixed with appropriate amount of ⁇ -mercaptoethanol by pipetting up and down, followed by incubation of the samples for 10 minutes on a heat-block set at 100 °C. Samples were loaded into pre- casted SDS-polyacryamide gels and protein separation was accomplished by tris/glycine gel electrophoresis, followed by protein transfer to nitrocellulose membranes using Invitrogen iBlot Transfer Apparatus, according to the manufacturer's instructions.
  • Western Blotting was accomplished by first placing the membrane in a square petri dish and rinsing it in washing buffer for 10 minutes while continuously shaking at low speed and at room temperature, followed by the addition of Blocking Buffer (2% BSA in washing buffer) with continuous shaking for 1 hour. Blocking Buffer was then discarded, followed by the addition of 10 ml Blocking Buffer and 10 ⁇ of 1 : 10 diluted anti-FLAG antibody (final dilution: 1 : 10,000) and incubation with shaking for 1 hour. Washing Buffer was added for 5 minutes and repeated 3 times.
  • Blocking Buffer 2% BSA in washing buffer
  • Anti-FLAG antibody binding to the Int recombinase indicated expression of the int gene in strains #1, #9 and #13, grown on spectinomycin and no expression of the int gene in AldhA: :attR_aacCl _attL grown on spectinomycin. Not shown here is the expression of Int in strain 4; however, since strain 4 was shown to grow on spectinomycin, the Int gene was expressed.
  • the fragment detected by pAQl F/ pAQl R was a wild type pAQl plasmid replacing pAQlEx during washing out in both strains.
  • the pAQlEx plasmid fragment was not available in either strain to be PCR amplified by the S_F/pEx_R primer pair.
  • strain #9 showed an attB BOB' nucleotide scar in its genome indicating an Int-mediated
  • control groups use genomic DNA templates of JCC138 and the constructed plasmid vector AldhA : :attR_aacCl attL .
  • JCC138 AldhA :attB ⁇ pAQlEx_P cpcB _int_aadA host cell colonies were re-cultured on solid A + medium without the use of any antibiotics in order to wash out (eliminate) the modified pAQlEx_P cpcB _int_aadA plasmid.
  • the modified pAQlEx_P cpcB _int_aadA will tend to "wash out” and be replaced by the the wild-tpe pAQl plasmid during cell propogation without the selective pressure since wild-type pAQl is indispensible to the host cell.
  • Primers LH F and LH R were used to indicate the intervening nucleotide spacer size, and primers G_F and LH R were used to indicate absence or presence of the gentomycin genetic marker, also as represented in Fig. 1 and Fig. 2.
  • Primers pAQl F and pAQl R were used to confirm the presence of wild-type plasmid pAQl because the deleted region created by pAQlEx integration was regenerated from the reintroduction of pAQl into the host cells after washing out. Primers of S_F and pEx_R were used to confirm the absence of the engineered plasmid pAQlEx (Fig 2.).
  • strain #9 (predicted to be AldhAr. attB) was prepared grown on solid A + medium containing either 25 ⁇ g/ml gentamycin or 50 ⁇ g/ml spectinomycin. If both spectinomycin and gentamycin antibiotic markers were removed from the host cells, the strain would be sensitive to both antibiotics and would be unable to grow. Strain #9 could not grow at indicated antibiotic concentrations, indicating both the aacCl marker and
  • Example 1 The method described above in Example 1 was performed to delete the nifJ gene in Synechococcus sp. PCC 7002.
  • an attR-aacC7(gent)-attL site comprising a gentomycin marker was inserted in an nifJ gene in Synechococcus PCC 7002 to form a AnifJ_attR- aacCl-attL mutant.
  • the AnifJ_attR- aacCl-attL mutant was selected for on gentomycin.
  • the gentomycin marker was removed by incorporating the HK022 integrase and selecting the culture on spectinomycin, which is the marker used to incorporate the integrase. The integrase was then successfully removed by selecting without any antibiotics.

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Abstract

La présente invention concerne des compositions et des procédés pour le retrait de marqueurs génétiques dans des cyanobactéries. Elle concerne des cyanobactéries portant un marqueur génétique qui peut être excisé par voie endogène avec une protéine recombinase. Une ségrégation de plasmides consécutive retire de la cellule hôte le ou les marqueurs génétiques et les séquences nucléotidiques exprimant la recombinase.
PCT/US2012/031696 2011-04-01 2012-03-30 Procédé pour retirer des marqueurs génétiques WO2012135766A1 (fr)

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Citations (2)

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US20040033608A1 (en) * 2001-10-10 2004-02-19 Purdue Research Foundation Plasmids, strains, and methods of use
US20110008861A1 (en) * 2008-03-03 2011-01-13 Joule Unlimited, Inc. Engineered CO2 Fixing Microorganisms Producing Carbon-Based Products of Interest

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040033608A1 (en) * 2001-10-10 2004-02-19 Purdue Research Foundation Plasmids, strains, and methods of use
US20110008861A1 (en) * 2008-03-03 2011-01-13 Joule Unlimited, Inc. Engineered CO2 Fixing Microorganisms Producing Carbon-Based Products of Interest

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MELNIKOV ET AL.: "Site-specific recombination in the cyanobacterium Anabaena sp. strain PCC 7120 catalyzed by the integrase of coliphage HK022.", J. BACTERIOL., vol. 191, no. 13, July 2009 (2009-07-01), pages 4459 - 4464 *
PARDO.: "BT-engineered bugs versus insect pests.", BIOENG. BUGS., vol. 1, no. 5, September 2010 (2010-09-01), pages 367 - 368 *
ZARITSKY ET AL.: "Transgenic organisms expressing genes from Bacillus thuringiensis to combat insect pests.", BIOENG. BUGS., vol. 1, no. 5, September 2010 (2010-09-01), pages 341 - 344 *

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