WO2019079135A1 - Production of heme-containing proteins in cyanobacteria - Google Patents

Production of heme-containing proteins in cyanobacteria Download PDF

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Publication number
WO2019079135A1
WO2019079135A1 PCT/US2018/055785 US2018055785W WO2019079135A1 WO 2019079135 A1 WO2019079135 A1 WO 2019079135A1 US 2018055785 W US2018055785 W US 2018055785W WO 2019079135 A1 WO2019079135 A1 WO 2019079135A1
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heme
cyanobacterium
seq
genetically modified
protein
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PCT/US2018/055785
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French (fr)
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Michael Mcconnell
Frank ULICZKA-OPITZ
Ulf DÜHRING
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Algenol Biotech LLC
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Publication of WO2019079135A1 publication Critical patent/WO2019079135A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/008—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/20—Proteins from microorganisms or unicellular algae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

Definitions

  • This application contains a sequence listing submitted by EFS-Web, thereby satisfying the requirements of 37 C.F.R. ⁇ 1.821-1.825.
  • the sequence listing, created on October 9, 2018, contains 60 sequences and is 195 KB in size.
  • the present invention relates generally to the recombinant production of heme-containing proteins in cyanobacterial host cells.
  • the heme-containing proteins can be used, for example, in meat substitute products.
  • a vegetarian diet is preferred by many consumers.
  • Non-animal derived meat-like material that tastes and looks like red meat but is made entirely of non-animal ingredients, would be a valuable consumer product.
  • many vegetarian meat-like products are lacking in taste and color.
  • a red meat-like product would have a pleasing taste and would look and process like an actual meat product.
  • a vegetarian-based ground meat or sausage meat that is similar to real meat would be desirable.
  • Modified cyanobacteria can be used to produce many types of proteins and other products in an environmentally friendly manner.
  • the transformation of the cyanobacterial genus Synechococcus with genes of interest has been described (U.S. Patent Nos. 6,699,696 and 6,306,639, both to Woods et al).
  • the transformation of the cyanobacterial genus Synechocystis has been described, for example, in PCT/EP2009/000892 and in PCT/EP2009/060526.
  • the transformation of the cyanobacterial genus Cyanobacterium sp. has been described (U.S. Patent No. 8,846,369, U.S. Patent No. 9,315,832, and PCT/US2013/077364).
  • a genetically modified cyanobacterium having a polynucleotide encoding a heterologous heme-containing protein is provided.
  • the protein can be, for example, a leghemoglobin or a cyanoglobin.
  • the polynucleotide can be, for example, codon optimized for optimal expression in the cyanobacterium, and can be linked to a constitutive or inducible (regulatable) promoter.
  • the polynucleotide can be located on an endogenous or heterologous extrachromosomal plasmid, or can be integrated into the
  • the cyanobacterium can also have a modification that increases heme levels. This can be accomplished, for example, by a knockdown of endogenous heme oxygenase HOI . This can also be accomplished by a modification that increases the flux through ferrochelatase, such as by a knockdown of the gene encoding magnesium chelatase (MgCh), the overexpression of ferrochelatase (FeCh), the knockdown of gun4 (MgCh activator), a gun4(W192A) mutation, magnesium-starvation, iron supplementation, or nitrogen- starvation.
  • MgCh magnesium chelatase
  • FeCh the overexpression of ferrochelatase
  • gun4 MgCh activator
  • W192A gun4(W192A) mutation
  • the cyanobacterium can also have a modification that results in a deficiency in phycocyanin.
  • a method of producing a heme-containing protein in a cyanobacterium by growing the genetically modified cyanobacterium under conditions to produce the heme-containing protein; and obtaining the heme-containing protein from the cyanobacterium.
  • a method of producing a heme-containing protein in a cyanobacterium by growing the genetically modified cyanobacterium under conditions to produce the heme-containing protein and additionally under conditions to increase heme production in the cyanobacterium, then obtaining the heme-containing protein from the cyanobacterium.
  • a biomass containing the genetically modified cyanobacterium is provided.
  • a dried meal (or bulk protein) obtained from the genetically modified cyanobacterium is provided.
  • a partially purified heme-containing protein obtained from the genetically modified cyanobacterium is provided.
  • a food product prepared from the heme- containing protein made from the genetically modified cyanobacterium is provided.
  • the food product can be, for example, a protein-rich product such as a meat substitute.
  • FIG. 1 is a map of the endogenous 6.8 kb plasmid isolated from Cyanobacterium sp. ABICyanol (SEQ ID NO: 17).
  • FIG. 2 depicts a map of the 10403 BP circular plasmid construct and sequence annotation of plasmid #2884 (pAB l_6.8: :PsmtA-legH_Gm(ABlopt)-TpsaB) (SEQ ID NO: 18).
  • FIG. 3 depicts a map of the 10412 BP circular plasmid construct and sequence annotation of plasmid #2885 (pAB l_6.8: :PsmtA-legH_As(AB lopt)-TpsaB) (SEQ ID NO: 19).
  • FIG. 4 depicts a map of the 10406 BP circular plasmid construct and sequence annotation of plasmid # #2886 (pABl_6.8: :PsmtA-legH_Cc(ABlopt)-TpsaB) (SEQ ID NO: 20).
  • FIG. 5 depicts a map of the 10340 BP circular plasmid construct and sequence annotation of plasmid # 2887 (pABl_6.8: :PsmtA-glbN_S6803(ABlopt)-TpsaB) (SEQ ID NO: 21).
  • FIG. 6 depicts a map of the 10340 BP circular plasmid construct and sequence annotation of plasmid #2888 (pAB l_6.8: :PsmtA-glbN_Ap8005(ABlopt)-TpsaB) (SEQ ID NO: 22).
  • FIG. 8 depicts a map of the 10393 BP circular plasmid construct and sequence annotation of plasmid #2890 (pAB l_6.8: :PsmtA-glbN_S6803(ABlopt)-TpsaB-PnirA-anti_HOl_vl) (SEQ ID NO: 24).
  • FIG. 9 depicts a map of the 10634 BP circular plasmid construct and sequence annotation of plasmid #2942 (pABICyanol_6.8: :PcpcB-glbN_S6803(ABICyanolopt)-TpsaB-Porf0316'- anti HOl vl) (SEQ ID NO: 56).
  • FIG. 10 depicts a map of the 10491 BP circular plasmid construct and sequence annotation of plasmid #2994 (pABl_6.8::PcpcB-glbN_Ap8005(AB lopt)-TpsaB) (SEQ ID NO: 57).
  • FIG. 11 depicts a map of the 11432 BP circular plasmid construct and sequence annotation of plasmid #3022 (pABICyanol_6.8: :PcpcB-glbN_S6803(ABICyanolopt)-TpsaB- PcpcB-glbN_S6803(ABICyanolopt)-TpsaB-Porf0316'-anti_HOl_vl) (SEQ ID NO: 58).
  • FIG. 12 depicts a map of the 7033 BP circular plasmid construct and sequence annotation of plasmid #2949 (nblA2: :PcpcB 1918-glbN_Ap8005-TpsaB-PrbcL2293-ble-TB1002) (SEQ ID NO: 59).
  • FIG. 13 depicts a map of the 8060 BP circular plasmid construct and sequence annotation of plasmid #3057 (nblA2: :PcpcB 1918-glbN_Ap8005-PcpcB1918_glbN_Ap8005-TpsaB- PrbcL2293-ble-TB1002) (SEQ ID NO: 60).
  • FIG. 14 is a diagram of the biosynthetic pathway from glutamate to protoporphyrin, heme, chlorophyll, biliverdin, phycobilin, and leghemoglobin. Also shown is the location of inhibition of heme oxygenase (HOI) using an inducible sRNA for gene knock-down.
  • HOI heme oxygenase
  • FIG. 15 is a stained protein gel showing the expression of the cyanoglobin (GlbN) protein from Synechocystis strain PCC 6803 in Cyanobacterium sp. ABICyanol . Expression of the cyanoglobin (black arrow) was induced with 20 ⁇ zinc sulfate. Protein extracts were separated on a 16% native PAGE and stained with Coomassie Brilliant Blue.
  • GlbN cyanoglobin
  • FIG. 16 is a photograph of a stained protein gel showing the expression of the cyanoglobin (GlbN) protein from Synechocystis strain PCC 6803 in Cyanobacterium sp.
  • FIG. 17 is a photograph of a stained protein gel showing the expression of the leghemoglobin C2 protein (black arrow) in Cyanobacterium sp. ABICyanol . Expression of leghemoglobin C2 was induced with different zinc concentrations in the presence of nitrate to knockdown expression of the heme oxygenase ABICyanol . l_orf29000 via sRNA. Protein extracts were separated by denaturing electrophoresis on a 16% Tricine-SDS-PAGE including 6 M urea and subjected to a Coomassie Brilliant Blue staining. [0031] FIG. 18A and FIG.
  • FIG. 19 is a line graph showing the relative absorbance spectra of Cyanobacterium sp. ABICyanol and a Phycocyanin-deficient strain AB0492 (AcpcA).
  • FIG. 20 is a photograph of a stained protein gel showing the constitutive expression of the cyanoglobin (GlbN) protein from Synechocystis strain PCC 6803 in the Cyanobacterium sp. ABICyanol and in a phycocyanin-deficient derivative AB0492 (AcpcA). The strains were grown in the absence and presence of 10 ⁇ copper-EDTA to induce the knockdown of the heme oxygenase via anti-HOl sRNA.
  • AB4011 has a single copy of the PcpcB-glbN_S6803, whereas AB4012 comprises two PcpcB-glbN_S6803 copies on the same plasmid. Protein extracts were separated on a 16% native PAGE and stained with Coomassie Brilliant Blue.
  • FIG. 21 is a photograph of a stained protein gel showing the constitutive expression of the cyanoglobin (GlbN) protein from Arthrospira platensis PCC 8005 in Cyanobacterium sp. ABICyanol . Protein extracts were separated on a 16% native PAGE and stained with Coomassie Brilliant Blue.
  • GlbN cyanoglobin
  • FIG. 22A and FIG. 22B are photographs of a protein gel showing the expression of the cyanoglobin (GlbN) protein from Arthrospira platensis PCC 8005 in Arthrospira platensis PCC9108 (AB4041, AB4065) and the derivative strain Arthrospira platensis AB 1298 (AB4066).
  • AB4041 has a single copy of the PcpcB-glbN_Ap8005
  • AB4065 and AB4066 comprise two PcpcB-glbN_Ap8005 copies on the same plasmid.
  • Protein extracts were separated on a 16% native PAGE (FIG. 22A, unstained) and stained with Coomassie Brilliant Blue (FIG. 22B).
  • FIG. 23 A is a photograph of a purification column showing the purification of the cyanoglobin (GlbN) protein from Synechocystis strain PCC 6803 expressed in Cyanobacterium sp. ABICyanol strain AB4005 via gel filtration with a Sephacryl 100-HR column.
  • GlbN cyanoglobin
  • FIG. 23B is a photograph of several of the elution fractions (El to E14) which were collected for further analysis. The reddish heme coloration can be seen in the samples.
  • FIG. 24A and FIG. 24B are photpgraphs of protein gels showing the purification of the cyanoglobin (GlbN) protein from Synechocystis strain PCC 6803 expressed in Cyanobacterium sp. ABICyanol strain AB4005 via gel filtration with a Sephacryl 100-HR column. Elution fractions were collected and separated on a 16% native PAGE (FIG. 24 A, unstained) and stained with Coomassie Brilliant Blue (FIG. 24B). CE water-soluble cell extract, E2 - E14 elution fractions.
  • FIG. 25 is a line graph depicting the relative absorbance spectra of cell extracts from Cyanobacterium sp.
  • AB4005 exhibits 2 PcpcB-glbN copies in tandem and an inducible sRNA to knockdown expression of the heme oxygenase ABICyanol . l_orf29000.
  • AB4005 was grown in the absence and presence of 10 ⁇ copper-EDTA to induce the anti-HOl sRNA.
  • the ferric form of GlbN from Synechocystis strain PCC 6803 has a main absorbance peak at 410 nm.
  • a cyanobacterium that is capable of producing a heterologous heme- containing protein.
  • the biomass produced from the modified cyanobacterium can be used to produce various valuable products, such as a protein-rich meal, iron-rich meal, nutritional supplements, food ingredients, or food supplements.
  • the heme-containing protein can be processed to form a meat-like material.
  • the cyanobacterium can be grown outdoors using light, CO2, and nutrients, and can then be processed as needed to produce a red meat-like material which contains a heme-containing protein.
  • the nucleic acid encoding the heme-containing protein can be derived from, for example, an animal sequence, a plant sequence, a bacterial sequence, or a cyanobacterial sequence.
  • the nucleic acid encoding the heme-containing protein can also be a synthetic nucleic acid sequence.
  • cyanobacterium refers to a member from the group of photoautotrophic prokaryotic microorganisms which can utilize solar energy and fix carbon dioxide.
  • Cyanobacteria are also referred to as blue-green algae.
  • host cell and "recombinant host cell” are intended to include a cell suitable for metabolic manipulation, e.g., which can incorporate heterologous polynucleotide sequences, e.g., which can be transformed.
  • the term is intended to include progeny of the cell originally transformed.
  • the cell is a prokaryotic cell, e.g., a cyanobacterial cell.
  • recombinant host cell is intended to include a cell that has already been selected or engineered to have certain desirable properties and to be suitable for further genetic
  • Computer to express refers to a host cell that provides a sufficient cellular
  • the term "genetically modified” refers to any change in the endogenous genome of a wild type cell or to the addition of non-endogenous genetic code to a wild type cell, e.g., the introduction of a heterologous gene. More specifically, such changes are made by the hand of man through the use of recombinant DNA technology or mutagenesis.
  • the changes can involve protein coding sequences or non-protein coding sequences, including regulatory sequences such as promoters or enhancers.
  • polynucleotide and “nucleic acid” also refer to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof.
  • nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs.
  • nucleic acids of this present invention may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide
  • nucleic acid also referred to as polynucleotide
  • nucleic acid molecules having an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences and introns.
  • the terms are intended to include one or more genes that map to a functional locus.
  • the terms are intended to include a specific gene for a selected purpose. The gene can be endogenous to the host cell or can be recombinantly introduced into the host cell.
  • the invention also provides nucleic acids which are at least 60%, 70%, 80%> 90%), 95%), 99%o, or 99.5% identical to the nucleic acids disclosed herein.
  • the percentage of identity of two nucleic acid sequences or two amino acid sequences can be determined using the algorithm of Thompson et al. (CLUSTALW, 1994, Nucleic Acids Research 22: 4673-4680).
  • a nucleotide sequence or an amino acid sequence can also be used as a so-called "query sequence” to perform a search against public nucleic acid or protein sequence databases in order, for example, to identify further unknown homologous promoters, which can also be used in embodiments of this invention.
  • any nucleic acid sequences or protein sequences disclosed in this patent application can also be used as a "query sequence" in order to identify yet unknown sequences in public databases, which can encode for example new enzymes, which could be useful in this invention.
  • Such searches can be performed using the algorithm of Karlin and Altschul (1990, Proceedings of the National Academy of Sciences U.S.A. 87: 2,264 to 2,268), modified as in Karlin and Altschul (1993, Proceedings of the National Academy of Sciences U.S.A. 90: 5,873 to 5,877).
  • Such an algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al. (1990, Journal of Molecular Biology 215: 403 to 410).
  • Suitable parameters for these database searches with these programs are, for example, a score of 100 and a word length of 12 for BLAST nucleotide searches as performed with the NBLAST program.
  • BLAST protein searches are performed with the XBLAST program with a score of 50 and a word length of 3. Where gaps exist between two sequences, gapped BLAST is utilized as described in Altschul et al. (1997, Nucleic Acids Research, 25: 3,389 to 3,402).
  • the invention also provides peptides, polypeptides, and proteins having an amino acid sequence that is at least 50%, 55%, 60%, 70%, 80% 90%, 95%, 99%, or 99.5% or more identical to the amino acid sequence of a protein disclosed herein.
  • the enzyme commission numbers (EC numbers) cited throughout this patent application are numbers which are a numerical classification scheme for enzymes based on the chemical reactions which are catalyzed by the enzymes.
  • Recombinant refers to polynucleotides synthesized or otherwise manipulated in vitro ("recombinant polynucleotides”) and to methods of using recombinant polynucleotides to produce gene products encoded by those polynucleotides in cells or other biological systems.
  • a cloned polynucleotide may be inserted into a suitable expression vector, such as a bacterial plasmid, and the plasmid can be used to transform a suitable host cell.
  • a host cell that comprises the recombinant polynucleotide is referred to as a "recombinant host cell” or a “recombinant bacterium” or a “recombinant cyanobacterium.”
  • the gene is then expressed in the recombinant host cell to produce, e.g., a "recombinant protein.”
  • a recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
  • homologous recombination refers to the process of recombination between two nucleic acid molecules based on nucleic acid sequence similarity.
  • the term embraces both reciprocal and nonreciprocal recombination (also referred to as gene conversion).
  • the recombination can be the result of equivalent or non- equivalent cross-over events. Equivalent crossing over occurs between two equivalent sequences or chromosome regions, whereas nonequivalent crossing over occurs between identical (or substantially identical) segments of nonequivalent sequences or chromosome regions. Unequal crossing over typically results in gene duplications and deletions.
  • non-homologous or random integration refers to any process by which DNA is integrated into the genome that does not involve homologous recombination. It appears to be a random process in which incorporation can occur at any of a large number of genomic locations.
  • expressed endogenously refers to polynucleotides that are native to the host cell and are naturally expressed in the host cell.
  • operably linked refers to a functional relationship between two parts in which the activity of one part (e.g., the ability to regulate transcription) results in an action on the other part (e.g., transcription of the sequence).
  • a polynucleotide is "operably linked to a promoter" when there is a functional linkage between a polynucleotide expression control sequence (such as a promoter or other transcription regulation sequences) and a second polynucleotide sequence (e.g., a native or a heterologous polynucleotide), where the expression control sequence directs transcription of the polynucleotide.
  • vector as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector is a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector is a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid which generally refers to a circular double stranded DNA molecule into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme.
  • PCR polymerase chain reaction
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain 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 “promoter” is an array of nucleic acid control sequences that direct transcription of an associated polynucleotide, which may be a heterologous or native polynucleotide.
  • a promoter includes nucleic acid sequences near the start site of transcription, such as a polymerase binding site. The promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
  • the term “promoter” is intended to include a polynucleotide segment that can transcriptionally control a gene of interest, e.g., a pyruvate decarboxylase gene that it does or does not transcriptionally control in nature.
  • the transcriptional control of a promoter results in an increase in expression of the gene of interest.
  • a promoter is placed 5' to the gene of interest.
  • a heterologous promoter can be used to replace the natural promoter, or can be used in addition to the natural promoter.
  • a promoter can be endogenous with regard to the host cell in which it is used or it can be a heterologous polynucleotide sequence introduced into the host cell, e.g., exogenous with regard to the host cell in which it is used.
  • Promoters of the invention may also be inducible, meaning that certain exogenous stimuli (e.g., nutrient starvation, heat shock, mechanical stress, light exposure, etc.) will induce the promoter leading to the transcription of the gene.
  • recombinant nucleic acid molecule includes a nucleic acid molecule (e.g., a DNA molecule) that has been altered, modified or engineered such that it differs in nucleotide sequence from the native or natural nucleic acid molecule from which the recombinant nucleic acid molecule was derived (e.g., by addition, deletion or substitution of one or more nucleotides).
  • the recombinant nucleic acid molecule e.g., a recombinant DNA molecule
  • Gene refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to a nucleic acid fragment that expresses a specific protein or polypeptide, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
  • endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • a “foreign” gene or “heterologous” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer.
  • Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
  • a “transgene” is a gene that has been introduced into the genome by a transformation procedure.
  • fragment refers to a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence substantially identical to the reference nucleic acid.
  • a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from at least about 6, 50, 100, 200, 500, 1,000, to about 1,500 or more consecutive nucleotides of a polynucleotide according to the invention.
  • ORF open reading frame
  • upstream refers to a nucleotide sequence that is located 5' to reference nucleotide sequence.
  • upstream nucleotide sequences generally relate to sequences that are located on the 5' side of a coding sequence or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.
  • downstream refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence.
  • downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.
  • homology refers to the percent of identity between two polynucleotide or two polypeptide moieties.
  • the correspondence between the sequence from one moiety to another can be determined by techniques known to the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded- specific nuclease(s) and size determination of the digested fragments.
  • substantially similar refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence.
  • substantially similar also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript.
  • restriction endonuclease and “restriction enzyme” refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.
  • expression refers to the transcription and stable accumulation mRNA derived from a nucleic acid or polynucleotide. Expression may also refer to translation of mRNA into a protein or polypeptide.
  • an "expression cassette” or “construct” refers to a series of polynucleotide elements that permit transcription of a gene in a host cell.
  • the expression cassette includes a promoter and a heterologous or native polynucleotide sequence that is transcribed.
  • Expression cassettes or constructs may also include, e.g., transcription termination signals, polyadenylation signals, and enhancer elements.
  • cognate refers to a triplet of nucleotides coding for a single amino acid.
  • codon-anticodon recognition refers to the interaction between a codon on an mRNA molecule and the corresponding anticodon on a tRNA molecule.
  • codon bias refers to the fact that different organisms use different codon frequencies.
  • the term "codon optimization” refers to the modification of at least some of the codons present in a heterologous gene sequence from a triplet code that is infrequently used in the host organism to a triplet code that is more common in the particular host organism. This can result in a higher expression level of the gene of interest.
  • transformation is used herein to mean the insertion of heterologous genetic material into the host cell. Typically, the genetic material is DNA on a plasmid vector, but other means can also be employed. General transformation methods and selectable markers for bacteria and cyanobacteria are known in the art (Wirth, Mol Gen Genet.
  • selectable marker means an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and the like, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest.
  • selectable marker genes include: genes providing resistance to ampicillin, streptomycin, gentamycin, spectinomycin, kanamycin, zeocin, chloramphenicol, hygromycin, and the like.
  • a "polypeptide” is a polymeric compound comprised of covalently linked amino acid residues.
  • a “protein” is a polypeptide that performs a structural or functional role in a living cell.
  • a "heterologous protein” refers to a protein not naturally produced in the cell.
  • fragment of a polypeptide refers to a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide. Such fragments of a polypeptide according to the invention may have a length of at least about 2, 50, 100, 200, or 300 or more amino acids.
  • a "variant" of a polypeptide or protein is any analogue, fragment, derivative, or mutant which is derived from a polypeptide or protein and which retains at least one biological property of the polypeptide or protein.
  • Different variants of the polypeptide or protein may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post-translational modification. The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements.
  • primer is an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double stranded nucleic acid region that can serve as an initiation point for DNA synthesis under suitable conditions. Such primers may be used in a polymerase chain reaction.
  • PCR polymerase chain reaction
  • oligonucleotide primer to the template nucleic acid, and extension of the annealed primer(s) by DNA polymerase.
  • PCR provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.
  • sRNA refers to small non-coding regulatory RNA (typically ⁇ 200bp), which can form a stable secondary structure and is able to bind and affect stability and translation of specific mRNAs in a targeted manner.
  • globin refers to a type of heme-containing globular protein involved in reversible binding and transporting of oxygen utilizing a heme prosthetic group. Globins incorporate a series of alpha helical segments known as globin folds.
  • heme-containing protein or "heme-binding protein” refer to a polypeptide that can bind to a heme group.
  • heme-containing proteins include but are not limited to hemoglobin, cyanoglobin, and leghemoglobin.
  • heme refers to a heterocyclic organic compound cofactor made up of four joined pyrrolic groups, having an iron (Fe 2+ ) group located in the center of the molecule.
  • the heme iron often acts as an electron source or sink during electron transfer or redox chemistry.
  • heme oxygenase refers to an enzyme that catalyzes the degradation of the heme molecule to form other cellular components, such as biliverdin and phycobilins.
  • selection-free refers to a growth medium that does not include a selection agent, such as an antibiotic, that would allow only cells having a functional selectable marker gene to survive.
  • cyanobacterial cells can be modified to express a heterologous heme-containing protein.
  • the modified cells can then be cultured at a large scale, such as in outdoor photobioreactors, using CO2, light, and nutrients, in order to produce large quantities of the heme-containing protein.
  • heme-containing protein can be made without being obtained from animal sources.
  • leghemoglobin is naturally synthesized in the root nodules of certain types of plants, such as leguminous plants that utilize the root nodules for nitrogen fixation.
  • One such plant is soybean.
  • U.S. Patent No. 8,021,695, to Gruber describes a personal care composition (soaps, shampoos, skin care medicaments, cosmetics, and therapeutic formulations) that contains, in part, leghemoglobin extracted from nitrogen fixing root nodules.
  • U.S. Patent Application No. 20110286992A1 describes methods of isolating the leghemoglobin from root nodules in order to prepare such compositions. The nodules have a reddish color due to the concentration of leghemoglobin. This extraction method is time consuming and expensive, however. The crop is planted and grown for a period of time, then the root nodules are collected and washed. The leghemoglobin is then isolated and purified from the washed nodules. This is a time consuming process.
  • WO/2015/153666 discloses the production of ground meat replicas that include a certain amount of a heme-containing protein, such as a leghemoglobin from soybean, pea, or cowpea.
  • the leghemoglobin can be recombinantly produced, for example, in E. coli or yeast.
  • leghemoglobin or other globins or heme-containing proteins
  • cyanobacteria Production of Heme-containing Proteins in Cyanobacterial Cells
  • cyanobacterial cells can be modified to produce the heme-containing protein.
  • the cyanobacterial cell is modified with a gene encoding the heme-containing protein, as well genes encoding other enzymes involved in assembling the heme-protein complex.
  • any suitable cyanobacterial strain can be modified to produce a heme-containing protein.
  • the cyanobacterial host cell is
  • Cyanobacterium sp. ATCC accession number PTA- 13311 (herein termed "ABICyanol” or “AB 1"). This strain has been found to grow well under various outdoor conditions, and can be genetically modified to produce a compound of interest.
  • the strain, as well as its endogenous plasmid p6.8 (FIG. 1, SEQ ID NO: 17), has been described, for example, in U.S. Patent No. 8,846,369, U.S. 9,315,832, and U.S. Patent No. 9, 157,101, all of which are hereby incorporated by reference in their entireties.
  • the cyanobacterial host cell for production of a heme-containing protein is another cyanobacterial strain, such as Arthrospira sp., Synechocystis sp., or Synechococcus sp.
  • a culture of the cyanobacterial cell is grown in an outdoor photobioreactor system, and the resulting cellular material is collected.
  • the entire protein can be separated from the remaining cell contents, or the heme-containing protein itself can be separated from the cellular material.
  • the separated protein can then be dried, frozen, or further processed as desired.
  • leghemoglobin (or other heme-containing proteins) can be recombinantly produced in cyanobacteria.
  • CO2 and inorganic nutrients can be used to convert light to heme-like proteins that can then be further processed, for example, to a meat-like material.
  • One benefit of using cyanobacteria is that CO2, rather than organic carbon- based materials, can be used as the starting material. This lowers the cost of producing the protein, as organic carbon sources are not needed.
  • heme-containing proteins can be heterologously produced in cyanobacteria. Many heme-containing proteins are found in plants.
  • One exemplary heme-containing protein that can be expressed in cyanobacteria is leghemoglobin C2 from soybean ⁇ Glycine max) GenBank No. AAA33980.1.
  • the native DNA sequence is shown in SEQ ID NO: 1.
  • the codon optimized gene sequence (codon optimized for optimal expression in Cyanobacterium sp. ABICyanol) is SEQ ID NO: 2.
  • the protein sequence is SEQ ID NO: 3.
  • cyanobacteria is Leghemoglobin from Astragalus sinicus GenBank No. ABB 13622.1.
  • the native DNA sequence is shown in SEQ ID NO: 4.
  • the codon optimized gene sequence (codon optimized for optimal expression in Cyanobacterium sp. ABICyanol) is SEQ ID NO: 5.
  • the protein sequence is SEQ ID NO: 6.
  • Yet another exemplary heme-containing protein that can be expressed in cyanobacteria is Leghemoglobin from Cajanus cajan GenBank No. KYP60668.1.
  • the native DNA sequence is shown in SEQ ID NO: 7.
  • the codon optimized gene sequence (codon optimized for optimal expression in Cyanobacterium sp. ABICyanol) is SEQ ID NO: 8.
  • the protein sequence is SEQ ID NO: 9.
  • Cyanobacterial organisms also naturally produce certain types of heme-containing proteins.
  • Native heme-containing proteins present in cyanobacteria include, for example, leghemoglobin and cyanoglobin.
  • cyanoglobin from Synechocystis sp. PCC6803 (GenBank No. BAA17991.1).
  • the native DNA sequence is shown in SEQ ID NO: 10.
  • the codon optimized gene sequence (codon optimized for optimal expression in Cyanobacterium sp. ABICyanol) is SEQ ID NO: 11.
  • the protein sequence is SEQ ID NO: 12.
  • cyanoglobin from Arthrospira sp. PCC 8005 (GenBank No. CCE20200.1).
  • the native DNA sequence is shown in SEQ ID NO: 13.
  • the codon optimized gene sequence (codon optimized for optimal expression in Cyanobacterium sp. ABICyanol) is SEQ ID NO: 14.
  • the protein sequence is SEQ ID NO: 15.
  • Cyanobacteria can be modified to add genes of interest as shown herein in order to produce heme-containing proteins.
  • the DNA sequences encoding the genes can be amplified by polymerase chain reaction (PCR) using specific primers.
  • PCR polymerase chain reaction
  • the amplified PCR fragments were digested with the appropriate restriction enzymes and cloned into either a self-replicating plasmid or an integrative plasmid.
  • An antibiotic resistance cassette for selection of positive clones can be present on the appropriate plasmid.
  • the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques.
  • PCR can be used to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.
  • PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, and for nucleic acid sequencing.
  • DNA vectors suitable for transformation of cyanobacteria can be prepared. Techniques for transformation are well known and described in the technical and scientific literature. For example, a DNA sequence encoding one or more of the genes described herein can be combined with
  • the genes of interest are inserted into the cyanobacterial chromosome.
  • the gene insertions can be present in all of the copies of the chromosome, or in some of the copies of the chromosome.
  • the inserted genes are present on an extrachromosomal plasmid.
  • the extrachromosomal plasmid can be derived from an outside source, such as, for example, RSFlOlO-based plasmid vectors, or it can be derived from an endogenous plasmid from the cyanobacterial cell or from another species of cyanobacteria.
  • the inserted genes are present on an extrachromosomal plasmid, wherein the plasmid has multiple copies per cell.
  • the plasmid can be present, for example, at about 1, 3, 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or more copies per host cyanobacterial cell.
  • the plasmid containing the inserted gene is fully segregated in the cell.
  • the inserted genes are present on one cassette driven by one promoter. In another embodiment, the inserted genes are present on separate plasmids, or on multiple cassettes on one plasmid. [00122] In another embodiment, the inserted genes are modified for optimal expression by modifying the nucleic acid sequence to accommodate the cyanobacterial cell's protein translation system. Modifying the nucleic acid sequences in this manner can result in an increased expression of the genes.
  • At least some of the heterologous nucleic acid sequences to be expressed in cyanobacterial host cells can be codon improved for optimal expression in the target
  • codon usage frequency of highly expressed genes is generally correlated to the host cognate tRNA abundance. (Bulmer, Nature 325:728-730; 1987). Codon improvement (sometimes referred to as codon optimization or codon adaptation) can be performed to increase the expression level of foreign genes.
  • Codon improvement of the gene encoding the heme-containing protein can be performed for improved expression in the cyanobacterial host cell. Codon improvement can also be performed by adapting the codon usage of the at least one recombinant gene to the codon usage in Cyanobacterium sp., in particular ABICyanol .
  • the G and/or C wobble bases in the codons for the amino acids in the at least one recombinant gene can be replaced by A and/or T because the G+C content of the genome of ABICyanol is relatively low at about 36%.
  • codon improved In an embodiment, only 2% to 6% or 1% to 10% of the codons of variants of recombinant genes are codon improved. In another embodiment, highly codon improved variants of recombinant genes, at least 25%, to at least 50%, 65% or even at least 70% of the codons have been changed. In another embodiment, recombinant genes are used which are not codon improved.
  • ABICyanol codon usage table derived from ribosomal proteins and highly expressed genes (such as photosynthesis genes).
  • OPTIMIZER a web server for optimizing the codon usage of DNA sequences Nucleic Acids Research 35(suppl 2):W126-W131) based on the codon-usage table derived from ABICyanol genome.
  • the codon adaptation index is a measure of directional synonymous codon usage bias, and its potential applications, (see Nucleic Acids Research 15(3): 1281-1295).
  • the effective number of codons are designed match those of highly expressed genes (such as ribosomal proteins) in the ABICyanol genome.
  • the resulting polynucleotides using improved codons were further modified and optimized to avoid the presence of any known or predicted putative ABICyanol endonuclease restriction sites ⁇ Aval, BsaRI, KasI, Xhol, etc.); internal Shine-Dalgarno sequence and RNA destabilizing sequences; an internal terminator sequence; and a repeat sequence of greater than about 10 bp (see, Welch et al., PLOS One 4, e7002; 2009; and Welch et al., Journal of the Royal Society; Interface 6 (Suppl 4), S467-S476; 2009).
  • the selectable marker genes are also modified so that they will have improved expression in cyanobacteria.
  • the selectable marker gene that confers gentamycin or kanamycin resistance was codon optimized for higher expression in
  • the G+C % of the antibiotic resistance genes decreased from 40-53% to 33-40%, which is similar to that of ABICyanol coding genes (about 36% on average).
  • the codon adaptation index of the codon improved antibiotic resistance genes is significantly improved from less than 0.4 to greater than 0.8, which is similar to that of ABICyanol endogenous genes. Table 1, below, depicts the codon usage statistics within the cyanobacterial strain ABICyanol .
  • the inserted genes can be controlled by one promoter, or they can be controlled by different individual promoters.
  • the promoters can be constitutive or regulatable.
  • the promoters can be, for example, inducible.
  • the promoter sequences can be derived, for example, from the host cell, from another organism, or can be synthetically derived.
  • Any desired promoter can be used to regulate the expression of the inserted genes.
  • Exemplary promoter types include but are not limited to, for example, constitutive promoters, regulatable promoters such as inducible promoters (e.g., by nutrient source, nutrient starvation, heat shock, mechanical stress, environmental stress, metal concentration, specific metabolites, light exposure, etc.), endogenous promoters, heterologous promoters, and the like.
  • Suitable promoter sequences are also disclosed, for example, in U.S. Patent No. 9,315, 820, U.S. Patent No. 9,551,014, PCT/EP2012/067534, U.S. Patent No. 9,476,067, U.S. Patent No. 9,157, 101, PCT/US2013/077364, U.S. Patent No. 9,493,794, and PCT/US2015/000210, all of which are hereby incorporated by reference in their entireties.
  • the recombinant gene(s) can be under the transcriptional control of a constitutive promoter.
  • a constitutive promoter can be endogenous to the cyanobacterial cell. This has the advantage that no recombinant transcription factor has to be present in the host cell.
  • the endogenous promoter is usually well-recognized by the metabolically enhanced cyanobacterial cell without the need to introduce further genetic modifications.
  • Suitable constitutive promoters include, without limitation, the PrpsL promoter
  • ABICyanol_orf2107 PcpcB promoter (ABICyanol _orf2472), PatpG (ABICyanol_orfl814), PrbcL promoter (ABICyanol_orfl369), and variations thereof.
  • suitable endogenous constitutive promoters from genes with unknown function exhibiting appropriate transcriptional activity include, without limitation, the promoters of Gene IDs ABICyano_orfl924,
  • the promoters can be derived from the cyanobacterial strain Cyanobacterium sp. PTA-13311, or they can be derived from another cyanobacterium or from another organism. In an embodiment, the promoters can be about 60%, 70%, 75%, 80%, 85%, 90%), 95%), 97%), 99%), or 100 %> identical to the promoter sequences described herein.
  • the promoters can be regulatable promoters, such as inducible promoters. For example, certain promoters are up-regulated by the presence of a compound, while other promoters can be up-regulated by the absence of a compound (also termed "repressible").
  • promoters that can be used include promoters that are regulatable by the presence (or in other promoters, by the absence) of inductors such as different metal ions, different nutrient sources, different metabolites, different external stimuli such as heat, cold, salinity or light.
  • the regulatable or inducible promoters are induced under conditions such as nutrient starvation, stationary growth phase, heat shock, cold shock, oxidative stress, salt stress, light, darkness, metal ions, organic chemical compounds, and combinations thereof.
  • a particularly tight control of the expression of gene can be achieved if a gene is under the transcriptional control of a Zn-, Ni-, or Co-inducible promoter.
  • the regulatable or inducible promoter is inducible by a change of a metal-ion concentration.
  • a change of metal-ion concentration includes for instance the addition or depletion of certain metal ions.
  • Suitable inducible promoters include, without limitation, the PziaA promoter, the PsmtA promoter, PaztA promoter, the PcorT promoter, the PnrsB promoter, the PpetJ promoter, PpetE promoter, the Porf0316 promoter, the Porf0221 promoter, the Porf0223 promoter, the Porf3126 promoter, the PmntC promoter, and variations thereof.
  • the regulatable or inducible promoter is endogenous to the cells
  • cyanobacterial cell An endogenous inducible promoter is usually well-recognized by the metabolically enhanced cyanobacterial cell without the need to introduce further genetic modifications.
  • the choice of regulatable or inducible promoters can include, but are not limited to, PntcA, PnblA, PisiA, PpetJ, PggpS, PpsbA2, PsigB, PlrtA, PhtpG, PnirA, PnarB, PnrtA, PhspA, PclpBl, PhliB, PcrhC, PziaA, PsmtA, PcorT, PnrsB, PnrsB916, PaztA, PbmtA, Pbxal, PzntA, PczrB, PnmtA, PpstS, and the like.
  • the regulatable or inducible promoter can, for instance, also be a nitrate inducible promoter.
  • Suitable nitrate inducible promoters include, without limitation, the PnirA promoter, the PnrtA promoter, the PnarB promoter, and variations thereof.
  • truncated or partially truncated versions of these promoters including only a small portion of the native promoters upstream of the transcription start point, such as the region ranging from -35 to the transcription start can often be used.
  • introducing nucleotide changes into the promoter sequence e.g. into the TATA box, the operator sequence, 5 '-untranslated region and/or the ribosomal binding site (RBS) can be used to tailor or optimize the promoter strength and/or its induction conditions, e.g. the concentration of inductor required for induction.
  • the different inducible promoters are inducible by different metal ions.
  • the promoters hspA, clpBl, and hliB can be induced by heat shock (raising the growth temperature of the host cell culture from 30°C to 40°C), cold shock (such as, for example, reducing the growth temperature of the cell culture from 30°C to 20°C), oxidative stress (for example by adding oxidants such as hydrogen peroxide to the culture), or osmotic stress (for example by increasing the salinity).
  • the promoter sigB can be induced by stationary growth, heat shock, and osmotic stress.
  • the promoters ntcA and nblA can be induced by decreasing the concentration of nitrogen in the growth medium and the promoters psaA and psbA2 can be induced by low light or high light conditions.
  • the promoter htpG can be induced by osmotic stress and heat shock.
  • the promoter crhC can be induced by cold shock.
  • An increase in copper concentration can be used in order to induce the promoter petE, whereas the promoter petJ is induced by decreasing the copper concentration. Additional details of these promoters can be found, for example, in PCT/EP2009/060526, which is incorporated by reference herein in its entirety.
  • the promoters of any of the above embodiments may be selected from the endogenous inducible promoters identified in Cyanobacterium sp. with the ATCC accession number PTA-13311 ("ABICyanol”) as listed in Table 2 below, and variants thereof.
  • truncated or partially truncated versions of these promoters including only a small portion of the native promoters upstream of the transcription start point, such as the region ranging from -35 to the transcription start can often be used.
  • introducing nucleotide changes into the promoter sequence e.g. into the TATA box, the operator sequence and/or the ribosomal binding site (RBS) can be used to tailor or optimize the promoter strength and/or its induction conditions, e.g. the concentration of inductor required for induction.
  • the cyanobacterial strain ABICyanol is transformed to produce the heme-containing protein.
  • Methods for producing a genetically enhanced, non- naturally occurring cyanobacterial host cells are disclosed herein.
  • methods include introducing a recombinant nucleic acid sequence into a cyanobacterial host cell.
  • At least one recombinant gene can be introduced into the host cells through the transformation of the host cell by an extrachromosomal plasmid.
  • the extrachromosomal plasmid can independently replicate in the host cell.
  • at least one recombinant gene can be introduced into the genome of the host cell.
  • at least one recombinant gene is introduced into the genome of the host cell by homologous recombination.
  • Genetically enhanced cyanobacterial cells can include further genetic
  • genetic enhancements of the genes encoding enzymes of the carbon fixation and subsequent carbohydrate metabolism can be genetically enhanced to further increase the production of a compound of interest.
  • Genetic enhancement targets include, but are not limited to, components of the photosystems (antennas and pigment modification), and components of the photosynthetic and respiratory electron transport systems.
  • enhancement targets include local and global regulatory factors including, but not limited to, the two component system, sigma factors, small regulating RNAs and antisense RNAs.
  • a recombinant nucleic acid sequence can be provided as part of an extrachromosomal plasmid containing cyanobacterial nucleic acid sequences in order to increase the likelihood of success for the transformation.
  • the method for producing a genetically enhanced cyanobacterial cell uses an extrachromosomal plasmid derived from an endogenous plasmid of the host cell to introduce a recombinant nucleic acid sequence into the host cell.
  • This endogenous plasmid can be, for example, an extrachromosomal plasmid derived from the 6.8 kb endogenous plasmid of ABICyanol .
  • the cyanobacterial strain ABICyanol is transformed to produce the heme-containing protein. It has been found that the use of modified endogenous plasmids improves the stability of the plasmid in the host cell.
  • the cyanobacterial strain ABICyanol is transformed to produce the heme-containing protein. It has been found that the use of modified endogenous plasmids improves the stability of the plasmid in the host cell.
  • ABICyanol contains three endogenous plasmids. In combination with other genotypic and phenotypic attributes, these endogenous plasmids differentiate ABICyanol from other cyanobacterial species.
  • One plasmid is 6,826 base pairs, another is 39,702 base pairs, and a third plasmid is 28,554 base pairs.
  • the 6,826 bp endogenous plasmid is alternatively referred to herein as pABICyanol, p6.8 or 6.8.
  • plasmid 6.8 has been modified in vivo and in vitro for use as a plasmid vector containing genes of interest for the production of compounds of interest.
  • a modified endogenous vector derived from p6.8 from ABICyanol was developed.
  • the modified endogenous vector from ABICyanol can be used to transform cyanobacteria from a broad range of genera, including ABICyanol itself.
  • the present invention includes the p6.8 plasmid and modified vectors comprising sequences of the p6.8 plasmid.
  • the modified endogenous vector contains at least one of the following: a recombinant gene that encodes a heme-containing protein; and an origin of replication suitable for replication in ABICyanol .
  • RSFlOlO-based shuttle plasmids extrachromosomal broad-host vector
  • pVZ321-6 series described, for example, in PCT/EP2009/060526 and in
  • a gene coding for a replication initiation factor that binds to the origin of replication can either be present on the modified endogenous vector or can be present in the chromosomes or other extrachromosomal plasmids of ABICyanol .
  • An origin of replication suitable for replication in ABICyanol and the gene coding for the replication initiation factor binding to that origin of replication ensure that the modified endogenous vector can be replicated in ABICyanol .
  • the nucleotide sequence of an origin of replication of the modified endogenous plasmid vector can have at least 80%, 90%, and 95% identity or can be identical to the nucleotides 3375 to 3408 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 17).
  • the sequence of the gene coding for the replication initiation factor has at least 80%, 90%, and 95% identity or is identical to nucleotides 594 to 3779 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 17).
  • the gene coding for the replication initiation factor codes for a protein having at least 80%, 90%, and 95% sequence identity or is identical to the protein coded by nucleotides 594 to 3779 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 17) of ABICyanol .
  • This putative initiation replication factor is thought to bind to the putative origin of replication, thereby ensuring the replication of a plasmid containing the initiation factor in ABICyanol .
  • a modified endogenous plasmid vector can contain a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%), at least 85%, at least 90% or at least 95% identity to the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 17).
  • the modified endogenous vector contains the entire p6.8 endogenous plasmid from ABICyanol .
  • gene delivery vehicles that are developed using the endogenous 6.8 kb plasmid (or a portion of the plasmid) containing characteristic portions of the endogenous 6.8 kb plasmid may be able to be efficiently transformed into a wide range of cyanobacteria.
  • characteristic portions of the 6.8 kb endogenous plasmid from ABICyanol include portions that enable it to replicate in a host cell (origin of replication and replication initiation factor, for example) and can be referred to as the backbone of the endogenous 6.8 kb plasmid.
  • Such vectors may also be able to efficiently produce heterologous proteins and other compounds of interest in cyanobacterial cultures.
  • modifications starting with the backbone of the 6.8 kb endogenous plasmid from ABICyanol are performed individually or together to increase transformation efficiency, increase the replication rate within the cell, and to increase the production of a desired product from the cyanobacterial cell.
  • Suitable modifications include, for example, insertion of selection markers (such as antibiotic resistance genes), recombinant genes or cassettes for the production of a desired compound, and other modifications to increase the expression or stability of the plasmid in the cyanobacterial cell.
  • the invention includes cyanobacteria, e.g. ABICyanol, comprising a modified p6.8 plasmid having any of these improved characteristics.
  • the modular design of the p6.8 derived vector allows complex sequence manipulation in cyanobacteria.
  • the method for producing a genetically enhanced cyanobacterial cell involves protecting the recombinant nucleic acid sequence, for example a plasmid, against endogenous restriction endonucleases of the host cell by methylating at least a part of the recombinant nucleic acid sequence or modifying and/or eliminating the recognition sequences of the endogenous restriction endonucleases.
  • endogenous restriction endonucleases of ABICyanol for example, can cut an extrachromosomal plasmid carrying recombinant genes, thereby preventing a genetic transformation event of this host cell.
  • methyltransferases for example Aval and Acyl
  • the plasmids can either be incubated with the methyltransferases in vitro or a helper plasmid can be present in a helper E. coli strain in order to methylate the extrachromosomal plasmids in vivo before conjugation takes place during the transformation of ABICyanol .
  • recognition sequences for the restriction enzymes can be modified or deleted.
  • plasmid pRL528 can be used as a helper plasmid for conjugal transfer.
  • the indicated genes are M.
  • Aval coding for the methyltransferase protecting against the restriction endonuclease Aval and the respective gene coding for M.
  • Avail The latter is not required for transformation of ABICyanol, as it lacks any endonuclease activity of Avail.
  • the vector to be transformed into cyanobacterial host cells can be modified to integrate into the cyanobacterial chromosome by adding an appropriate DNA sequence homologous to the target region of the host genome.
  • the vector to be transformed can be modified to integrate into the cyanobacterial chromosome through in vivo transposition by introducing mosaic ends to the vector. Once the plasmid is established in the host cell, it can be present, for example, at a range of from 1 to many copies per cell.
  • an endogenous plasmid derived from ABICyanol can be modified, either in vivo or in vitro, to be a plasmid vector capable of introducing exogenous genes encoding enzymes for the production of a compound or compounds of interest into a wide range of host cyanobacterial cells such as ABICyanol, Cyanobacterium sp., or other
  • cyanobacterial genera such as Synechocystis and Synechococcus.
  • the ABICyanol 6.8 kb endogenous plasmid is used as a backbone for a plasmid vector used for transformation of Cyanobacterium sp. Since this is the endogenous vector from the species, it is likely to be more stable when transformed into the cell than plasmids derived from completely different organisms.
  • the entire p6.8 endogenous plasmid is inserted into a vector used for transformation.
  • a sequence of about 50%, 70%, 75%, 80% 85%, 90%, 95%, 98%, 99%, or 99.5% identity to the entire endogenous plasmid sequence is inserted into the extrachromosomal plasmid vector.
  • a modified p6.8 vector is designed to have several modular units that can be swapped out using specific restriction enzymes. Promoters, genes of interest, selectable markers, and other desired sequences can be moved in and out of the vector as desired. This modular design makes genetic experiments faster and more efficient.
  • the modified p6.8 vector can replicate in both cyanobacteria and in E. coli.
  • the vector contains a replication unit that can function in a broad range of cyanobacterial genera.
  • the vector also contains a replicon for propagation in E. coli for ease of cloning and genetic manipulation using E. coli.
  • a plasmid shuttle vector which is characterized by being replicable in both is. coli and cyanobacterial species.
  • the plasmid comprises a promoter capable of functioning in cyanobacteria and E. coli and a DNA sequence encoding a sequence capable of functioning as a selective marker for both E. coli and cyanobacteria.
  • the shuttle vector includes two different promoter systems, one functioning in cyanobacteria and the other one functional in E. coli.
  • the plasmid shuttle vector contains at least 50% of the p6.8 plasmid. The plasmid shuttle vector enables the efficient transformation of cyanobacteria and the expression of recombinant genes of interest.
  • the p6.8 derived plasmid vector also contains an origin of transfer (oriT) which is suitable for conjugation.
  • the plasmid vector can contain a combined origin of replication and an origin of transfer (oriVT), which enables replication in Enterobacteriaceae, in particular E. coli, and which also enables conjugation with, for example, an E. coli donor strain and Cyanobacterium sp., in particular ABICyanol as a recipient strain.
  • Such an plasmid vector can be used for triparental mating wherein a conjugative plasmid present in one bacterial strain assists the transfer of a mobilizable plasmid, for example a plasmid vector disclosed herein, present in a second bacterial strain into a third recipient bacterial strain, which can be ABICyanol.
  • a non-naturally occurring plasmid vector in which a gene encoding a heme-containing protein of interest is operably linked to a shuttle vector.
  • cyanobacterial cells are transformed with the recombinant shuttle vector.
  • the recombinant shuttle vector is relatively small in size, relatively stable in a cyanobacterial host cell, and can replicate in a variety of cyanobacterial species. This recombinant vector is useful for expressing a variety of heterologous genes in cyanobacteria.
  • a shuttle vector expresses a codon-optimized antibiotic resistance gene (Ab R ), such as codon improved kanamycin or gentamycin resistance genes.
  • the shuttle vector is constructed based on a modular basis so that all of the key elements (replication ori, Ab R gene and reporter gene) are exchangeable via unique restriction sites thus providing versatile cloning options and facilitating the delivery of genes of interest to target organisms.
  • Other antibiotic resistance genes can be used if desired.
  • genes conferring resistance to ampicillin, chloramphenicol, erythromycin, zeocin, kanamycin, gentamycin, spectinomycin or other antibiotics can be inserted into the vector, under the control of a suitable promoter.
  • the vector contains more than one antibiotic resistance gene.
  • the plasmid is modified by several factors so that it is capable of efficient replication in multiple types of cyanobacterial species.
  • the vector has also been organized so that various sequences can be easily replaced with other desired sequences as needed.
  • a construct having a different gene (or genes) of interest, a different antibiotic, a different promoter, etc. can be made with relative ease.
  • the modified vector allows for rapid testing of various heterologous constructs in a cyanobacterial cell.
  • RSF1010 vectors having a backbone from a broad-host range bacterial plasmid, such as RSF1010.
  • the RSFlOlO-based vector has been widely used as a conjugation vector for transforming bacteria, including cyanobacteria (Mermet-Bouvier et al. (1993) "Transfer and Replication of RSFlOlO-derived Plasmids in Several Cyanobacteria of the Genera Synechocystis and Synechococcus" Current Microbiology 27:323-327).
  • RSF1010 has an E. coli origin of replication, but does not have a cyanobacterial origin of replication.
  • cyanobacteria disclosed herein can be transformed to add biochemical pathways to produce heme-containing proteins of interest.
  • the recombinant nucleic acids of interest can be amplified from nucleic acid samples using known amplification techniques. PCR can be used to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, and for nucleic acid sequencing.
  • recombinant genes are present on an extrachromosomal plasmid having multiple copies per cell.
  • the plasmid can be present, for example, at about 1, 3, 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or more copies per cyanobacterial host cell.
  • the recombinant plasmids are fully segregated from the non-recombinant plasmids.
  • recombinant genes are present on one cassette driven by one promoter.
  • the recombinant genes are present on separate plasmids, or on different cassettes.
  • recombinant genes are modified for optimal expression by modifying the nucleic acid sequence to accommodate the cyanobacterial cell's protein translation system. Modifying the nucleic acid sequences in this manner can result in an increased expression of the genes.
  • Some strains of cyanobacteria can be transformed through natural uptake of exogenous DNA.
  • Other cyanobacterial strains can be transformed, for example, by the use of conjugation or electroporation.
  • Some cyanobacterial strains are difficult to transform by any known means. For many of these types of difficult to transform strains, specific methods of preparing the cells for transformation, as well as specific methods of allowing entry of the foreign DNA into the cells, need to be designed de novo.
  • Exemplary cyanobacteria that can be transformed include, but are not limited to,
  • Synechocystis Synechococcus, Synechococcus, Acaryochloris, Anabaena, Arthrospira, Thermosynechococcus, Chamae siphon, Chroococcus, Cyanobacterium, Cyanobium, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus, Prochloron, Chroococcidiopsis, Cyanocystis, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria, Xenococcus, Borzia, Crinalium, Geitlerinema, Halospirulina, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Cyanodictyon, Aphanocapsa, Oscillatoria, Planktothrix, Prochlorothrix, Pseudan
  • Chlorogloeopsis Fischerella, Geitleria, Nostochopsis, Iyengariella, Stigonema, Rivularia, Scytonema, Tolypothrix, Cyanothece, Phormidium, Adrianema, and the like.
  • Exemplary methods suitable for transformation of Cyanobacteria include, as nonlimiting examples, natural DNA uptake (Chung, et al. (1998) FEMS Microbiol. Lett. 164: 353-361; Frigaard, et al. (2004) Methods Mol. Biol. 274: 325-40; Zang, et al. (2007) J.
  • Microbiol. Biotechnol. 78: 729-35 laser-mediated transformation, or incubation with DNA in the presence of or after pre-treatment with any of poly(amidoamine) dendrimers (Pasupathy, et al. (2008) Biotechnol. J. 3 : 1078-82), polyethylene glycol (Ohnuma, et al. (2008) Plant Cell Physiol. 49: 117-120), cationic lipids (Muradawa, et al. (2008) J. Biosci. Bioeng. 105: 77-80), dextran, calcium phosphate, or calcium chloride (Mendez-Alvarez, et al. (1994) J. Bacteriol.
  • the cyanobacterial strain ABICyanol is used as the host cell for production of the heme-containing protein.
  • electroporation can also be used for successful transformation of ABICyanol, although at a somewhat lower efficiency. Strain-specific adaptations of standard electroporation protocols may be made to avoid DNA digestion by endogenous restriction enzymes and to allow DNA entry through the EPS layer of ABICyanol host cells. To achieve successful electroporation, DNA may be protected against endogenous restriction enzymes by methylation.
  • ABICyanol cells Prior to electroporation using techniques well known in the art, ABICyanol cells may be pretreated with positively charged polyaminoacids such as poly-L-lysine hydrobromide or poly-L-ornithine hydrochloride or combinations thereof (in particular poly-L-lysine hydrobromide) in order to increase the DNA uptake efficiency.
  • positively charged polyaminoacids such as poly-L-lysine hydrobromide or poly-L-ornithine hydrochloride or combinations thereof (in particular poly-L-lysine hydrobromide)
  • the presence of a foreign gene encoding antibiotic resistance in a cell is selected by placing putatively transformed cells into a media containing an amount of the corresponding antibiotic and selecting cells that survive. The selected cells are then grown in the appropriate culture medium to allow for further testing.
  • colony PCR methods are used to confirm transformants.
  • three primer sets are used and are directed against parts of the shuttle vectors to detect specific fragments of the shuttle vector. Transformants exhibiting the predicted PCR products are analyzed further by plasmid rescue.
  • a 25 mL liquid culture is subjected to DNA isolation.
  • plasmid rescue 500 ng to ⁇ g of isolated DNA from transformants containing the transformed plasmids is re-transformed into E. coli and usually results in approximately 10-20 transformants per transformation implemented. Plasmid DNA of ten E. coli colonies is isolated and analyzed by PCR using specific primers for the transformed plasmids. The plasmid DNA is further analyzed with specific restriction enzymes and then sequenced.
  • Another method of increasing the heme-containing protein level is by additional modifications that result in an increase in the flux through ferrochelatase. This can be accomplished, for example, by a knockdown of the gene encoding magnesium chelatase
  • FIG. 14 provides a schematic diagram showing the pathway interactions that can affect the levels of production of the heme-containing protein.
  • Culture medium modifications can also be used to increase the level of the heme-containing protein produced in the cyanobacterial cells. Examples of these modifications include but are not limited to magnesium-starvation, iron supplementation, and nitrogen-starvation. A combination of these methods and genetic modifications can also be used.
  • Heme-containing proteins can be produced at even higher levels when the host cells have a phycocyanin-deficient background. This is demonstrated in Examples 13 and 14.
  • the phycocyanin deficiency can be obtained, for example, by a knockout of the phycocyanin gene.
  • the phycocyanin deficiency can also be obtained, for example, by an inducible or constitutive knockdown of the phycocyanin gene, such as by the use of a sRNA or anti sense molecule that targets phycocyanin levels.
  • a heme-containing protein is synthesized in cyanobacterial cultures by preparing host cyanobacterial cells having the gene constructs discussed herein, and growing cultures of the cells.
  • Methods of growing cyanobacteria in liquid media and on agarose-containing plates are well known to those skilled in the art (see, e.g., websites associated with ATCC). Any of these methods or media maybe used to culture the cyanobacterial cells.
  • a number of known recipes for cyanobacterial growth medium can be used.
  • BG11 medium is used, see Stanier, R.Y., et al., Bacteriol. Rev. 1971, 35: 171-205, which is hereby incorporated by reference.
  • the cyanobacterial strain is a fresh water strain, and BG11 is used.
  • the cyanobacteria culture grows best in a marine (salt water) medium, by adding an amount of salt to the BG11 medium.
  • marine BG11 mBGl 1
  • the cells are grown autotrophically, and the only carbon source is CO2.
  • the cells are grown mixotrophically, for example with the addition of another carbon source such as glycerol or glucose.
  • the cultures can be grown indoors or outdoors.
  • the light cycle can be set as for continuous light, or for periodic exposure to light, e.g., 16 hours on and 8 hours off, or 14 hours on and 10 hours off, or 12 hours on and 2 hours off, or any alternative variation of on and off hours of light comprising about a day.
  • the cultures can be axenic, or the cultures can also contain other contaminating species.
  • the cyanobacteria are grown in enclosed bioreactors in quantities of at least about 1 L, 20L, 50L, 100 L, 500 L, 1,000 L, 2,000 L, 5,000 L, or more. In a preferred embodiment the bioreactors are about 20 L to about 100 L. In an embodiment, the cyanobacterial cell cultures are grown in disposable, flexible, and tubular photobioreactors made of a clear plastic material.
  • cultures are grown indoors or outdoors with continuous light, in a sterile environment.
  • the cultures are grown outdoors in an open pond type of photobioreactor.
  • the choice of culture medium can depend on the cyanobacterial species.
  • the following BG11 medium for growing cyanobacteria can be used.
  • NaCl is added to the culture medium.
  • PCR was performed using an Eppendorf Mastercycler thermocycler (Hauppauge, NY), using Phire II Hot Start polymerase or Taq DNA polymerase (NEB) for diagnostic amplifications, and Phusion polymerase or Crimson LongAmp Taq Polymerase (NEB) for high fidelity amplifications. PCR temperature profiles were set up as recommended by the Taq manufacturer. Cloning was performed in E. coli using XLIO-Gold Ultracompetent cells (Agilent Technologies, Santa Clara, CA) following the manufacturer's protocol. TOPO cloning kits (Zero Blunt TOPO PCR Cloning kit) were purchased from
  • E. coli strains HB101 Promega, Madison, WI
  • XLIO-Gold Stratagene, San Diego, CA
  • a-select Bioline
  • LB Luria-Bertani
  • Ampicillin 50 ⁇ g/mL
  • kanamycin 50 ⁇ g/mL
  • chloramphenicol 34 ⁇ g/mL
  • Cultures were continuously shaken overnight at 200 rpm and at 100 rpm when used for conjugation.
  • ABICyanol was cultured at from 28 °C to 37 °C in liquid BG11 fresh water medium on a reciprocal shaker at 150 rpm under continuous illumination of approximately 30 - 40 ⁇ photons*m -2 *sec _1 .
  • Plasmid DNA from E. coli strains was isolated using a GeneJet Plasmid Miniprep
  • Kit Fermentas according to the manufacture's protocol.
  • total DNA was prepared according to Saha et al. (2005), World Jour. Microbiol Biotechnol 21 :877-881.
  • BG-11 stock solution was purchased from Sigma
  • Marine BG-11 (MBG-11) was prepared by dissolving 35 g Instant Ocean (United Pet Group, Inc, Cincinnati, OH) in 1 L water and supplementing with BG-11 stock solution.
  • Vitamin B12 (Sigma Aldrich) was supplemented to MBG-11 to achieve a final concentration of 1 ⁇ g/L, as needed.
  • Stock solutions of the antibiotics spectinomycin (100 mg/ml) and kanamycin (50 mg/ml) were purchased from Teknova (Hollister, CA).
  • Stock solution of the antibiotic gentamycin (10 mg/ml) was purchased from MP Biomedicals (Solon, OH).
  • the ABICyanol transformants were selected on solid BG1 1 medium containing 10 - 20 ⁇ g/mL kanamycin and were maintained on BG11 plates containing 40 ⁇ g/mL kanamycin. For growth in liquid freshwater BG11 medium, 30-40 ⁇ g/mL of kanamycin was applied.
  • Example 3
  • the endogenous 6.8 kb plasmid of ABICyanol was used as a means of shuttling exogenous DNA to cyanobacterial host cells.
  • an origin of replication that is effective in E. coli such as R6KOri
  • the p6.8 kb plasmid DNA was able to be manipulated in bacteria, such as E. coli, to incorporate genes and sequences of interest into a recombinant p6.8 kb.
  • modifications to decrease the effectiveness of endogenous restriction systems that are present in ABICyanol, such as methylation were performed.
  • the cell pellet was resuspended in 0.5 M sucrose and incubated 60 minutes at room temperature (RT) with slow shaking (85 rpm). Cells were then centrifuged and resuspended in 40 mL of a solution containing 50mM Tris (pH 8.0), 10 mM EDTA (pH 8.0), 4% sucrose, and 20-40 ⁇ g/mL lysozyme.
  • Triparental mating was performed as follows: E. coli strain J53 bearing a conjugative RP4 plasmid and E. coli strain HB101 bearing the cargo to be introduced into ABICyanol and the pRL528 helper plasmid (for in vivo methylation) were used.
  • E. coli strains were grown in LB broth supplemented with the appropriate antibiotic overnight at 37 °C with shaking at 100 rpm. An aliquot of 3 - 5 mL of each culture was centrifuged, washed twice with LB medium and resuspended in 200 ⁇ iL LB medium. Subsequently, the E. coli strains were mixed, centrifuged and resuspended in 100 [iL BG11 medium. Two hundred mL of
  • cyanobacterial culture (OD750nm of greater than 0.5 and less than 1.0) was centrifuged (3,000 rpm, 10 min), pretreated to degrade the EPS layer as described above in Example 4, and subsequently washed and resuspended in 400 ⁇ BG11 culture medium containing Tris/sucrose buffer.
  • a 100 ⁇ L aliquot of resuspended cyanobacterial and E. coli cultures was mixed and applied onto a membrane filter (Millipore GVWP, 0.22 ⁇ m. pore size) placed on the surface of solid BG11 medium supplemented with 5% LB. Petri dishes were incubated under dim light (5 ⁇ m -2 s -1 ) for 2 days.
  • Electroporation can also be used for successful transformation of
  • Cyanobacterium sp. ABICyanol or other strains such as Arthrospira, Synechococcus, and Synechocystis, using, for example, the same plasmids as for conjugation, but with lower efficiency.
  • strain-specific adaptations of standard electroporation protocols can be made to avoid DNA digestion by endogenous restriction enzymes and to allow DNA entry through the EPS layer.
  • DNA is protected against endogenous restriction enzymes by methylation.
  • ABICyanol cells Prior to electroporation, ABICyanol cells are pretreated with positively charged polyaminoacids such as poly-L-lysine hydrobromide or poly-L-ornithine hydrochloride or combinations thereof (in particular poly-L-lysine hydrobromide) in order to increase the DNA uptake efficiency.
  • ABICyanol culture corresponding to a cell density of approximately 2xl0 7 cells/mL
  • ABICyanol culture was harvested, washed and resuspended in 0.9 % NaCl containing 25 mM Tris-HCl (pH 8.0).
  • Poly-L-lysine hydrobromide was added to the resuspended cells to obtain a final concentration of 50 ⁇ g/mL.
  • ABICyanol cells were then incubated for several hours or overnight before electroporation.
  • the cells were washed sequentially once more with 1 mM HEPES and ETMT buffer containing 0.1 mM HEPES, 0.2 mM K2HPO4 and 0.2 mM MgCh.
  • the cells were harvested by centrifugation at 15,000 x g for 5 minutes. All of the washes and centrifugations were carried out on ice or in a pre-chilled centrifuge (4 °C). For each electroporation procedure, 3 ⁇ g methylated DNA was added to 100 ⁇ of concentrated cells.
  • BGl 1 medium was immediately added to the cyanobactenal suspension, which was subsequently transferred to a 50 mL flask containing 15 mL fresh BGl 1 medium.
  • coli culture was centrifuged, washed twice with Zarrouk' s medium and resuspended in 100 ⁇ L Zarrouk' s medium.
  • 10 mL of a late exponentially growing Arthrospira platensis culture (OD750nm between 0.5 and 1.0) was centrifuged (3,000 rpm, 10 min), washed and resuspended in 200 ⁇ L Zarrouk's medium.
  • a 100 ⁇ L aliquot of resuspended cyanobactenal and E. coli cultures was then mixed and incubated under dim light (5 ⁇ m -2 s -1 ) for 2-3 days.
  • cyanoglobin (glbN) gene from Synechocystis PCC 6803 (SEQ ID NO: 11), which was codon optimized for optimal expression, was inserted to the p6.8 modified endogenous plasmid of Cyanobacterium sp. ABICyanol, under the control of an endogenous zinc-inducible smtA promoter. This created a 10,340 bp construct which was designated
  • the antisense sRNA lessened the presence of heme oxygenase, causing a higher content of the heme groups to be available to bind to the heterologous heme-containing protein being expressed in the cyanobacteria. This resulted in higher production of the mature heme-protein complex (See Example 10, below).
  • the inducible sRNA is designed to increase the flux through the ferrochelatase.
  • An increased flux through the ferrochelatase is accomplished by different means such as, for example, a knockdown of the gene encoding magnesium chelatase (MgCh), the overexpression of ferrochelatase (FeCh), the knockdown of gun4 (MgCh activator), a gun4(W192A) mutation, magnesium-starvation, iron supplementation, or nitrogen- starvation.
  • the cells were transformed with the plasmid construct, and were then grown in culture medium. Cells were isolated from the medium, and the protein was extracted from the cell material. The protein extracts were separated on a 16% native PAGE and stained with Coomassie Brilliant Blue (FIG. 16). The data shows that when nitrate was added to the culture to induce production of the anti-heme oxygenase sRNA, less heme oxygenase and thereby more heme-containing protein was produced (black arrow, right lane, FIG. 16).
  • leghemoglobin C2 gene from soybean (SEQ ID NO: 1) was codon optimized (SEQ ID NO: 2) for optimal expression in Cyanobacterium sp. ABICyanol .
  • the gene was placed under the control of an endogenous zinc- inducible smtA promoter on the p6.8 modified endogenous ABICyanol plasmid.
  • the plasmid also encoded an sRNA directed against the ribosomal binding site of the heme oxygenase ABICyanol .
  • Transformed cells were then grown in culture medium and examined for leghemoglobin production under various induction conditions. Protein extracts were separated by denaturing electrophoresis on a 16% Tricine-SDS-PAGE including 6 M urea and subjected to a Coomassie Brilliant Blue staining.
  • the protein gel (FIG. 17) demonstrated that the zinc-inducible promoter delayed expression of the soybean leghemoglobin C2 protein (arrow) until zinc was added to the medium. Upon addition of zinc, the leghemoglobin was produced. Upon addition of nitrate, the sRNA against heme oxygenase was also induced. The combination of the induction of the leghemoglobin gene and the anti-heme oxygenase sRNA resulted in strong production of the leghemoglobin protein.
  • Cyanobacterium sp. ABICyanol following the method described in the above example, except that the cyanoglobin gene was placed under the regulatory control of an endogenous, strong constitutive promoter PcpcB, rather than an inducible smtA promoter.
  • the plasmid #2942 ⁇ pABICyanol_6.8: :PcpcB-glbN_S6803(ABICyanolopt)-TpsaB-Porf0316'-anti_HOl_vl allowed constitutive and high expression of the glbN gene during the full length of the culture growth. This was found to result in greater cyanoglobin production per day.
  • the p6.8 plasmid that carried the heterologous gene is generally present in many copies per cell.
  • an additional construct #3022 ⁇ pABICyanol_6.8: :PcpcB-glbN_S6803(ABICyanolopt)- TpsaB-PcpcB-glbN_S6803(ABICyanolopt)-TpsaB-Porf0316'-anti_HOl_vl (SEQ ID NO: 58) was created that has two copies of the gene encoding the cyanoglobin glbN protein, each with its own PcpcB promoter.
  • Both plasmids encode the anti-HOl sRNA sequence downstream of the endogenous copper-inducible promoter from Cyanobacterium sp. ABICyanol .
  • the plasmids were transformed to Cyanobacterium sp. ABICyanol following the method described in Examples 3-5.
  • the cells were then grown in culture medium. Cells were isolated from the medium, and the protein was extracted from the cell material. Protein extracts were separated by denaturing electrophoresis on a 16% Tricine-SDS-PAGE including 6 M urea.
  • the protein gel of FIG. 18A was unstained, and the heme-containing protein can be seen as a reddish band.
  • FIG. 18B shows a duplicate protein gel, which was subjected to Coomassie Brilliant Blue stain to visualize the total proteins.
  • the Cyanobacterium sp. ABICyanol strain was grown under high-light stress in order to isolate derivatives with altered phycocyanin expression.
  • the applied conditions were as follows: a BGl 1-agar plate with a cell lawn of ABICyanol was exposed to high-light (3,000 ⁇ m -2 s -1 ) for 90 minutes, resulting in a cell death rate of about 99.99 %. Individual colonies that survived the treatment were picked and patched onto new plates for later characterization. UV- VIS absorbance spectra from individual isolates identified cell lines with reduced phycocyanin content. FIG.
  • heme-containing protein was compared with that of host cells that had a normal amount of phycocyanin.
  • the strains were grown in the absence and presence of 10 ⁇ copper-EDTA to induce the knockdown of the heme oxygenase via anti-HOl sRNA.
  • AB4011 has a single copy of the PcpcB-glbN_S6803, whereas AB4012 comprises two PcpcB-glbN_S6803 copies on the same plasmid. Protein extracts were separated on a 16% native PAGE and stained with Coomassie Brilliant Blue.
  • Synechocystis strain PCC 6803 in the AcpcA derivative AB4011 (lx PcpcB-glbN) was generally lower compared to the expression in the phycocyanin-rich ABICyanol derivative AB1336.
  • the copper-induced knockdown of the heme oxygenase in AB4011 had a much stronger effect on the expression of glbN, compared to the relatively mild effects of the heme oxygenase knockdown in AB1336 on the glbN expression (FIG. 20).
  • AB4012 (2x PcpcB-glbN copies) expressed a significant amount of apo-GlbN (cyanoglobin without heme), indicating a lack of sufficient heme caused by a potential higher turnover of heme via the heme oxygenase.
  • knockdown of the heme oxygenase in AB4012 resulted in strongly expressed and fully assembled heme protein.
  • Construct #2994, strain AB1364 A codon-optimized cyanoglobin (glbN) gene
  • Construct #2949 (SEQ ID NO: 59), strain AB4041 : The codon optimized cyanoglobin (glbN) gene from Arthrospira sp. PCC 8005 (SEQ ID NO: 14) was inserted into chromosome of Arthrospira platensis PCC9108 via homologous recombination.
  • the cyanoglobin gene was placed under the control of the endogenous constitutive promoter PcpcB from Arthrospira platensis PCC9108.
  • the gene cassette was inserted into the chromosome of Arthrospira platensis PCC9108 via homologous recombination following the methods described in Examples 6-7.
  • Construct #3057 (SEQ ID NO: 60), strains AB4065 and AB4066: In order to maximize production of the heterologous heme-containing protein, an additional construct #3057 ⁇ oriVT-nblA2_2293_up-PcpcB1918-glbN_Ap8005-PcpcB1918_glbN_Ap8005-TpsaB- PrbcL2293-ble-TB1002-nblA2_2293_down was created that has two copies of the codon- optimized gene encoding the cyanoglobin glbN protein Arthrospira sp.
  • PCC 8005 (SEQ ID NO: 14), each with its own PcpcB promoter from Arthrospira platensis PCC9108.
  • the gene cassette was inserted into chromosome of Arthrospira platensis PCC9108 and the derivative strain Arthrospira platensis AB 1298 via homologous recombination following the methods described in Examples 6-7.
  • FIG. 22A is a photograph of the unstained protein gel.
  • FIG. 22B is a photograph of an identical gel, stained with Coomassie Brilliant Blue to identify total protein.
  • AB4041 has a single copy of the PcpcB-glbN_Ap8005, whereas AB4065 and AB4066 comprise two PcpcB-glbN_Ap8005 copies on the same plasmid.
  • Cyanobacterium sp. ABICyanol strain AB4005 was grown in stirred 1 L photobioreactors (12/12 h day/night cycle) in artificial seawater, supplemented with BG11 trace elements and 100 ⁇ Fe(II)NH4 sulfate. The pH was maintained at pH 7.3 by addition of 5 % CO2 on demand with a flow-rate of 20 - 40 ml/min. AB4005 exhibited constitutive expression of a codon-optimized cyanoglobin from Synechocystis sp. PCC6803 (2 copies in tandem). 10 ⁇ CuEDT A was added to induce the sRNA knockdown of heme oxygenase
  • ABICyanol . l_orf29000 200 ml of the culture was harvested via centrifugation for preparation of a soluble protein extract (Tris-HCl pH 8.0, 10 mM EDTA, 10 mM sodium dithionite), followed by a concentration step with lOkDa centrifugal filters. 100 ⁇ _, of the soluble protein concentrate was loaded on a 30 mL Sephacryl 100-HR gel filtration column driven by gravity flow (FIG. 23 A). Elution fractions El through E14 (FIG. 23B) were collected for further analysis.
  • a soluble protein extract Tris-HCl pH 8.0, 10 mM EDTA, 10 mM sodium dithionite
  • 100 ⁇ _ of the soluble protein concentrate was loaded on a 30 mL Sephacryl 100-HR gel filtration column driven by gravity flow (FIG. 23 A). Elution fractions El through E14 (FIG. 23B) were collected for further analysis.
  • FIG. 24A A photograph of the unstained gel (showing the reddish-colored heme fractions) is shown in FIG. 24A.
  • FIG. 24B A duplicate gel that has been stained with Coomassie Brilliant Blue stain to identify total protein is shown in FIG. 24B.
  • the left lane of both gels is the control sample (water soluble extract; labeled "CE”.
  • Two methods can be used for determination of the concentration of heme- containing proteins in the bulk extraction.
  • One method is the use of absorbance spectroscopy with known extinction coefficients.
  • the ferric form of GlbN from Synechocystis strain PCC 6803 has a main absorbance peak at 410 nm. In this way, one can determine the relative induction of the heme-containing protein in the induced versus either non-induced or wild type strains.
  • FIG. 25 depicts the relative absorbance spectra of cell extracts from Cyanobacterium sp. ABICyanol expressing the cyanoglobin (GlbN) protein from
  • AB4005 contains two PcpcB-glbN copies in tandem and an inducible sRNA to knockdown expression of the heme oxygenase ABICyanol . I_orf29000.
  • AB4005 cells were grown in either the absence or presence of 10 ⁇ copper-EDTA to induce the anti-HOl sRNA.
  • the absorbance peak at 410 nm shows the ferric form of GlbN from Synechocystis strain PCC 6803.
  • Another method of analysis involves heme staining following gel electrophoresis.
  • This method is used to determine accumulation levels in producers since the heme component of heme-containing proteins remains bound to the protein as it migrates through the gel. Molecular weights of all heme proteins present can also be determined using this method.
  • a culture of modified cyanobacteria is scaled-up to flasks, then 1 liter containers, then 5 liter containers, then to outdoor vertical photobioreactors.
  • the host cells are then grown without induction of the inserted genes for most of the length of the culture period (such as, for example, about 20 days). Near the end of the culture period (approximately 2 to 4 days prior to culture harvest), the production of the heme-containing protein is induced. At this time, other modifications can also be induced, such as modifications to increase the heme that is available for binding.
  • the culture can then be harvested, and the cells can be dried to produce a dried biomass material containing the heme-containing protein. Alternatively, the total protein can be isolated from the cell material. Further, the heme-containing protein can be substantially purified from the total protein fraction, if desired.
  • Protein concentration in the cellular extracts is determined using the Bio-Rad DC protein assay Kit (Bio-Rad; Hercules, CA; Cat# 500-0111) according to the manufacturer's instructions. Bovine serum albumin is used as the standard.
  • a modified strain of Arthrospira platensis is modified as described in one of the above examples, to produce large amounts of an exogenous heme-containing protein.
  • the culture is grown for 3 weeks in an outdoor photobioreactor.
  • the culture is harvested, and the biomass is dried.
  • the resulting biomass has a heme-bound iron content that is much higher than the iron content of wild type Arthrospira, and can thus be used as an iron-rich food supplement that is also rich in other nutrients.
  • the food supplement can be formed into flakes, a powder, a tablet, or a capsule.
  • a modified strain of Arthrospira platensis is modified as described in the above example, except that a substantial amount of the phycocyanin is isolated from the biomass before it is processed for its use as an iron-rich food supplement. In this way, two valuable products, phycocyanin and an iron-rich food supplement, can be obtained from the cyanobacterial culture. If desired, the product can be pasteurized or sterilized during the processing step.
  • Example 23
  • Extraction and isolation of a protein product enriched with a heme-containing protein is achieved by removal of the lipid and hydrophobic fraction of the biomass with a nonpolar solvent. The remaining material is then precipitated using a pH shift. The protein-rich fraction is collected. The protein-rich material is desalted by filtration or buffer exchange. Optionally, the desalted protein-rich material is then dried to a fine powder.
  • the protein-rich material of the above example is obtained and formulated into a vegetarian, meat-like food product.
  • the gene encoding the heme-containing protein can be derived from a cyanobacterial source, an algal source, a plant source, or from an animal source.
  • the heme-containing protein can be first produced in the cell as an apoprotein without bound heme, and can then be extracted from the cell biomass, purified, then further modified chemically to add in the heme-containing moiety.

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Abstract

Modified cyanobacterial cells that are capable of producing various heme-containing proteins (such as cyanoglobin or leghemoglobin) are described, as well as methods of producing the heme-containing proteins from the cells, and methods of use for protein isolates and food products. Further disclosed are cyanobacterial genera that can be used for producing various heme-containing proteins.

Description

TITLE
PRODUCTION OF HEME-CONTAINING PROTEINS IN CYANOBACTERIA CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 62/573,088, filed on October 16, 2017, the disclosure of which is hereby incorporated by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] Not Applicable.
REFERENCE TO SEQUENCE LISTING
[0003] This application contains a sequence listing submitted by EFS-Web, thereby satisfying the requirements of 37 C.F.R. §§ 1.821-1.825. The sequence listing, created on October 9, 2018, contains 60 sequences and is 195 KB in size.
FIELD OF THE INVENTION
[0004] The present invention relates generally to the recombinant production of heme-containing proteins in cyanobacterial host cells. The heme-containing proteins can be used, for example, in meat substitute products.
BACKGROUND OF THE INVENTION
[0005] A vegetarian diet is preferred by many consumers. Non-animal derived meat-like material, that tastes and looks like red meat but is made entirely of non-animal ingredients, would be a valuable consumer product. However, many vegetarian meat-like products are lacking in taste and color. Ideally, a red meat-like product would have a pleasing taste and would look and process like an actual meat product. For example, a vegetarian-based ground meat or sausage meat that is similar to real meat would be desirable.
[0006] Modified cyanobacteria can be used to produce many types of proteins and other products in an environmentally friendly manner. The transformation of the cyanobacterial genus Synechococcus with genes of interest has been described (U.S. Patent Nos. 6,699,696 and 6,306,639, both to Woods et al). The transformation of the cyanobacterial genus Synechocystis has been described, for example, in PCT/EP2009/000892 and in PCT/EP2009/060526. The transformation of the cyanobacterial genus Cyanobacterium sp. has been described (U.S. Patent No. 8,846,369, U.S. Patent No. 9,315,832, and PCT/US2013/077364).
SUMMARY OF THE INVENTION
[0007] In an embodiment of the invention, a genetically modified cyanobacterium having a polynucleotide encoding a heterologous heme-containing protein is provided. The protein can be, for example, a leghemoglobin or a cyanoglobin. The polynucleotide can be, for example, codon optimized for optimal expression in the cyanobacterium, and can be linked to a constitutive or inducible (regulatable) promoter. The polynucleotide can be located on an endogenous or heterologous extrachromosomal plasmid, or can be integrated into the
chromosome.
[0008] In an embodiment of the invention, the cyanobacterium can also have a modification that increases heme levels. This can be accomplished, for example, by a knockdown of endogenous heme oxygenase HOI . This can also be accomplished by a modification that increases the flux through ferrochelatase, such as by a knockdown of the gene encoding magnesium chelatase (MgCh), the overexpression of ferrochelatase (FeCh), the knockdown of gun4 (MgCh activator), a gun4(W192A) mutation, magnesium-starvation, iron supplementation, or nitrogen- starvation.
[0009] In another embodiment, the cyanobacterium can also have a modification that results in a deficiency in phycocyanin.
[0010] In yet another embodiment of the invention, a method of producing a heme-containing protein in a cyanobacterium is provided, by growing the genetically modified cyanobacterium under conditions to produce the heme-containing protein; and obtaining the heme-containing protein from the cyanobacterium.
[0011] In another embodiment of the invention, a method of producing a heme-containing protein in a cyanobacterium is provided, by growing the genetically modified cyanobacterium under conditions to produce the heme-containing protein and additionally under conditions to increase heme production in the cyanobacterium, then obtaining the heme-containing protein from the cyanobacterium. [0012] In yet another embodiment of the invention, a biomass containing the genetically modified cyanobacterium is provided. In yet another embodiment of the invention, a dried meal (or bulk protein) obtained from the genetically modified cyanobacterium is provided. In a further embodiment of the invention, a partially purified heme-containing protein obtained from the genetically modified cyanobacterium is provided.
[0013] In a further embodiment of the invention, a food product prepared from the heme- containing protein made from the genetically modified cyanobacterium is provided. The food product can be, for example, a protein-rich product such as a meat substitute.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a map of the endogenous 6.8 kb plasmid isolated from Cyanobacterium sp. ABICyanol (SEQ ID NO: 17).
[0015] FIG. 2 depicts a map of the 10403 BP circular plasmid construct and sequence annotation of plasmid #2884 (pAB l_6.8: :PsmtA-legH_Gm(ABlopt)-TpsaB) (SEQ ID NO: 18).
[0016] FIG. 3 depicts a map of the 10412 BP circular plasmid construct and sequence annotation of plasmid #2885 (pAB l_6.8: :PsmtA-legH_As(AB lopt)-TpsaB) (SEQ ID NO: 19).
[0017] FIG. 4 depicts a map of the 10406 BP circular plasmid construct and sequence annotation of plasmid # #2886 (pABl_6.8: :PsmtA-legH_Cc(ABlopt)-TpsaB) (SEQ ID NO: 20).
[0018] FIG. 5 depicts a map of the 10340 BP circular plasmid construct and sequence annotation of plasmid # 2887 (pABl_6.8: :PsmtA-glbN_S6803(ABlopt)-TpsaB) (SEQ ID NO: 21).
[0019] FIG. 6 depicts a map of the 10340 BP circular plasmid construct and sequence annotation of plasmid #2888 (pAB l_6.8: :PsmtA-glbN_Ap8005(ABlopt)-TpsaB) (SEQ ID NO: 22).
[0020] FIG. 7 depicts a map of the 10456 BP circular plasmid construct and sequence annotation of plasmid #2889 (pAB l_6.8: :PsmtA-legH_Gm(ABlopt)-TpsaB-PnirA-anti_H01_vl) (SEQ ID
NO: 23).
[0021] FIG. 8 depicts a map of the 10393 BP circular plasmid construct and sequence annotation of plasmid #2890 (pAB l_6.8: :PsmtA-glbN_S6803(ABlopt)-TpsaB-PnirA-anti_HOl_vl) (SEQ ID NO: 24).
[0022] FIG. 9 depicts a map of the 10634 BP circular plasmid construct and sequence annotation of plasmid #2942 (pABICyanol_6.8: :PcpcB-glbN_S6803(ABICyanolopt)-TpsaB-Porf0316'- anti HOl vl) (SEQ ID NO: 56). [0023] FIG. 10 depicts a map of the 10491 BP circular plasmid construct and sequence annotation of plasmid #2994 (pABl_6.8::PcpcB-glbN_Ap8005(AB lopt)-TpsaB) (SEQ ID NO: 57).
[0024] FIG. 11 depicts a map of the 11432 BP circular plasmid construct and sequence annotation of plasmid #3022 (pABICyanol_6.8: :PcpcB-glbN_S6803(ABICyanolopt)-TpsaB- PcpcB-glbN_S6803(ABICyanolopt)-TpsaB-Porf0316'-anti_HOl_vl) (SEQ ID NO: 58).
[0025] FIG. 12 depicts a map of the 7033 BP circular plasmid construct and sequence annotation of plasmid #2949 (nblA2: :PcpcB 1918-glbN_Ap8005-TpsaB-PrbcL2293-ble-TB1002) (SEQ ID NO: 59).
[0026] FIG. 13 depicts a map of the 8060 BP circular plasmid construct and sequence annotation of plasmid #3057 (nblA2: :PcpcB 1918-glbN_Ap8005-PcpcB1918_glbN_Ap8005-TpsaB- PrbcL2293-ble-TB1002) (SEQ ID NO: 60).
[0027] FIG. 14 is a diagram of the biosynthetic pathway from glutamate to protoporphyrin, heme, chlorophyll, biliverdin, phycobilin, and leghemoglobin. Also shown is the location of inhibition of heme oxygenase (HOI) using an inducible sRNA for gene knock-down.
[0028] FIG. 15 is a stained protein gel showing the expression of the cyanoglobin (GlbN) protein from Synechocystis strain PCC 6803 in Cyanobacterium sp. ABICyanol . Expression of the cyanoglobin (black arrow) was induced with 20 μΜ zinc sulfate. Protein extracts were separated on a 16% native PAGE and stained with Coomassie Brilliant Blue.
[0029] FIG. 16 is a photograph of a stained protein gel showing the expression of the cyanoglobin (GlbN) protein from Synechocystis strain PCC 6803 in Cyanobacterium sp.
ABICyanol in the presence and absence of the anti-HOl sRNA. Expression of the cyanoglobin was induced with 20 μΜ zinc sulfate (black arrow). Expression of the anti-HO sRNA was induced in the presence of nitrate and repressed in the presence of urea. Protein extracts were separated on a 16% native PAGE and stained with Coomassie Brilliant Blue.
[0030] FIG. 17 is a photograph of a stained protein gel showing the expression of the leghemoglobin C2 protein (black arrow) in Cyanobacterium sp. ABICyanol . Expression of leghemoglobin C2 was induced with different zinc concentrations in the presence of nitrate to knockdown expression of the heme oxygenase ABICyanol . l_orf29000 via sRNA. Protein extracts were separated by denaturing electrophoresis on a 16% Tricine-SDS-PAGE including 6 M urea and subjected to a Coomassie Brilliant Blue staining. [0031] FIG. 18A and FIG. 18B show the constitutive expression of the cyanoglobin (GlbN) protein (black arrows) from Synechocystis strain PCC 6803 in Cyanobacterium sp. ABICyanol in the presence and absence of the anti-HOl sRNA, induced with 10 μΜ copper-EDTA. AB 1336 has a single copy of the PcpcB-glbN_S6803, whereas AB4005 exhibits two PcpcB-glbN_S6803 copies on the same plasmid. Protein extracts were separated on a 16% native PAGE (FIG. 18 A, unstained) and stained with Coomassie Brilliant Blue (FIG. 18B).
[0032] FIG. 19 is a line graph showing the relative absorbance spectra of Cyanobacterium sp. ABICyanol and a Phycocyanin-deficient strain AB0492 (AcpcA).
[0033] FIG. 20 is a photograph of a stained protein gel showing the constitutive expression of the cyanoglobin (GlbN) protein from Synechocystis strain PCC 6803 in the Cyanobacterium sp. ABICyanol and in a phycocyanin-deficient derivative AB0492 (AcpcA). The strains were grown in the absence and presence of 10 μΜ copper-EDTA to induce the knockdown of the heme oxygenase via anti-HOl sRNA. AB4011 has a single copy of the PcpcB-glbN_S6803, whereas AB4012 comprises two PcpcB-glbN_S6803 copies on the same plasmid. Protein extracts were separated on a 16% native PAGE and stained with Coomassie Brilliant Blue.
[0034] FIG. 21 is a photograph of a stained protein gel showing the constitutive expression of the cyanoglobin (GlbN) protein from Arthrospira platensis PCC 8005 in Cyanobacterium sp. ABICyanol . Protein extracts were separated on a 16% native PAGE and stained with Coomassie Brilliant Blue.
[0035] FIG. 22A and FIG. 22B are photographs of a protein gel showing the expression of the cyanoglobin (GlbN) protein from Arthrospira platensis PCC 8005 in Arthrospira platensis PCC9108 (AB4041, AB4065) and the derivative strain Arthrospira platensis AB 1298 (AB4066). AB4041 has a single copy of the PcpcB-glbN_Ap8005, whereas AB4065 and AB4066 comprise two PcpcB-glbN_Ap8005 copies on the same plasmid. Protein extracts were separated on a 16% native PAGE (FIG. 22A, unstained) and stained with Coomassie Brilliant Blue (FIG. 22B).
[0036] FIG. 23 A is a photograph of a purification column showing the purification of the cyanoglobin (GlbN) protein from Synechocystis strain PCC 6803 expressed in Cyanobacterium sp. ABICyanol strain AB4005 via gel filtration with a Sephacryl 100-HR column.
[0037] FIG. 23B is a photograph of several of the elution fractions (El to E14) which were collected for further analysis. The reddish heme coloration can be seen in the samples. [0038] FIG. 24A and FIG. 24B are photpgraphs of protein gels showing the purification of the cyanoglobin (GlbN) protein from Synechocystis strain PCC 6803 expressed in Cyanobacterium sp. ABICyanol strain AB4005 via gel filtration with a Sephacryl 100-HR column. Elution fractions were collected and separated on a 16% native PAGE (FIG. 24 A, unstained) and stained with Coomassie Brilliant Blue (FIG. 24B). CE water-soluble cell extract, E2 - E14 elution fractions.
[0039] FIG. 25 is a line graph depicting the relative absorbance spectra of cell extracts from Cyanobacterium sp. ABICyanol expressing the cyanoglobin (GlbN) protein from Synechocystis strain PCC 6803. AB4005 exhibits 2 PcpcB-glbN copies in tandem and an inducible sRNA to knockdown expression of the heme oxygenase ABICyanol . l_orf29000. AB4005 was grown in the absence and presence of 10 μΜ copper-EDTA to induce the anti-HOl sRNA. The ferric form of GlbN from Synechocystis strain PCC 6803 has a main absorbance peak at 410 nm.
DETAILED DESCRIPTION
[0040] Disclosed herein is a cyanobacterium that is capable of producing a heterologous heme- containing protein. The biomass produced from the modified cyanobacterium can be used to produce various valuable products, such as a protein-rich meal, iron-rich meal, nutritional supplements, food ingredients, or food supplements. In an embodiment, the heme-containing protein can be processed to form a meat-like material. The cyanobacterium can be grown outdoors using light, CO2, and nutrients, and can then be processed as needed to produce a red meat-like material which contains a heme-containing protein. The nucleic acid encoding the heme-containing protein can be derived from, for example, an animal sequence, a plant sequence, a bacterial sequence, or a cyanobacterial sequence. The nucleic acid encoding the heme-containing protein can also be a synthetic nucleic acid sequence.
[0041] Aspects of the invention utilize techniques and methods common to the fields of molecular biology, microbiology and cell culture. Useful laboratory references for these types of methodologies are readily available to those skilled in the art. See, for example, Molecular Cloning: A Laboratory Manual (Third Edition), Sambrook, J., et al. (2001) Cold Spring Harbor Laboratory Press; Current Protocols in Microbiology (2007) Edited by Coico, R, et al., John Wiley and Sons, Inc.; The Molecular Biology of Cyanobacteria (1994) Donald Bryant (Ed.), Springer Netherlands; Handbook Of Microalgal Culture Biotechnology And Applied Phycology (2003) Richmond, A.; (ed.), Blackwell Publishing; and "The cyanobacteria, molecular Biology, Genomics and Evolution", Edited by Antonia Herrero and Enrique Flores, Caister Academic Press, Norfolk, UK, 2008.
[0042] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Definitions
[0043] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
[0044] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical value/range, it modifies that value/range by extending the boundaries above and below the numerical value(s) set forth. In general, the term "about" is used herein to modify a numerical value(s) above and below the stated value(s) by a variance of 20%.
[0045] The term "cyanobacterium" refers to a member from the group of photoautotrophic prokaryotic microorganisms which can utilize solar energy and fix carbon dioxide.
Cyanobacteria are also referred to as blue-green algae.
[0046] The terms "host cell" and "recombinant host cell" are intended to include a cell suitable for metabolic manipulation, e.g., which can incorporate heterologous polynucleotide sequences, e.g., which can be transformed. The term is intended to include progeny of the cell originally transformed. In particular embodiments, the cell is a prokaryotic cell, e.g., a cyanobacterial cell. The term recombinant host cell is intended to include a cell that has already been selected or engineered to have certain desirable properties and to be suitable for further genetic
enhancement.
[0047] "Competent to express" refers to a host cell that provides a sufficient cellular
environment for expression of endogenous and/or exogenous polynucleotides.
[0048] As used herein, the term "genetically modified" refers to any change in the endogenous genome of a wild type cell or to the addition of non-endogenous genetic code to a wild type cell, e.g., the introduction of a heterologous gene. More specifically, such changes are made by the hand of man through the use of recombinant DNA technology or mutagenesis. The changes can involve protein coding sequences or non-protein coding sequences, including regulatory sequences such as promoters or enhancers.
[0049] The terms "polynucleotide" and "nucleic acid" also refer to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs. It will be understood that, where required by context, when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T." [0050] The nucleic acids of this present invention may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide
modifications such as uncharged linkages, charged linkages, alkylators, intercalators, pendent moieties, modified linkages, and chelators. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
[0051] The term "nucleic acid" (also referred to as polynucleotide) is also intended to include nucleic acid molecules having an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences and introns. In addition, the terms are intended to include one or more genes that map to a functional locus. In addition, the terms are intended to include a specific gene for a selected purpose. The gene can be endogenous to the host cell or can be recombinantly introduced into the host cell.
[0052] In one aspect the invention also provides nucleic acids which are at least 60%, 70%, 80%> 90%), 95%), 99%o, or 99.5% identical to the nucleic acids disclosed herein.
[0053] The percentage of identity of two nucleic acid sequences or two amino acid sequences can be determined using the algorithm of Thompson et al. (CLUSTALW, 1994, Nucleic Acids Research 22: 4673-4680). A nucleotide sequence or an amino acid sequence can also be used as a so-called "query sequence" to perform a search against public nucleic acid or protein sequence databases in order, for example, to identify further unknown homologous promoters, which can also be used in embodiments of this invention. In addition, any nucleic acid sequences or protein sequences disclosed in this patent application can also be used as a "query sequence" in order to identify yet unknown sequences in public databases, which can encode for example new enzymes, which could be useful in this invention. Such searches can be performed using the algorithm of Karlin and Altschul (1990, Proceedings of the National Academy of Sciences U.S.A. 87: 2,264 to 2,268), modified as in Karlin and Altschul (1993, Proceedings of the National Academy of Sciences U.S.A. 90: 5,873 to 5,877). Such an algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al. (1990, Journal of Molecular Biology 215: 403 to 410). Suitable parameters for these database searches with these programs are, for example, a score of 100 and a word length of 12 for BLAST nucleotide searches as performed with the NBLAST program. BLAST protein searches are performed with the XBLAST program with a score of 50 and a word length of 3. Where gaps exist between two sequences, gapped BLAST is utilized as described in Altschul et al. (1997, Nucleic Acids Research, 25: 3,389 to 3,402).
[0054] In one aspect the invention also provides peptides, polypeptides, and proteins having an amino acid sequence that is at least 50%, 55%, 60%, 70%, 80% 90%, 95%, 99%, or 99.5% or more identical to the amino acid sequence of a protein disclosed herein.
[0055] Database entry numbers given in the following are for the CyanoBase, the genome database for cyanobacteria (available on the world wide web at
genome.microbedb.jp/cyanobase/) Fujisawa et al. "CyanoBase: a large-scale update on its 20th anniversary. Nucleic Acids Research. 2017, Vol. 45, p. D551.
[0056] The enzyme commission numbers (EC numbers) cited throughout this patent application are numbers which are a numerical classification scheme for enzymes based on the chemical reactions which are catalyzed by the enzymes.
[0057] "Recombinant" refers to polynucleotides synthesized or otherwise manipulated in vitro ("recombinant polynucleotides") and to methods of using recombinant polynucleotides to produce gene products encoded by those polynucleotides in cells or other biological systems. For example, a cloned polynucleotide may be inserted into a suitable expression vector, such as a bacterial plasmid, and the plasmid can be used to transform a suitable host cell. A host cell that comprises the recombinant polynucleotide is referred to as a "recombinant host cell" or a "recombinant bacterium" or a "recombinant cyanobacterium." The gene is then expressed in the recombinant host cell to produce, e.g., a "recombinant protein." A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
[0058] The term "homologous recombination" refers to the process of recombination between two nucleic acid molecules based on nucleic acid sequence similarity. The term embraces both reciprocal and nonreciprocal recombination (also referred to as gene conversion). In addition, the recombination can be the result of equivalent or non- equivalent cross-over events. Equivalent crossing over occurs between two equivalent sequences or chromosome regions, whereas nonequivalent crossing over occurs between identical (or substantially identical) segments of nonequivalent sequences or chromosome regions. Unequal crossing over typically results in gene duplications and deletions. For a description of the enzymes and mechanisms involved in homologous recombination see Court et al., "Genetic engineering using homologous
recombination," Annual Review of Genetics 36:361-388; 2002.
[0059] The term "non-homologous or random integration" refers to any process by which DNA is integrated into the genome that does not involve homologous recombination. It appears to be a random process in which incorporation can occur at any of a large number of genomic locations.
[0060] The term "expressed endogenously" refers to polynucleotides that are native to the host cell and are naturally expressed in the host cell.
[0061] The term "operably linked" refers to a functional relationship between two parts in which the activity of one part (e.g., the ability to regulate transcription) results in an action on the other part (e.g., transcription of the sequence). Thus, a polynucleotide is "operably linked to a promoter" when there is a functional linkage between a polynucleotide expression control sequence (such as a promoter or other transcription regulation sequences) and a second polynucleotide sequence (e.g., a native or a heterologous polynucleotide), where the expression control sequence directs transcription of the polynucleotide.
[0062] The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a
"plasmid," which generally refers to a circular double stranded DNA molecule into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme.
[0063] Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain 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").
[0064] A "promoter" is an array of nucleic acid control sequences that direct transcription of an associated polynucleotide, which may be a heterologous or native polynucleotide. A promoter includes nucleic acid sequences near the start site of transcription, such as a polymerase binding site. The promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. The term "promoter" is intended to include a polynucleotide segment that can transcriptionally control a gene of interest, e.g., a pyruvate decarboxylase gene that it does or does not transcriptionally control in nature. In one embodiment, the transcriptional control of a promoter results in an increase in expression of the gene of interest. In an embodiment, a promoter is placed 5' to the gene of interest. A heterologous promoter can be used to replace the natural promoter, or can be used in addition to the natural promoter. A promoter can be endogenous with regard to the host cell in which it is used or it can be a heterologous polynucleotide sequence introduced into the host cell, e.g., exogenous with regard to the host cell in which it is used. Promoters of the invention may also be inducible, meaning that certain exogenous stimuli (e.g., nutrient starvation, heat shock, mechanical stress, light exposure, etc.) will induce the promoter leading to the transcription of the gene.
[0065] The term "recombinant nucleic acid molecule" includes a nucleic acid molecule (e.g., a DNA molecule) that has been altered, modified or engineered such that it differs in nucleotide sequence from the native or natural nucleic acid molecule from which the recombinant nucleic acid molecule was derived (e.g., by addition, deletion or substitution of one or more nucleotides). The recombinant nucleic acid molecule (e.g., a recombinant DNA molecule) also includes an isolated nucleic acid molecule or gene of the present invention. [0066] The term "gene" refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids. "Gene" also refers to a nucleic acid fragment that expresses a specific protein or polypeptide, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
[0067] The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene or "heterologous" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.
[0068] The term "fragment" refers to a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence substantially identical to the reference nucleic acid. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from at least about 6, 50, 100, 200, 500, 1,000, to about 1,500 or more consecutive nucleotides of a polynucleotide according to the invention.
[0069] The term "open reading frame," abbreviated as "ORF," refers to a length of nucleic acid sequence, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.
[0070] The term "upstream" refers to a nucleotide sequence that is located 5' to reference nucleotide sequence. In particular, upstream nucleotide sequences generally relate to sequences that are located on the 5' side of a coding sequence or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.
[0071] The term "downstream" refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In particular, downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.
[0072] The term "homology" refers to the percent of identity between two polynucleotide or two polypeptide moieties. The correspondence between the sequence from one moiety to another can be determined by techniques known to the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded- specific nuclease(s) and size determination of the digested fragments.
[0073] As used herein, "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence.
[0074] The term "substantially similar" also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript.
[0075] The terms "restriction endonuclease" and "restriction enzyme" refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.
[0076] The term "expression", as used herein, refers to the transcription and stable accumulation mRNA derived from a nucleic acid or polynucleotide. Expression may also refer to translation of mRNA into a protein or polypeptide.
[0077] An "expression cassette" or "construct" refers to a series of polynucleotide elements that permit transcription of a gene in a host cell. Typically, the expression cassette includes a promoter and a heterologous or native polynucleotide sequence that is transcribed. Expression cassettes or constructs may also include, e.g., transcription termination signals, polyadenylation signals, and enhancer elements.
[0078] The term "codon" refers to a triplet of nucleotides coding for a single amino acid.
[0079] The term "codon-anticodon recognition" refers to the interaction between a codon on an mRNA molecule and the corresponding anticodon on a tRNA molecule.
[0080] The term "codon bias" refers to the fact that different organisms use different codon frequencies.
[0081] The term "codon optimization" refers to the modification of at least some of the codons present in a heterologous gene sequence from a triplet code that is infrequently used in the host organism to a triplet code that is more common in the particular host organism. This can result in a higher expression level of the gene of interest. [0082] The term "transformation" is used herein to mean the insertion of heterologous genetic material into the host cell. Typically, the genetic material is DNA on a plasmid vector, but other means can also be employed. General transformation methods and selectable markers for bacteria and cyanobacteria are known in the art (Wirth, Mol Gen Genet. 216: 175-177 (1989); Koksharova, Appl Microbiol Biotechnol 58: 123-137 (2002). Additionally, transformation methods and selectable markers for use in bacteria are well known (see, e.g., Sambrook et al, supra).
[0083] The term "selectable marker" means an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and the like, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest.
Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, spectinomycin, kanamycin, zeocin, chloramphenicol, hygromycin, and the like.
[0084] A "polypeptide" is a polymeric compound comprised of covalently linked amino acid residues. A "protein" is a polypeptide that performs a structural or functional role in a living cell.
[0085] A "heterologous protein" refers to a protein not naturally produced in the cell.
[0086] An "isolated polypeptide" or "isolated protein" is a polypeptide or protein that is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids).
[0087] The term "fragment" of a polypeptide refers to a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide. Such fragments of a polypeptide according to the invention may have a length of at least about 2, 50, 100, 200, or 300 or more amino acids.
[0088] A "variant" of a polypeptide or protein is any analogue, fragment, derivative, or mutant which is derived from a polypeptide or protein and which retains at least one biological property of the polypeptide or protein. Different variants of the polypeptide or protein may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post-translational modification. The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements.
[0089] The term "primer" is an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double stranded nucleic acid region that can serve as an initiation point for DNA synthesis under suitable conditions. Such primers may be used in a polymerase chain reaction.
[0090] The term "polymerase chain reaction," also termed "PCR," refers to an in vitro method for enzymatically amplifying specific nucleic acid sequences. PCR involves a repetitive series of temperature cycles with each cycle comprising three stages: denaturation of the template nucleic acid to separate the strands of the target molecule, annealing a single stranded PCR
oligonucleotide primer to the template nucleic acid, and extension of the annealed primer(s) by DNA polymerase. PCR provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.
[0091] The term "sRNA" refers to small non-coding regulatory RNA (typically < 200bp), which can form a stable secondary structure and is able to bind and affect stability and translation of specific mRNAs in a targeted manner.
[0092] The term "globin" refers to a type of heme-containing globular protein involved in reversible binding and transporting of oxygen utilizing a heme prosthetic group. Globins incorporate a series of alpha helical segments known as globin folds.
[0093] The terms "heme-containing protein" or "heme-binding protein" refer to a polypeptide that can bind to a heme group. Examples of heme-containing proteins include but are not limited to hemoglobin, cyanoglobin, and leghemoglobin.
[0094] The term "heme" refers to a heterocyclic organic compound cofactor made up of four joined pyrrolic groups, having an iron (Fe2+) group located in the center of the molecule. The heme iron often acts as an electron source or sink during electron transfer or redox chemistry.
[0095] The term "heme oxygenase" or "HOI" refers to an enzyme that catalyzes the degradation of the heme molecule to form other cellular components, such as biliverdin and phycobilins.
[0096] The term "selection-free" refers to a growth medium that does not include a selection agent, such as an antibiotic, that would allow only cells having a functional selectable marker gene to survive.
[0097] The term "plurality" means more than one. Heterologous Production of Heme-containing Proteins
[0098] In an embodiment of the invention, cyanobacterial cells can be modified to express a heterologous heme-containing protein. The modified cells can then be cultured at a large scale, such as in outdoor photobioreactors, using CO2, light, and nutrients, in order to produce large quantities of the heme-containing protein. By use of this method, heme-containing protein can be made without being obtained from animal sources.
[0099] Leghemoglobin is naturally synthesized in the root nodules of certain types of plants, such as leguminous plants that utilize the root nodules for nitrogen fixation. One such plant is soybean. U.S. Patent No. 8,021,695, to Gruber, describes a personal care composition (soaps, shampoos, skin care medicaments, cosmetics, and therapeutic formulations) that contains, in part, leghemoglobin extracted from nitrogen fixing root nodules. U.S. Patent Application No. 20110286992A1 describes methods of isolating the leghemoglobin from root nodules in order to prepare such compositions. The nodules have a reddish color due to the concentration of leghemoglobin. This extraction method is time consuming and expensive, however. The crop is planted and grown for a period of time, then the root nodules are collected and washed. The leghemoglobin is then isolated and purified from the washed nodules. This is a time consuming process.
[00100] International Patent Application Publication No. WO1998012913A1 discloses plants that have been transformed with a nucleic acid sequence that encodes a globin protein. The patent application also discloses that certain globin proteins can be produced from a host cell that is a bacterium or an algae. However, the patent application does not teach the production the protein from cyanobacteria.
[00101] WO/2015/153666 discloses the production of ground meat replicas that include a certain amount of a heme-containing protein, such as a leghemoglobin from soybean, pea, or cowpea. The leghemoglobin can be recombinantly produced, for example, in E. coli or yeast. However, there is no discussion of the recombinant production of leghemoglobin (or other globins or heme-containing proteins) in cyanobacteria. Production of Heme-containing Proteins in Cyanobacterial Cells
[00102] In an embodiment of the invention, cyanobacterial cells can be modified to produce the heme-containing protein. In another embodiment, the cyanobacterial cell is modified with a gene encoding the heme-containing protein, as well genes encoding other enzymes involved in assembling the heme-protein complex.
[00103] In an embodiment of the invention, any suitable cyanobacterial strain can be modified to produce a heme-containing protein.
[00104] In an embodiment of the invention, the cyanobacterial host cell is
Cyanobacterium sp. ATCC accession number PTA- 13311 (herein termed "ABICyanol" or "AB 1"). This strain has been found to grow well under various outdoor conditions, and can be genetically modified to produce a compound of interest. The strain, as well as its endogenous plasmid p6.8 (FIG. 1, SEQ ID NO: 17), has been described, for example, in U.S. Patent No. 8,846,369, U.S. 9,315,832, and U.S. Patent No. 9, 157,101, all of which are hereby incorporated by reference in their entireties.
[00105] In another embodiment of the invention, the cyanobacterial host cell for production of a heme-containing protein is another cyanobacterial strain, such as Arthrospira sp., Synechocystis sp., or Synechococcus sp.
[00106] In an embodiment, a culture of the cyanobacterial cell is grown in an outdoor photobioreactor system, and the resulting cellular material is collected. The entire protein can be separated from the remaining cell contents, or the heme-containing protein itself can be separated from the cellular material. The separated protein can then be dried, frozen, or further processed as desired.
[00107] In an embodiment, leghemoglobin (or other heme-containing proteins) can be recombinantly produced in cyanobacteria. Using this method, CO2 and inorganic nutrients can be used to convert light to heme-like proteins that can then be further processed, for example, to a meat-like material. One benefit of using cyanobacteria is that CO2, rather than organic carbon- based materials, can be used as the starting material. This lowers the cost of producing the protein, as organic carbon sources are not needed.
[00108] Many types of heme-containing proteins can be heterologously produced in cyanobacteria. Many heme-containing proteins are found in plants. [00109] One exemplary heme-containing protein that can be expressed in cyanobacteria is leghemoglobin C2 from soybean {Glycine max) GenBank No. AAA33980.1. The native DNA sequence is shown in SEQ ID NO: 1. The codon optimized gene sequence (codon optimized for optimal expression in Cyanobacterium sp. ABICyanol) is SEQ ID NO: 2. The protein sequence is SEQ ID NO: 3.
[00110] Another exemplary heme-containing protein that can be expressed in
cyanobacteria is Leghemoglobin from Astragalus sinicus GenBank No. ABB 13622.1. The native DNA sequence is shown in SEQ ID NO: 4. The codon optimized gene sequence (codon optimized for optimal expression in Cyanobacterium sp. ABICyanol) is SEQ ID NO: 5. The protein sequence is SEQ ID NO: 6.
[00111] Yet another exemplary heme-containing protein that can be expressed in cyanobacteria is Leghemoglobin from Cajanus cajan GenBank No. KYP60668.1. The native DNA sequence is shown in SEQ ID NO: 7. The codon optimized gene sequence (codon optimized for optimal expression in Cyanobacterium sp. ABICyanol) is SEQ ID NO: 8. The protein sequence is SEQ ID NO: 9.
[00112] Cyanobacterial organisms also naturally produce certain types of heme-containing proteins. Native heme-containing proteins present in cyanobacteria include, for example, leghemoglobin and cyanoglobin.
[00113] An example of a native cyanobacterial heme-containing protein is cyanoglobin from Synechocystis sp. PCC6803 (GenBank No. BAA17991.1). The native DNA sequence is shown in SEQ ID NO: 10. The codon optimized gene sequence (codon optimized for optimal expression in Cyanobacterium sp. ABICyanol) is SEQ ID NO: 11. The protein sequence is SEQ ID NO: 12.
[00114] Another example of a native cyanobacterial heme-containing protein is cyanoglobin from Arthrospira sp. PCC 8005 (GenBank No. CCE20200.1). The native DNA sequence is shown in SEQ ID NO: 13. The codon optimized gene sequence (codon optimized for optimal expression in Cyanobacterium sp. ABICyanol) is SEQ ID NO: 14. The protein sequence is SEQ ID NO: 15.
[00115] Cyanobacteria can be modified to add genes of interest as shown herein in order to produce heme-containing proteins. The DNA sequences encoding the genes can be amplified by polymerase chain reaction (PCR) using specific primers. The amplified PCR fragments were digested with the appropriate restriction enzymes and cloned into either a self-replicating plasmid or an integrative plasmid. An antibiotic resistance cassette for selection of positive clones can be present on the appropriate plasmid.
[00116] In an embodiment, the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. PCR can be used to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, and for nucleic acid sequencing.
[00117] To use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of cyanobacteria can be prepared. Techniques for transformation are well known and described in the technical and scientific literature. For example, a DNA sequence encoding one or more of the genes described herein can be combined with
transcriptional and other regulatory sequences which will direct the transcription of the sequence from the gene in the transformed cyanobacteria.
[00118] In an embodiment, the genes of interest are inserted into the cyanobacterial chromosome. When the cell is polyploid, the gene insertions can be present in all of the copies of the chromosome, or in some of the copies of the chromosome.
[00119] In another embodiment, the inserted genes are present on an extrachromosomal plasmid. The extrachromosomal plasmid can be derived from an outside source, such as, for example, RSFlOlO-based plasmid vectors, or it can be derived from an endogenous plasmid from the cyanobacterial cell or from another species of cyanobacteria.
[00120] In an embodiment, the inserted genes are present on an extrachromosomal plasmid, wherein the plasmid has multiple copies per cell. The plasmid can be present, for example, at about 1, 3, 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or more copies per host cyanobacterial cell. In an embodiment, the plasmid containing the inserted gene is fully segregated in the cell.
[00121] In another embodiment, the inserted genes are present on one cassette driven by one promoter. In another embodiment, the inserted genes are present on separate plasmids, or on multiple cassettes on one plasmid. [00122] In another embodiment, the inserted genes are modified for optimal expression by modifying the nucleic acid sequence to accommodate the cyanobacterial cell's protein translation system. Modifying the nucleic acid sequences in this manner can result in an increased expression of the genes.
[00123] Methods to modify cyanobacterial cells with genetic modifications to produce compounds of interest are also described in U.S. Patent No. 9,315,820, U.S. Patent No.
9,965,364, U.S. Patent No. 9,551,014, U.S. Patent No. 9,476,067, U.S. Patent No. 9,493,794, and U.S. Patent No. 9,353,400, all of which are incorporated by reference herein in their entireties.
Codon Improvement of Recombinant Genes
[00124] At least some of the heterologous nucleic acid sequences to be expressed in cyanobacterial host cells can be codon improved for optimal expression in the target
cyanobacterial strain. The underlying rationale is that the codon usage frequency of highly expressed genes is generally correlated to the host cognate tRNA abundance. (Bulmer, Nature 325:728-730; 1987). Codon improvement (sometimes referred to as codon optimization or codon adaptation) can be performed to increase the expression level of foreign genes.
[00125] Codon improvement of the gene encoding the heme-containing protein can be performed for improved expression in the cyanobacterial host cell. Codon improvement can also be performed by adapting the codon usage of the at least one recombinant gene to the codon usage in Cyanobacterium sp., in particular ABICyanol . In an embodiment, the G and/or C wobble bases in the codons for the amino acids in the at least one recombinant gene can be replaced by A and/or T because the G+C content of the genome of ABICyanol is relatively low at about 36%.
[00126] In an embodiment, only 2% to 6% or 1% to 10% of the codons of variants of recombinant genes are codon improved. In another embodiment, highly codon improved variants of recombinant genes, at least 25%, to at least 50%, 65% or even at least 70% of the codons have been changed. In another embodiment, recombinant genes are used which are not codon improved.
[00127] Codon improvement of heterologously derived genes (such as genes encoding antibiotic resistance genes, and the recombinant production genes) was guided by the
ABICyanol codon usage table derived from ribosomal proteins and highly expressed genes (such as photosynthesis genes). To improve heterologous gene expression, original sequences of interest were assessed with the online software OPTIMIZER (Puigbo P, Guzman E, Romeu A, & Garcia- Vallve S (2007) OPTIMIZER: a web server for optimizing the codon usage of DNA sequences Nucleic Acids Research 35(suppl 2):W126-W131) based on the codon-usage table derived from ABICyanol genome.
[00128] The codon adaptation index is a measure of directional synonymous codon usage bias, and its potential applications, (see Nucleic Acids Research 15(3): 1281-1295). The effective number of codons (see, Wright F (1990) Gene 87(l):23-29) are designed match those of highly expressed genes (such as ribosomal proteins) in the ABICyanol genome. The resulting polynucleotides using improved codons were further modified and optimized to avoid the presence of any known or predicted putative ABICyanol endonuclease restriction sites {Aval, BsaRI, KasI, Xhol, etc.); internal Shine-Dalgarno sequence and RNA destabilizing sequences; an internal terminator sequence; and a repeat sequence of greater than about 10 bp (see, Welch et al., PLOS One 4, e7002; 2009; and Welch et al., Journal of the Royal Society; Interface 6 (Suppl 4), S467-S476; 2009).
[00129] In an embodiment, the selectable marker genes are also modified so that they will have improved expression in cyanobacteria. For example, the selectable marker gene that confers gentamycin or kanamycin resistance was codon optimized for higher expression in
cyanobacteria. In an embodiment, as a result of codon improvement, the G+C % of the antibiotic resistance genes decreased from 40-53% to 33-40%, which is similar to that of ABICyanol coding genes (about 36% on average). The codon adaptation index of the codon improved antibiotic resistance genes is significantly improved from less than 0.4 to greater than 0.8, which is similar to that of ABICyanol endogenous genes. Table 1, below, depicts the codon usage statistics within the cyanobacterial strain ABICyanol .
Table 1: Codon Usage - ABICyanol
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Choice of Promoters
[00130] The inserted genes can be controlled by one promoter, or they can be controlled by different individual promoters. The promoters can be constitutive or regulatable. The promoters can be, for example, inducible. The promoter sequences can be derived, for example, from the host cell, from another organism, or can be synthetically derived.
[00131] Any desired promoter can be used to regulate the expression of the inserted genes. Exemplary promoter types include but are not limited to, for example, constitutive promoters, regulatable promoters such as inducible promoters (e.g., by nutrient source, nutrient starvation, heat shock, mechanical stress, environmental stress, metal concentration, specific metabolites, light exposure, etc.), endogenous promoters, heterologous promoters, and the like. Suitable promoter sequences are also disclosed, for example, in U.S. Patent No. 9,315, 820, U.S. Patent No. 9,551,014, PCT/EP2012/067534, U.S. Patent No. 9,476,067, U.S. Patent No. 9,157, 101, PCT/US2013/077364, U.S. Patent No. 9,493,794, and PCT/US2015/000210, all of which are hereby incorporated by reference in their entireties.
[00132] The recombinant gene(s) can be under the transcriptional control of a constitutive promoter. In this way, a sustained level of transcription and, therefore, enzymatic activity of the corresponding protein can be maintained during the whole period of cultivation. For example, the constitutive promoter can be endogenous to the cyanobacterial cell. This has the advantage that no recombinant transcription factor has to be present in the host cell. The endogenous promoter is usually well-recognized by the metabolically enhanced cyanobacterial cell without the need to introduce further genetic modifications.
[00133] Suitable constitutive promoters include, without limitation, the PrpsL promoter
(Gene ID: ABICyanol_orfl758), PpsaA promoter (ABICyanol_orf3243), PpsbB
(ABICyanol_orf2107), PcpcB promoter (ABICyanol _orf2472), PatpG (ABICyanol_orfl814), PrbcL promoter (ABICyanol_orfl369), and variations thereof. Further suitable endogenous constitutive promoters from genes with unknown function exhibiting appropriate transcriptional activity include, without limitation, the promoters of Gene IDs ABICyano_orfl924,
ABICyano_orfl997, ABICyano_orf3446, ABICyano_orf0865, ABICyano_orfl919,
ABICyano_orf3278, ABICyano orfl 181, ABICyano_orfl627, ABICyano_orf0265 and
ABICyano_orf2536, ABICyano_orf0615, and variants thereof.
[00134] In an embodiment, the promoters can be derived from the cyanobacterial strain Cyanobacterium sp. PTA-13311, or they can be derived from another cyanobacterium or from another organism. In an embodiment, the promoters can be about 60%, 70%, 75%, 80%, 85%, 90%), 95%), 97%), 99%), or 100 %> identical to the promoter sequences described herein.
[00135] The promoters can be regulatable promoters, such as inducible promoters. For example, certain promoters are up-regulated by the presence of a compound, while other promoters can be up-regulated by the absence of a compound (also termed "repressible").
[00136] Various promoters that can be used include promoters that are regulatable by the presence (or in other promoters, by the absence) of inductors such as different metal ions, different nutrient sources, different metabolites, different external stimuli such as heat, cold, salinity or light. In some embodiments, the regulatable or inducible promoters are induced under conditions such as nutrient starvation, stationary growth phase, heat shock, cold shock, oxidative stress, salt stress, light, darkness, metal ions, organic chemical compounds, and combinations thereof. For example, a particularly tight control of the expression of gene can be achieved if a gene is under the transcriptional control of a Zn-, Ni-, or Co-inducible promoter. Exemplary Zn- regulatable promoters and their variants are described, for example, in International Application No. PCT/EP2013/077496. Exemplary Zn, Ni, and Co-regulatable promoters are described, for example, in International Application No. PCT/2012/076790, both of which are incorporated by reference herein in their entireties. [00137] In a further embodiment, the regulatable or inducible promoter is inducible by a change of a metal-ion concentration. Such a change of metal-ion concentration includes for instance the addition or depletion of certain metal ions. Suitable inducible promoters include, without limitation, the PziaA promoter, the PsmtA promoter, PaztA promoter, the PcorT promoter, the PnrsB promoter, the PpetJ promoter, PpetE promoter, the Porf0316 promoter, the Porf0221 promoter, the Porf0223 promoter, the Porf3126 promoter, the PmntC promoter, and variations thereof.
[00138] Preferably, the regulatable or inducible promoter is endogenous to the
cyanobacterial cell. An endogenous inducible promoter is usually well-recognized by the metabolically enhanced cyanobacterial cell without the need to introduce further genetic modifications.
[00139] In further embodiments, the choice of regulatable or inducible promoters can include, but are not limited to, PntcA, PnblA, PisiA, PpetJ, PggpS, PpsbA2, PsigB, PlrtA, PhtpG, PnirA, PnarB, PnrtA, PhspA, PclpBl, PhliB, PcrhC, PziaA, PsmtA, PcorT, PnrsB, PnrsB916, PaztA, PbmtA, Pbxal, PzntA, PczrB, PnmtA, PpstS, and the like.
[00140] The regulatable or inducible promoter can, for instance, also be a nitrate inducible promoter. Suitable nitrate inducible promoters include, without limitation, the PnirA promoter, the PnrtA promoter, the PnarB promoter, and variations thereof.
[00141] In certain other preferred embodiments, truncated or partially truncated versions of these promoters including only a small portion of the native promoters upstream of the transcription start point, such as the region ranging from -35 to the transcription start can often be used. Furthermore, introducing nucleotide changes into the promoter sequence, e.g. into the TATA box, the operator sequence, 5 '-untranslated region and/or the ribosomal binding site (RBS) can be used to tailor or optimize the promoter strength and/or its induction conditions, e.g. the concentration of inductor required for induction. In some preferred variants, the different inducible promoters are inducible by different metal ions.
[00142] The promoters hspA, clpBl, and hliB can be induced by heat shock (raising the growth temperature of the host cell culture from 30°C to 40°C), cold shock (such as, for example, reducing the growth temperature of the cell culture from 30°C to 20°C), oxidative stress (for example by adding oxidants such as hydrogen peroxide to the culture), or osmotic stress (for example by increasing the salinity). The promoter sigB can be induced by stationary growth, heat shock, and osmotic stress. The promoters ntcA and nblA can be induced by decreasing the concentration of nitrogen in the growth medium and the promoters psaA and psbA2 can be induced by low light or high light conditions. The promoter htpG can be induced by osmotic stress and heat shock. The promoter crhC can be induced by cold shock. An increase in copper concentration can be used in order to induce the promoter petE, whereas the promoter petJ is induced by decreasing the copper concentration. Additional details of these promoters can be found, for example, in PCT/EP2009/060526, which is incorporated by reference herein in its entirety.
[00143] In an embodiment, the promoters of any of the above embodiments may be selected from the endogenous inducible promoters identified in Cyanobacterium sp. with the ATCC accession number PTA-13311 ("ABICyanol") as listed in Table 2 below, and variants thereof.
Table 2: Cyanobacterium sp. ABICyanol endogenous promoter sequences
Figure imgf000029_0001
Figure imgf000030_0001
[00144] In certain other preferred embodiments, truncated or partially truncated versions of these promoters including only a small portion of the native promoters upstream of the transcription start point, such as the region ranging from -35 to the transcription start can often be used. Furthermore, introducing nucleotide changes into the promoter sequence, e.g. into the TATA box, the operator sequence and/or the ribosomal binding site (RBS) can be used to tailor or optimize the promoter strength and/or its induction conditions, e.g. the concentration of inductor required for induction.
Plasmid Vector Construction
[00145] In an embodiment, the cyanobacterial strain ABICyanol is transformed to produce the heme-containing protein. Methods for producing a genetically enhanced, non- naturally occurring cyanobacterial host cells are disclosed herein. In an embodiment, methods include introducing a recombinant nucleic acid sequence into a cyanobacterial host cell. At least one recombinant gene can be introduced into the host cells through the transformation of the host cell by an extrachromosomal plasmid. In an embodiment, the extrachromosomal plasmid can independently replicate in the host cell. In another embodiment, at least one recombinant gene can be introduced into the genome of the host cell. In yet another embodiment, at least one recombinant gene is introduced into the genome of the host cell by homologous recombination.
[00146] Genetically enhanced cyanobacterial cells can include further genetic
enhancements such as partial deletions of endogenous genes or other recombinant genes which can increase the overall yield of the compound being produced by the host cells. Examples of such genetic enhancements are described in PCT patent publication number WO 2009/098089 A2, which is hereby incorporated by reference in its entirety.
[00147] In another embodiment, genetic enhancements of the genes encoding enzymes of the carbon fixation and subsequent carbohydrate metabolism can be genetically enhanced to further increase the production of a compound of interest. Genetic enhancement targets include, but are not limited to, components of the photosystems (antennas and pigment modification), and components of the photosynthetic and respiratory electron transport systems. Genetic
enhancement targets include local and global regulatory factors including, but not limited to, the two component system, sigma factors, small regulating RNAs and antisense RNAs.
[00148] In an embodiment, a recombinant nucleic acid sequence can be provided as part of an extrachromosomal plasmid containing cyanobacterial nucleic acid sequences in order to increase the likelihood of success for the transformation.
[00149] In another embodiment, the method for producing a genetically enhanced cyanobacterial cell uses an extrachromosomal plasmid derived from an endogenous plasmid of the host cell to introduce a recombinant nucleic acid sequence into the host cell. This endogenous plasmid can be, for example, an extrachromosomal plasmid derived from the 6.8 kb endogenous plasmid of ABICyanol .
[00150] In an embodiment, the cyanobacterial strain ABICyanol is transformed to produce the heme-containing protein. It has been found that the use of modified endogenous plasmids improves the stability of the plasmid in the host cell. The cyanobacterial strain
ABICyanol contains three endogenous plasmids. In combination with other genotypic and phenotypic attributes, these endogenous plasmids differentiate ABICyanol from other cyanobacterial species. One plasmid is 6,826 base pairs, another is 39,702 base pairs, and a third plasmid is 28,554 base pairs. The 6,826 bp endogenous plasmid is alternatively referred to herein as pABICyanol, p6.8 or 6.8. As disclosed herein, plasmid 6.8 has been modified in vivo and in vitro for use as a plasmid vector containing genes of interest for the production of compounds of interest. In an embodiment, a modified endogenous vector derived from p6.8 from ABICyanolwas developed. The modified endogenous vector from ABICyanol can be used to transform cyanobacteria from a broad range of genera, including ABICyanol itself.
[00151] In certain embodiments, the present invention includes the p6.8 plasmid and modified vectors comprising sequences of the p6.8 plasmid. In an embodiment, the modified endogenous vector contains at least one of the following: a recombinant gene that encodes a heme-containing protein; and an origin of replication suitable for replication in ABICyanol .
[00152] Other suitable plasmids that can be used to carry genes of interest in several strains of cyanobacteria (such as, for example, Synechocystis, Synechococcus 7002 and Anabaend), are the RSFlOlO-based shuttle plasmids (extrachromosomal broad-host vector) such as pVZ321-6 series (described, for example, in PCT/EP2009/060526 and in
PCT/EP2009/000892, both of which are incorporated herein by reference in their entireties). Further, the plasmid vector described in U.S. Patent No. 9,476,067 can be utilized to carry heterologous genes of interest in many cyanobacterial species.
[00153] In certain embodiments, a gene coding for a replication initiation factor that binds to the origin of replication can either be present on the modified endogenous vector or can be present in the chromosomes or other extrachromosomal plasmids of ABICyanol . An origin of replication suitable for replication in ABICyanol and the gene coding for the replication initiation factor binding to that origin of replication ensure that the modified endogenous vector can be replicated in ABICyanol .
[00154] In an embodiment, the nucleotide sequence of an origin of replication of the modified endogenous plasmid vector can have at least 80%, 90%, and 95% identity or can be identical to the nucleotides 3375 to 3408 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 17).
[00155] In an embodiment, the sequence of the gene coding for the replication initiation factor has at least 80%, 90%, and 95% identity or is identical to nucleotides 594 to 3779 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 17). In an embodiment, the gene coding for the replication initiation factor codes for a protein having at least 80%, 90%, and 95% sequence identity or is identical to the protein coded by nucleotides 594 to 3779 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 17) of ABICyanol . This putative initiation replication factor is thought to bind to the putative origin of replication, thereby ensuring the replication of a plasmid containing the initiation factor in ABICyanol .
[00156] In an embodiment, a modified endogenous plasmid vector can contain a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%), at least 85%, at least 90% or at least 95% identity to the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 17). In another embodiment, the modified endogenous vector contains the entire p6.8 endogenous plasmid from ABICyanol .
[00157] In another embodiment, gene delivery vehicles that are developed using the endogenous 6.8 kb plasmid (or a portion of the plasmid) containing characteristic portions of the endogenous 6.8 kb plasmid may be able to be efficiently transformed into a wide range of cyanobacteria. In an embodiment, characteristic portions of the 6.8 kb endogenous plasmid from ABICyanol include portions that enable it to replicate in a host cell (origin of replication and replication initiation factor, for example) and can be referred to as the backbone of the endogenous 6.8 kb plasmid. Such vectors may also be able to efficiently produce heterologous proteins and other compounds of interest in cyanobacterial cultures.
[00158] In another embodiment, modifications starting with the backbone of the 6.8 kb endogenous plasmid from ABICyanol are performed individually or together to increase transformation efficiency, increase the replication rate within the cell, and to increase the production of a desired product from the cyanobacterial cell. Suitable modifications include, for example, insertion of selection markers (such as antibiotic resistance genes), recombinant genes or cassettes for the production of a desired compound, and other modifications to increase the expression or stability of the plasmid in the cyanobacterial cell. In an embodiment, the invention includes cyanobacteria, e.g. ABICyanol, comprising a modified p6.8 plasmid having any of these improved characteristics.
[00159] In an embodiment, the modular design of the p6.8 derived vector allows complex sequence manipulation in cyanobacteria.
[00160] Several exemplary strains and plasmids carrying genes encoding heme-containing proteins are listed below in Table 3. The plasmid maps and SEQ ID NOs of these constructs are shown below.
Table 3: Cyanobacterial Strains and Plasmids for Production of Heme-containing Proteins
Figure imgf000033_0001
Figure imgf000034_0001
inducible sRNA
Figure imgf000035_0001
Figure imgf000036_0001
[00161] In yet another embodiment, the method for producing a genetically enhanced cyanobacterial cell involves protecting the recombinant nucleic acid sequence, for example a plasmid, against endogenous restriction endonucleases of the host cell by methylating at least a part of the recombinant nucleic acid sequence or modifying and/or eliminating the recognition sequences of the endogenous restriction endonucleases. By changing the nucleic acid sequence of potential recognition sites of restriction endonucleases, a digest of the recombinant nucleic acid sequence can be avoided. It was discovered that endogenous restriction endonucleases of ABICyanol, for example, can cut an extrachromosomal plasmid carrying recombinant genes, thereby preventing a genetic transformation event of this host cell.
[00162] In an embodiment, methyltransferases, for example Aval and Acyl, can be used to protect recombinant vector extrachromosomal plasmids. The plasmids can either be incubated with the methyltransferases in vitro or a helper plasmid can be present in a helper E. coli strain in order to methylate the extrachromosomal plasmids in vivo before conjugation takes place during the transformation of ABICyanol . In another embodiment, recognition sequences for the restriction enzymes can be modified or deleted. As described by Elhai and Wolk (Conjugal Transfer of DNA to Cyanobacteria in Methods in Enzymology 02/1988; 167:747-54, herein incorporated by reference), plasmid pRL528 can be used as a helper plasmid for conjugal transfer. The indicated genes are M. Aval coding for the methyltransferase protecting against the restriction endonuclease Aval and the respective gene coding for M. Avail. The latter is not required for transformation of ABICyanol, as it lacks any endonuclease activity of Avail. [00163] In an embodiment, the vector to be transformed into cyanobacterial host cells can be modified to integrate into the cyanobacterial chromosome by adding an appropriate DNA sequence homologous to the target region of the host genome. In another embodiment, the vector to be transformed can be modified to integrate into the cyanobacterial chromosome through in vivo transposition by introducing mosaic ends to the vector. Once the plasmid is established in the host cell, it can be present, for example, at a range of from 1 to many copies per cell.
[00164] In an embodiment, an endogenous plasmid derived from ABICyanol can be modified, either in vivo or in vitro, to be a plasmid vector capable of introducing exogenous genes encoding enzymes for the production of a compound or compounds of interest into a wide range of host cyanobacterial cells such as ABICyanol, Cyanobacterium sp., or other
cyanobacterial genera such as Synechocystis and Synechococcus.
[00165] In an embodiment, the ABICyanol 6.8 kb endogenous plasmid is used as a backbone for a plasmid vector used for transformation of Cyanobacterium sp. Since this is the endogenous vector from the species, it is likely to be more stable when transformed into the cell than plasmids derived from completely different organisms. In an embodiment, the entire p6.8 endogenous plasmid is inserted into a vector used for transformation. In another embodiment, a sequence of about 50%, 70%, 75%, 80% 85%, 90%, 95%, 98%, 99%, or 99.5% identity to the entire endogenous plasmid sequence is inserted into the extrachromosomal plasmid vector.
[00166] In an embodiment, a modified p6.8 vector is designed to have several modular units that can be swapped out using specific restriction enzymes. Promoters, genes of interest, selectable markers, and other desired sequences can be moved in and out of the vector as desired. This modular design makes genetic experiments faster and more efficient.
[00167] The modified p6.8 vector, according to certain embodiments of the disclosure, can replicate in both cyanobacteria and in E. coli. The vector contains a replication unit that can function in a broad range of cyanobacterial genera. The vector also contains a replicon for propagation in E. coli for ease of cloning and genetic manipulation using E. coli.
[00168] In an embodiment, a plasmid shuttle vector is provided which is characterized by being replicable in both is. coli and cyanobacterial species. The plasmid comprises a promoter capable of functioning in cyanobacteria and E. coli and a DNA sequence encoding a sequence capable of functioning as a selective marker for both E. coli and cyanobacteria. In another embodiment, the shuttle vector includes two different promoter systems, one functioning in cyanobacteria and the other one functional in E. coli. In an embodiment, the plasmid shuttle vector contains at least 50% of the p6.8 plasmid. The plasmid shuttle vector enables the efficient transformation of cyanobacteria and the expression of recombinant genes of interest.
[00169] In another embodiment, the p6.8 derived plasmid vector also contains an origin of transfer (oriT) which is suitable for conjugation. In particular, the plasmid vector can contain a combined origin of replication and an origin of transfer (oriVT), which enables replication in Enterobacteriaceae, in particular E. coli, and which also enables conjugation with, for example, an E. coli donor strain and Cyanobacterium sp., in particular ABICyanol as a recipient strain. Such an plasmid vector can be used for triparental mating wherein a conjugative plasmid present in one bacterial strain assists the transfer of a mobilizable plasmid, for example a plasmid vector disclosed herein, present in a second bacterial strain into a third recipient bacterial strain, which can be ABICyanol.
[00170] Also disclosed herein is a non-naturally occurring plasmid vector in which a gene encoding a heme-containing protein of interest is operably linked to a shuttle vector. In an embodiment, cyanobacterial cells are transformed with the recombinant shuttle vector. The recombinant shuttle vector is relatively small in size, relatively stable in a cyanobacterial host cell, and can replicate in a variety of cyanobacterial species. This recombinant vector is useful for expressing a variety of heterologous genes in cyanobacteria.
[00171] In an embodiment for transforming host cells with modified plasmid vectors, a shuttle vector expresses a codon-optimized antibiotic resistance gene (AbR), such as codon improved kanamycin or gentamycin resistance genes. In an embodiment, the shuttle vector is constructed based on a modular basis so that all of the key elements (replication ori, AbR gene and reporter gene) are exchangeable via unique restriction sites thus providing versatile cloning options and facilitating the delivery of genes of interest to target organisms. Other antibiotic resistance genes can be used if desired. For example, genes conferring resistance to ampicillin, chloramphenicol, erythromycin, zeocin, kanamycin, gentamycin, spectinomycin or other antibiotics can be inserted into the vector, under the control of a suitable promoter. In some embodiments, the vector contains more than one antibiotic resistance gene.
[00172] In yet another embodiment, the plasmid is modified by several factors so that it is capable of efficient replication in multiple types of cyanobacterial species. The vector has also been organized so that various sequences can be easily replaced with other desired sequences as needed. Thus, a construct having a different gene (or genes) of interest, a different antibiotic, a different promoter, etc. can be made with relative ease. The modified vector allows for rapid testing of various heterologous constructs in a cyanobacterial cell.
[00173] The transfer of exogenous genes into cyanobacteria often involves the
construction of vectors having a backbone from a broad-host range bacterial plasmid, such as RSF1010. The RSFlOlO-based vector has been widely used as a conjugation vector for transforming bacteria, including cyanobacteria (Mermet-Bouvier et al. (1993) "Transfer and Replication of RSFlOlO-derived Plasmids in Several Cyanobacteria of the Genera Synechocystis and Synechococcus" Current Microbiology 27:323-327). RSF1010 has an E. coli origin of replication, but does not have a cyanobacterial origin of replication.
[00174] As an example of additional tools used to transfer exogenous genes into cyanobacteria, several endogenous plasmids from Synechococcus sp. PCC 7002 were used as the backbone portions for plasmids to prepare vectors for heterologous gene expression, see Xu et al., Photosynthesis Research Protocols 684:273-293 (2011). Other vectors for transformation of cyanobacteria include the pDUl-based vectors. The pDUl origin of replication is best suited for filamentous cyanobacteria. Attempts to transform ABICyanol, with either RSF1010 or pDUl- based shuttle vectors were unsuccessful using the techniques as described in the art.
[00175] In an embodiment, cyanobacteria disclosed herein can be transformed to add biochemical pathways to produce heme-containing proteins of interest. In an embodiment, the recombinant nucleic acids of interest can be amplified from nucleic acid samples using known amplification techniques. PCR can be used to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, and for nucleic acid sequencing.
[00176] In an embodiment, recombinant genes are present on an extrachromosomal plasmid having multiple copies per cell. The plasmid can be present, for example, at about 1, 3, 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or more copies per cyanobacterial host cell. In an embodiment, the recombinant plasmids are fully segregated from the non-recombinant plasmids. [00177] In another embodiment, recombinant genes are present on one cassette driven by one promoter. In another embodiment, the recombinant genes are present on separate plasmids, or on different cassettes.
[00178] In yet another embodiment, recombinant genes are modified for optimal expression by modifying the nucleic acid sequence to accommodate the cyanobacterial cell's protein translation system. Modifying the nucleic acid sequences in this manner can result in an increased expression of the genes.
Transformation of Cyanobacterial Cells
[00179] Some strains of cyanobacteria can be transformed through natural uptake of exogenous DNA. Other cyanobacterial strains can be transformed, for example, by the use of conjugation or electroporation. Some cyanobacterial strains are difficult to transform by any known means. For many of these types of difficult to transform strains, specific methods of preparing the cells for transformation, as well as specific methods of allowing entry of the foreign DNA into the cells, need to be designed de novo.
[00180] Exemplary cyanobacteria that can be transformed include, but are not limited to,
Synechocystis, Synechococcus, Acaryochloris, Anabaena, Arthrospira, Thermosynechococcus, Chamae siphon, Chroococcus, Cyanobacterium, Cyanobium, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus, Prochloron, Chroococcidiopsis, Cyanocystis, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria, Xenococcus, Borzia, Crinalium, Geitlerinema, Halospirulina, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Cyanodictyon, Aphanocapsa, Oscillatoria, Planktothrix, Prochlorothrix, Pseudanabaena, Spirulina, Starria, Symploca, Trichodesmium, Tychonema, Anabaenopsis, Aphanizomenon, Calothrix, Cyanospira, Cylindrospermopsis, Cylindrospermum, Nodularia, Nostoc,
Chlorogloeopsis, Fischerella, Geitleria, Nostochopsis, Iyengariella, Stigonema, Rivularia, Scytonema, Tolypothrix, Cyanothece, Phormidium, Adrianema, and the like.
[00181] Exemplary methods suitable for transformation of Cyanobacteria, include, as nonlimiting examples, natural DNA uptake (Chung, et al. (1998) FEMS Microbiol. Lett. 164: 353-361; Frigaard, et al. (2004) Methods Mol. Biol. 274: 325-40; Zang, et al. (2007) J.
Microbiol. 45: 241-245), conjugation, transduction, glass bead transformation (Kindle, et al. (1989) J. Cell Biol. 109: 2589-601; Feng, et al. (2009) Mol. Biol. Rep. 36: 1433-9; U.S. Pat. No. 5,661,017), silicon carbide whisker transformation (Dunahay, et al. (1997) Methods Mol. Biol. (1997) 62: 503-9), biolistics (Dawson, et al. (1997) Curr. Microbiol. 35: 356-62; Hallmann, et al. (1997) Proc. Natl. Acad. USA 94: 7469-7474; Jakobiak, et al. (2004) Protist 155:381-93; Tan, et al. (2005) J. Microbiol. 43 : 361-365; Steinbrenner, et al. (2006) Appl Environ. Microbiol. 72: 7477-7484; Kroth (2007) Methods Mol. Biol. 390: 257-267; U.S. Pat. No. 5,661,017)
electroporation (Kjaerulff, et al. (1994) Photosynth. Res. 41 : 277-283; Iwai, et al. (2004) Plant Cell Physiol. 45: 171-5; Ravindran, et al. (2006) J. Microbiol. Methods 66: 174-6; Sun, et al. (2006) Gene 377: 140-149; Wang, et al. (2007) Appl. Microbiol. Biotechnol. 76: 651-657;
Chaurasia, et al. (2008) J. Microbiol. Methods 73 : 133-141; Ludwig, et al. (2008) Appl.
Microbiol. Biotechnol. 78: 729-35), laser-mediated transformation, or incubation with DNA in the presence of or after pre-treatment with any of poly(amidoamine) dendrimers (Pasupathy, et al. (2008) Biotechnol. J. 3 : 1078-82), polyethylene glycol (Ohnuma, et al. (2008) Plant Cell Physiol. 49: 117-120), cationic lipids (Muradawa, et al. (2008) J. Biosci. Bioeng. 105: 77-80), dextran, calcium phosphate, or calcium chloride (Mendez-Alvarez, et al. (1994) J. Bacteriol. 176: 7395-7397), optionally after treatment of the cells with cell wall-degrading enzymes (Perrone, et al. (1998) Mol. Biol. Cell 9: 3351-3365). Biolistic methods (see, for example, Ramesh, et al. (2004) Methods Mol. Biol. 274: 355-307; Doestch, et al. (2001) Curr. Genet. 39: 49-60; all incorporated herein by reference in their entireties).
[00182] In an embodiment, the cyanobacterial strain ABICyanol is used as the host cell for production of the heme-containing protein. In addition to the conjugation methods described below in the example section, electroporation can also be used for successful transformation of ABICyanol, although at a somewhat lower efficiency. Strain-specific adaptations of standard electroporation protocols may be made to avoid DNA digestion by endogenous restriction enzymes and to allow DNA entry through the EPS layer of ABICyanol host cells. To achieve successful electroporation, DNA may be protected against endogenous restriction enzymes by methylation. Prior to electroporation using techniques well known in the art, ABICyanol cells may be pretreated with positively charged polyaminoacids such as poly-L-lysine hydrobromide or poly-L-ornithine hydrochloride or combinations thereof (in particular poly-L-lysine hydrobromide) in order to increase the DNA uptake efficiency. Selecting for Successful Transformation
[00183] In an embodiment, the presence of a foreign gene encoding antibiotic resistance in a cell is selected by placing putatively transformed cells into a media containing an amount of the corresponding antibiotic and selecting cells that survive. The selected cells are then grown in the appropriate culture medium to allow for further testing.
[00184] In another embodiment, colony PCR methods are used to confirm transformants.
In certain embodiments of this procedure, three primer sets are used and are directed against parts of the shuttle vectors to detect specific fragments of the shuttle vector. Transformants exhibiting the predicted PCR products are analyzed further by plasmid rescue. In one
embodiment for plasmid rescue, a 25 mL liquid culture is subjected to DNA isolation.
[00185] In a non-limiting example of plasmid rescue, 500 ng to ^g of isolated DNA from transformants containing the transformed plasmids is re-transformed into E. coli and usually results in approximately 10-20 transformants per transformation implemented. Plasmid DNA of ten E. coli colonies is isolated and analyzed by PCR using specific primers for the transformed plasmids. The plasmid DNA is further analyzed with specific restriction enzymes and then sequenced.
Further Modifications to Improve Heme-containing Protein Production
[00186] Several additional modifications can be made to improve the levels of heme- containing protein present in a host cell. One suitable example is a modification that results in a lower level of heme oxygenase in the host cell. This can be achieved, for example, by use of an inducible sRNA designed to target the heme oxygenase. Examples 9 and 10 provide further details of this procedure. As shown in FIG. 16, this combined procedure results in a much higher level of heme-containing protein.
[00187] Another method of increasing the heme-containing protein level is by additional modifications that result in an increase in the flux through ferrochelatase. This can be accomplished, for example, by a knockdown of the gene encoding magnesium chelatase
(MgCh), the overexpression of ferrochelatase (FeCh), the knockdown of gun4 (MgCh activator), or a gun4(W192A) mutation. FIG. 14 provides a schematic diagram showing the pathway interactions that can affect the levels of production of the heme-containing protein. Culture medium modifications can also be used to increase the level of the heme-containing protein produced in the cyanobacterial cells. Examples of these modifications include but are not limited to magnesium-starvation, iron supplementation, and nitrogen-starvation. A combination of these methods and genetic modifications can also be used.
Phycocyanin Deficiency in the Host Cell Improves Heme Production Levels
[00188] Heme-containing proteins can be produced at even higher levels when the host cells have a phycocyanin-deficient background. This is demonstrated in Examples 13 and 14. The phycocyanin deficiency can be obtained, for example, by a knockout of the phycocyanin gene. The phycocyanin deficiency can also be obtained, for example, by an inducible or constitutive knockdown of the phycocyanin gene, such as by the use of a sRNA or anti sense molecule that targets phycocyanin levels.
Cultivation of Cyanobacterial Cells to Produce the Heme-containing Protein
[00189] In an embodiment, a heme-containing protein is synthesized in cyanobacterial cultures by preparing host cyanobacterial cells having the gene constructs discussed herein, and growing cultures of the cells.
[00190] Methods of growing cyanobacteria in liquid media and on agarose-containing plates are well known to those skilled in the art (see, e.g., websites associated with ATCC). Any of these methods or media maybe used to culture the cyanobacterial cells. A number of known recipes for cyanobacterial growth medium can be used. In an embodiment, BG11 medium is used, see Stanier, R.Y., et al., Bacteriol. Rev. 1971, 35: 171-205, which is hereby incorporated by reference.
[00191] In an embodiment, the cyanobacterial strain is a fresh water strain, and BG11 is used. In another embodiment, the cyanobacteria culture grows best in a marine (salt water) medium, by adding an amount of salt to the BG11 medium. In an embodiment, marine BG11 (mBGl 1) contains about 35 practical salinity units (psu), see Unesco, The Practical Salinity Scale 1978 and the International Equation of State of Seawater 1980. Tech. Pap. Mar. Sci., 1981, 36: 25 which is hereby incorporated by reference. [00192] In an embodiment, the cells are grown autotrophically, and the only carbon source is CO2. In another embodiment, the cells are grown mixotrophically, for example with the addition of another carbon source such as glycerol or glucose.
[00193] The cultures can be grown indoors or outdoors. The light cycle can be set as for continuous light, or for periodic exposure to light, e.g., 16 hours on and 8 hours off, or 14 hours on and 10 hours off, or 12 hours on and 2 hours off, or any alternative variation of on and off hours of light comprising about a day.
[00194] The cultures can be axenic, or the cultures can also contain other contaminating species.
[00195] In an embodiment, the cyanobacteria are grown in enclosed bioreactors in quantities of at least about 1 L, 20L, 50L, 100 L, 500 L, 1,000 L, 2,000 L, 5,000 L, or more. In a preferred embodiment the bioreactors are about 20 L to about 100 L. In an embodiment, the cyanobacterial cell cultures are grown in disposable, flexible, and tubular photobioreactors made of a clear plastic material.
[00196] In another embodiment, cultures are grown indoors or outdoors with continuous light, in a sterile environment. In another embodiment, the cultures are grown outdoors in an open pond type of photobioreactor.
[00197] The choice of culture medium can depend on the cyanobacterial species. In an embodiment of the invention, the following BG11 medium for growing cyanobacteria can be used. When salt water species are grown, NaCl is added to the culture medium.
Table 4: Culture Medium
Figure imgf000044_0001
Figure imgf000045_0001
[00198] The present disclosure is further described by the following non-limiting examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present disclosure.
EXAMPLES
Example 1 General Methods
[00199] Restriction endonucleases were purchased from New England Biolabs (NEB;
Ipswich, MA), unless otherwise noted. PCR was performed using an Eppendorf Mastercycler thermocycler (Hauppauge, NY), using Phire II Hot Start polymerase or Taq DNA polymerase (NEB) for diagnostic amplifications, and Phusion polymerase or Crimson LongAmp Taq Polymerase (NEB) for high fidelity amplifications. PCR temperature profiles were set up as recommended by the Taq manufacturer. Cloning was performed in E. coli using XLIO-Gold Ultracompetent cells (Agilent Technologies, Santa Clara, CA) following the manufacturer's protocol. TOPO cloning kits (Zero Blunt TOPO PCR Cloning kit) were purchased from
Invitrogen (Carlsbad, CA), and were used according to the manufacturer's protocol.
[00200] E. coli strains HB101 (Promega, Madison, WI), XLIO-Gold (Stratagene, San Diego, CA), and a-select (Bioline) were grown in Luria-Bertani (LB) medium at 37 °C.
Ampicillin (50 μg/mL), kanamycin (50 μg/mL), and chloramphenicol (34 μg/mL) were used when appropriate. Cultures were continuously shaken overnight at 200 rpm and at 100 rpm when used for conjugation. ABICyanol was cultured at from 28 °C to 37 °C in liquid BG11 fresh water medium on a reciprocal shaker at 150 rpm under continuous illumination of approximately 30 - 40 μπιοΐ photons*m-2 *sec_1.
[00201] Plasmid DNA from E. coli strains was isolated using a GeneJet Plasmid Miniprep
Kit (Fermentas) according to the manufacture's protocol. For plasmid isolation from putative ABICyanol transformants, total DNA was prepared according to Saha et al. (2005), World Jour. Microbiol Biotechnol 21 :877-881.
Example 2
Preparation of Cyanobacterial Culture Medium
[00202] For cyanobacterial growth, BG-11 stock solution was purchased from Sigma
Aldrich (St. Louis, MO). Marine BG-11 (MBG-11) was prepared by dissolving 35 g Instant Ocean (United Pet Group, Inc, Cincinnati, OH) in 1 L water and supplementing with BG-11 stock solution. Vitamin B12 (Sigma Aldrich) was supplemented to MBG-11 to achieve a final concentration of 1 μg/L, as needed. Stock solutions of the antibiotics spectinomycin (100 mg/ml) and kanamycin (50 mg/ml) were purchased from Teknova (Hollister, CA). Stock solution of the antibiotic gentamycin (10 mg/ml) was purchased from MP Biomedicals (Solon, OH).
[00203] Unless otherwise noted, the ABICyanol transformants were selected on solid BG1 1 medium containing 10 - 20 μg/mL kanamycin and were maintained on BG11 plates containing 40 μg/mL kanamycin. For growth in liquid freshwater BG11 medium, 30-40 μg/mL of kanamycin was applied. Example 3
Transformation of ABICyanol with a p6.8 kb Based Shuttle Vector
[00204] The endogenous 6.8 kb plasmid of ABICyanol was used as a means of shuttling exogenous DNA to cyanobacterial host cells. By inserting an origin of replication that is effective in E. coli (such as R6KOri), the p6.8 kb plasmid DNA was able to be manipulated in bacteria, such as E. coli, to incorporate genes and sequences of interest into a recombinant p6.8 kb. For example, modifications to decrease the effectiveness of endogenous restriction systems that are present in ABICyanol, such as methylation, were performed.
[00205] The presence of the endogenous origin of replication on the p6.8 plasmid allowed for replication of the recombinant p6.8 kb once it was transferred into a host cell. In some instances, multiple cloning sites were be added to allow for several different antibiotic resistance genes to be added, if desired. Multiple cloning sites were also inserted, for example, to allow for ease of insertion of various expression cassettes. In this way, various sequence segments of the plasmid could be replaced with other sequence segments as needed.
Example 4
Competent ABICyanol Cells for Transformation by Conjugation
[00206] The following method was used to decrease the EPS layer of the strain
Cyanobacterium sp. ABICyanol prior to conjugation. The method involved several steps:
treatment of cells with NAC, washing steps that utilize NaCl, treatment with lysozyme, and subsequent washing followed by a conjugation procedure.
[00207] Two hundred mL of an exponentially growing culture (OD750nm greater than about
0.5 and less than about 1.0) was incubated with NAC for 2 days at 16 °C (end concentration of NAC is about 0.1 mg/mL) without shaking. This pretreatment was followed by several steps to degrade the EPS and to weaken the cell wall. The pretreated culture was pelleted at 4,400 rpm and washed with 0.9% NaCl containing 8 mM EDTA.
[00208] For further treatment with lysozyme, the cell pellet was resuspended in 0.5 M sucrose and incubated 60 minutes at room temperature (RT) with slow shaking (85 rpm). Cells were then centrifuged and resuspended in 40 mL of a solution containing 50mM Tris (pH 8.0), 10 mM EDTA (pH 8.0), 4% sucrose, and 20-40 μg/mL lysozyme. After incubation at room temperature for 10-15 minutes, cells were centrifuged and washed three times using different washing solutions; i) 30 mM Tris containing 4% sucrose and 1 mM EDTA; ii) 100 mM Tris containing 2 % sucrose; and iii) BG11 medium. All centrifugation steps before lysozyme treatment were performed at 4,400 rpm for 10 minutes at 10 °C. All centrifugation steps after the lysozyme treatment were performed at 2,400 rpm for 5 minutes at 4 °C. Resuspended cells were used for conjugation.
Example 5
Transformation of ABICyanol by Conjugation
[00209] Gene transfer to ABICyanol (using modified plasmids containing oriVT) was performed using conjugation. The shuttle vectors were transformed into ABICyanol following a modified conjugation protocol which includes the pretreatment of ABICyanol to reduce its EPS layer as described in the above example.
[00210] Triparental mating was performed as follows: E. coli strain J53 bearing a conjugative RP4 plasmid and E. coli strain HB101 bearing the cargo to be introduced into ABICyanol and the pRL528 helper plasmid (for in vivo methylation) were used. E. coli strains were grown in LB broth supplemented with the appropriate antibiotic overnight at 37 °C with shaking at 100 rpm. An aliquot of 3 - 5 mL of each culture was centrifuged, washed twice with LB medium and resuspended in 200 \iL LB medium. Subsequently, the E. coli strains were mixed, centrifuged and resuspended in 100 [iL BG11 medium. Two hundred mL of
exponentially growing cyanobacterial culture (OD750nm of greater than 0.5 and less than 1.0) was centrifuged (3,000 rpm, 10 min), pretreated to degrade the EPS layer as described above in Example 4, and subsequently washed and resuspended in 400 μΕ BG11 culture medium containing Tris/sucrose buffer. A 100 μL aliquot of resuspended cyanobacterial and E. coli cultures was mixed and applied onto a membrane filter (Millipore GVWP, 0.22 μm. pore size) placed on the surface of solid BG11 medium supplemented with 5% LB. Petri dishes were incubated under dim light (5 μΕ m-2 s-1) for 2 days. Cells were then resuspended in fresh BG11 medium and plated onto selective medium containing 10 and 15 μg/mL kanamycin, respectively. The following selection conditions were used: light intensity of approximately 20 - 40 μΕ m-2 s-1 at a temperature of approximately 28 °C. Transformants were visible after approximately 7-10 days. The transformant colonies were then plated on BG11 media containing 15 μg/mL kanamycin and then transferred stepwise to higher kanamycin concentrations (up to 60 μg/mL) to aid in the selection process.
Example 6
Transformation of ABICyanol by Electroporation
[00211] Electroporation can also be used for successful transformation of
Cyanobacterium sp. ABICyanol, or other strains such as Arthrospira, Synechococcus, and Synechocystis, using, for example, the same plasmids as for conjugation, but with lower efficiency.
[00212] As with the conjugation transformation protocol (above), strain-specific adaptations of standard electroporation protocols can be made to avoid DNA digestion by endogenous restriction enzymes and to allow DNA entry through the EPS layer. To achieve successful electroporation, DNA is protected against endogenous restriction enzymes by methylation. Prior to electroporation, ABICyanol cells are pretreated with positively charged polyaminoacids such as poly-L-lysine hydrobromide or poly-L-ornithine hydrochloride or combinations thereof (in particular poly-L-lysine hydrobromide) in order to increase the DNA uptake efficiency.
[00213] As an example, a one hundred mL aliquot of exponentially growing ABICyanol culture (corresponding to a cell density of approximately 2xl07 cells/mL), was harvested, washed and resuspended in 0.9 % NaCl containing 25 mM Tris-HCl (pH 8.0). Poly-L-lysine hydrobromide was added to the resuspended cells to obtain a final concentration of 50 μg/mL. ABICyanol cells were then incubated for several hours or overnight before electroporation.
[00214] In a typical procedure, 50 mL of poly-L-lysine hydrobromide treated ABICyanol cells were harvested and treated with 30 mL ice-cold BG11 containing 6% DMSO. After incubation on ice for 20 minutes, cells were harvested and frozen in liquid nitrogen for 15 minutes. These pre-frozen cells were thawed by adding 15 mL ice-cold buffer containing 1 mM HEPES (pH7.5), 0.2 mM K2HPO4 and 0.2 mM MgCk. The cells were washed sequentially once more with 1 mM HEPES and ETMT buffer containing 0.1 mM HEPES, 0.2 mM K2HPO4 and 0.2 mM MgCh. The cells were harvested by centrifugation at 15,000 x g for 5 minutes. All of the washes and centrifugations were carried out on ice or in a pre-chilled centrifuge (4 °C). For each electroporation procedure, 3 μg methylated DNA was added to 100 μΕ of concentrated cells. Cells were electroporated in a cuvette with a 2 mm gap between the electrodes and pulsed once in a Gene Pulse X-cell (Bio-Rad) using an exponential decay protocol (electric field strength of 8 kV/cm, capacitance of 25 μF, resistance of 400 ohms, for a time of approximately 8-9 ms). After electroporation, 1-2 mL BGl 1 medium was immediately added to the cyanobactenal suspension, which was subsequently transferred to a 50 mL flask containing 15 mL fresh BGl 1 medium. After incubation for 1- 2 days under normal light (30 - 40 μΕ m-2 s-1) with gentle shaking at 30 °C, recovered cultures were centrifuged, resuspended in 500 μL BGl 1 medium and placed onto selective media (BGl 1 containing 20 μg/mL Km or 40-60 μg/mL of spectinomycin).
Example 7
Transformation of Arthrospira platensis PCC9108 by Conjugation
[00215] Gene transfer to Arthrospira platensis PCC9108 (using modified plasmids containing oriVT) was performed using conjugation. Biparental mating was performed as follows: E. coli strain ECIOODC bearing a modified conjugative RP4 plasmid (pRL443), the cargo plasmid to be introduced into Arthrospira platensis and the pRL528 helper plasmid (for in vivo methylation) was used. The E. coli strain was grown in LB broth supplemented with the appropriate antibiotics overnight at 37 °C with shaking at 100 rpm. The E. coli culture was centrifuged, washed twice with Zarrouk' s medium and resuspended in 100 μL Zarrouk' s medium. 10 mL of a late exponentially growing Arthrospira platensis culture (OD750nm between 0.5 and 1.0) was centrifuged (3,000 rpm, 10 min), washed and resuspended in 200 μL Zarrouk's medium. A 100 μL aliquot of resuspended cyanobactenal and E. coli cultures was then mixed and incubated under dim light (5 μΕ m-2 s-1) for 2-3 days. Cells were then plated onto selective Zarrouk' s plates containing 10 μg/mL zeocin, respectively. The following selection conditions were used: light intensity of approximately 10 - 20 μΕ m-2 s-1 at a temperature of approximately 28 °C. Transformants were visible after approximately 7-14 days. The transformant colonies were then transferred onto fresh Zarrouk' s plates containing 30 μg/mL zeocin and then transferred stepwise to higher zeocin concentrations (up to 100 μg/mL) to aid in the selection process. Example 8
Expression of a Synechocystis PCC 6803 Heme-containing Protein Cyanoglobin (GlbN) in
Cyanobacterium sp. ABICyanol
[00216] The cyanoglobin (glbN) gene from Synechocystis PCC 6803 (SEQ ID NO: 11), which was codon optimized for optimal expression, was inserted to the p6.8 modified endogenous plasmid of Cyanobacterium sp. ABICyanol, under the control of an endogenous zinc-inducible smtA promoter. This created a 10,340 bp construct which was designated
#2887\pABICyanol_6.8: :PsmtA-glbN_S6803(ABICyanolopt)-TpsaB, as shown in the plasmid map in FIG. 5, and present in Table 3 as Construct #2887, strain AB1307. The plasmid was transformed to Cyanobacterium sp. ABICyanol following the methods described in Examples 3- 5.
[00217] Transformed cells were then grown in culture medium and examined for cyanoglobin production under varied zinc concentrations. The cells were isolated from the medium, and protein extracts were separated on a 16% native PAGE and stained with Coomassie Brilliant Blue. Expression of the cyanoglobin protein (arrow) was induced with 20 μΜ zinc sulfate, as shown in FIG. 15. The protein gel shows that because of the zinc-inducible promoter, expression of the Synechocystis cyanoglobin did not begin until zinc was added to the medium.
Example 9
Additional Genetic Modification with an Inducible sRNA
[00218] In order to determine if one could increase the heme-containing protein production by lowering the level of the endogenous heme oxygenase enzyme in cyanobacterial cells (and thus divert heme production away from phycobilin production and towards production of the heme-containing protein), the following genetic modification was designed. An antisense construct (SEQ ID NO: 16) that targets the nucleic acid that encodes heme oxygenase, operably linked to an inducible promoter, was inserted into the plasmid. Upon induction, the antisense sRNA lessened the presence of heme oxygenase, causing a higher content of the heme groups to be available to bind to the heterologous heme-containing protein being expressed in the cyanobacteria. This resulted in higher production of the mature heme-protein complex (See Example 10, below). [00219] Alternatively, the inducible sRNA is designed to increase the flux through the ferrochelatase. An increased flux through the ferrochelatase is accomplished by different means such as, for example, a knockdown of the gene encoding magnesium chelatase (MgCh), the overexpression of ferrochelatase (FeCh), the knockdown of gun4 (MgCh activator), a gun4(W192A) mutation, magnesium-starvation, iron supplementation, or nitrogen- starvation.
Example 10
Production of Synechocystis PCC 6803 Heme-containing Protein Cyanoglobin (GlbN)
Increased by Decreasing the Level of Heme Oxygenase in the Cell
[00220] To determine whether the host cyanobacterial cells were able to produce more heme-containing protein when the level of the heme oxygenase ABICyanol . I_orf29000 was genetically reduced in the host cell, the following experiment was performed. An anti-HOl sRNA construct #2890\ pABICyanol_6.8: :PsmtA-glbN_S6803(ABICyanolopt)-TpsaB-PnirA- anti HOl vl (SEQ ID NO: 16) was prepared in order to inducibly decrease the heme oxygenase level in the cell. The plasmid is present in Table 3 as Construct #2890, strain AB1310. The sRNA sequence was placed upstream of the gene encoding the heme-containing protein, having its own endogenous nitrate-inducible promoter from Cyanobacterium sp. ABICyanol .
[00221] The cells were transformed with the plasmid construct, and were then grown in culture medium. Cells were isolated from the medium, and the protein was extracted from the cell material. The protein extracts were separated on a 16% native PAGE and stained with Coomassie Brilliant Blue (FIG. 16). The data shows that when nitrate was added to the culture to induce production of the anti-heme oxygenase sRNA, less heme oxygenase and thereby more heme-containing protein was produced (black arrow, right lane, FIG. 16).
Example 11
Expression of Leghemoglobin C2 from Soybean in Cyanobacterium sp. ABICyanol
[00222] To demonstrate that plant-derived leghemoglobin proteins can be produced in cyanobacteria, the following experiment was performed. The leghemoglobin C2 gene from soybean (SEQ ID NO: 1) was codon optimized (SEQ ID NO: 2) for optimal expression in Cyanobacterium sp. ABICyanol . The gene was placed under the control of an endogenous zinc- inducible smtA promoter on the p6.8 modified endogenous ABICyanol plasmid. [00223] The plasmid also encoded an sRNA directed against the ribosomal binding site of the heme oxygenase ABICyanol . I_orf29000, under control of the endogenous nitrate-inducible promoter from Cyanobacterium sp. ABICyanol . This resulted in a 10,456 bp plasmid designated as #2889\pABICyanol_6.8: :PsmtA-legH_GmC2(ABICyanolopt)-TpsaB-PnirA-anti_H01_vl, as shown in Table 3 as Construct #2889, strain AB1309, and in the plasmid map in FIG. 7. The plasmid was then transformed to Cyanobacterium sp. ABICyanol following the methods described in Examples 3-5.
[00224] Transformed cells were then grown in culture medium and examined for leghemoglobin production under various induction conditions. Protein extracts were separated by denaturing electrophoresis on a 16% Tricine-SDS-PAGE including 6 M urea and subjected to a Coomassie Brilliant Blue staining.
[00225] The protein gel (FIG. 17) demonstrated that the zinc-inducible promoter delayed expression of the soybean leghemoglobin C2 protein (arrow) until zinc was added to the medium. Upon addition of zinc, the leghemoglobin was produced. Upon addition of nitrate, the sRNA against heme oxygenase was also induced. The combination of the induction of the leghemoglobin gene and the anti-heme oxygenase sRNA resulted in strong production of the leghemoglobin protein.
Example 12
A Stronger Promoter and Multiple Copies of the Gene Encoding the Heme-containing
Protein on the Plasmid Construct Increased Protein Production
[00226] Constructs #2942 and #3022, strains AB 1336 and AB4005: The cyanoglobin
(glbN) gene from Synechocystis strain PCC 6803 (SEQ ID NO: 1 1), which was codon optimized for optimal expression, was inserted to the p6.8 modified endogenous plasmid of
Cyanobacterium sp. ABICyanol, following the method described in the above example, except that the cyanoglobin gene was placed under the regulatory control of an endogenous, strong constitutive promoter PcpcB, rather than an inducible smtA promoter. The plasmid #2942\ pABICyanol_6.8: :PcpcB-glbN_S6803(ABICyanolopt)-TpsaB-Porf0316'-anti_HOl_vl allowed constitutive and high expression of the glbN gene during the full length of the culture growth. This was found to result in greater cyanoglobin production per day. [00227] The p6.8 plasmid that carried the heterologous gene is generally present in many copies per cell. However, in order to maximize production of the heterologous heme-containing protein, an additional construct #3022\ pABICyanol_6.8: :PcpcB-glbN_S6803(ABICyanolopt)- TpsaB-PcpcB-glbN_S6803(ABICyanolopt)-TpsaB-Porf0316'-anti_HOl_vl (SEQ ID NO: 58) was created that has two copies of the gene encoding the cyanoglobin glbN protein, each with its own PcpcB promoter. Both plasmids encode the anti-HOl sRNA sequence downstream of the endogenous copper-inducible promoter from Cyanobacterium sp. ABICyanol .
[00228] The plasmids were transformed to Cyanobacterium sp. ABICyanol following the method described in Examples 3-5. The cells were then grown in culture medium. Cells were isolated from the medium, and the protein was extracted from the cell material. Protein extracts were separated by denaturing electrophoresis on a 16% Tricine-SDS-PAGE including 6 M urea. The protein gel of FIG. 18A was unstained, and the heme-containing protein can be seen as a reddish band. FIG. 18B shows a duplicate protein gel, which was subjected to Coomassie Brilliant Blue stain to visualize the total proteins.
[00229] The data show that by using a stronger promoter and two sets of heme protein genes, more protein is made. Further, when copper was added to induce production of the sRNA fragment, less heme oxygenase was produced and thereby more heme-containing protein was produced (FIG. 18A and FIG. 18B).
Example 13
Generation of a Phycocyanin-deficient Cyanobacterial Strain
[00230] The Cyanobacterium sp. ABICyanol strain was grown under high-light stress in order to isolate derivatives with altered phycocyanin expression. The applied conditions were as follows: a BGl 1-agar plate with a cell lawn of ABICyanol was exposed to high-light (3,000 μΕ m-2 s-1) for 90 minutes, resulting in a cell death rate of about 99.99 %. Individual colonies that survived the treatment were picked and patched onto new plates for later characterization. UV- VIS absorbance spectra from individual isolates identified cell lines with reduced phycocyanin content. FIG. 19 shows the absorbance spectrum of one of the isolated pale green cell lines, designated #AB0492, in comparison with the absorbance spectrum of wild type cells. Genome sequencing of the strain #AB0492 revealed that a spontaneous deletion within the cpcA gene was the cause of the phycocyanin-deficient phenotype. Example 14
Expression of the Heme-containing Protein in a Phycocyanin-deficient Background
[00231] Constructs #2942 and #3022, strains AB4011 and AB4012: To determine whether several gene modifications would work together to improve heterologous heme production even further, a construct was created that had constitutive expression of codon-optimized cyanoglobin from Synechocystis sp. PCC6803 (2 copies in tandem) and copper inducible sRNA knockdown of heme oxygenase ABICyanol . l_orf29000.
[00232] To determine whether more heme-containing protein would be made if the heterologous gene encoding the heme protein was inserted into a cell having a genetic phycocyanin deficiency (AB0492), the following method was used. The phycocyanin-deficient mutant host cell was prepared by the method described in the above example, and was then transformed with the two constructs #2942\pABICyanol_6.8: :PcpcB- glbN_S6803(ABICyanolopt)-TpsaB-Porf0316'-anti_HOl_vl (SEQ ID NO: 56) and
#3022\pABICyanol_6.8: :PcpcB-glbN_S6803(ABICyanolopt)-TpsaB-PcpcB- glbN_S6803(ABICyanolopt)-TpsaB-Porf0316'-anti_HOl_vl (SEQ ID NO: 58). Both plasmids lead to constitutive expression of codon-optimized cyanoglobin from Synechocystis sp. PCC6803 (1 and 2 copies in tandem) and a copper inducible sRNA knockdown of heme oxygenase ABICyanol . l_orf29000.
[00233] The production of the heme-containing protein was compared with that of host cells that had a normal amount of phycocyanin. The strains were grown in the absence and presence of 10 μΜ copper-EDTA to induce the knockdown of the heme oxygenase via anti-HOl sRNA. AB4011 has a single copy of the PcpcB-glbN_S6803, whereas AB4012 comprises two PcpcB-glbN_S6803 copies on the same plasmid. Protein extracts were separated on a 16% native PAGE and stained with Coomassie Brilliant Blue.
[00234] The results (FIG. 20) show that expression of the cyanoglobin (glbN) gene from
Synechocystis strain PCC 6803 in the AcpcA derivative AB4011 (lx PcpcB-glbN) was generally lower compared to the expression in the phycocyanin-rich ABICyanol derivative AB1336. However, the copper-induced knockdown of the heme oxygenase in AB4011 had a much stronger effect on the expression of glbN, compared to the relatively mild effects of the heme oxygenase knockdown in AB1336 on the glbN expression (FIG. 20). In alignment with this observation, AB4012 (2x PcpcB-glbN copies) expressed a significant amount of apo-GlbN (cyanoglobin without heme), indicating a lack of sufficient heme caused by a potential higher turnover of heme via the heme oxygenase. By contrast, knockdown of the heme oxygenase in AB4012 resulted in strongly expressed and fully assembled heme protein.
Example 15
Expression of an Arthrospira platensis PCC 8005 Cyanoglobin ( glbN) Gene in
Cyanobacterium sp. ABICyanol
[00235] Construct #2994, strain AB1364: A codon-optimized cyanoglobin (glbN) gene
(SEQ ID NO: 14) from Arthrospira platensis PCC 8005 was inserted to the p6.8 modified endogenous plasmid of Cyanobacterium sp. ABICyanol, under the control of the endogenous constitutive promoter PcpcB. This created a 10491 bp construct which was designated
#2994\pABICyanol_6.8: :PcpcB-glbN_Ap8005(ABICyanolopt)-TpsaB, as shown in the plasmid map in FIG. 10. The plasmid was transformed into Cyanobacterium sp. ABICyanol following the methods described in Examples 3-5.
[00236] Cell cultures were grown, and the cellular material was isolated. Protein extracts were prepared, separated on a 16% native PAGE, and stained with Coomassie Brilliant Blue (FIG. 21). The data shows that this modification resulted in constitutive and high expression of the glbN gene from Arthrospira platensis PCC 8005 during the full length of the culture growth (FIG. 21).
Example 16
Expression of an Arthrospira platensis Cyanoglobin (glbN) Gene in Arthrospira platensis
PCC9108
[00237] Construct #2949 (SEQ ID NO: 59), strain AB4041 : The codon optimized cyanoglobin (glbN) gene from Arthrospira sp. PCC 8005 (SEQ ID NO: 14) was inserted into chromosome of Arthrospira platensis PCC9108 via homologous recombination. The cyanoglobin gene was placed under the control of the endogenous constitutive promoter PcpcB from Arthrospira platensis PCC9108. The gene cassette was inserted into the chromosome of Arthrospira platensis PCC9108 via homologous recombination following the methods described in Examples 6-7. [00238] Construct #3057 (SEQ ID NO: 60), strains AB4065 and AB4066: In order to maximize production of the heterologous heme-containing protein, an additional construct #3057\ oriVT-nblA2_2293_up-PcpcB1918-glbN_Ap8005-PcpcB1918_glbN_Ap8005-TpsaB- PrbcL2293-ble-TB1002-nblA2_2293_down was created that has two copies of the codon- optimized gene encoding the cyanoglobin glbN protein Arthrospira sp. PCC 8005 (SEQ ID NO: 14), each with its own PcpcB promoter from Arthrospira platensis PCC9108. The gene cassette was inserted into chromosome of Arthrospira platensis PCC9108 and the derivative strain Arthrospira platensis AB 1298 via homologous recombination following the methods described in Examples 6-7.
[00239] Cell cultures were grown in Zarrouk's medium and protein was extracted from the cells. The protein extracts (FIG. 22A and FIG. 22B) were separated on a 16% native PAGE. FIG. 22A is a photograph of the unstained protein gel. FIG. 22B is a photograph of an identical gel, stained with Coomassie Brilliant Blue to identify total protein.
[00240] The results show the expression of the cyanoglobin (GlbN) protein from
Arthrospira platensis PCC 8005 in Arthrospira platensis PCC9108 (AB4041, AB4065) and the derivative strain Arthrospira platensis AB1298 (AB4066). AB4041 has a single copy of the PcpcB-glbN_Ap8005, whereas AB4065 and AB4066 comprise two PcpcB-glbN_Ap8005 copies on the same plasmid.
Example 17
Purification of the Heme-containing Protein from Cyanobacterial Cells
[00241] Cyanobacterium sp. ABICyanol strain AB4005 was grown in stirred 1 L photobioreactors (12/12 h day/night cycle) in artificial seawater, supplemented with BG11 trace elements and 100 μΜ Fe(II)NH4 sulfate. The pH was maintained at pH 7.3 by addition of 5 % CO2 on demand with a flow-rate of 20 - 40 ml/min. AB4005 exhibited constitutive expression of a codon-optimized cyanoglobin from Synechocystis sp. PCC6803 (2 copies in tandem). 10 μΜ CuEDT A was added to induce the sRNA knockdown of heme oxygenase
ABICyanol . l_orf29000. 200 ml of the culture was harvested via centrifugation for preparation of a soluble protein extract (Tris-HCl pH 8.0, 10 mM EDTA, 10 mM sodium dithionite), followed by a concentration step with lOkDa centrifugal filters. 100 μΙ_, of the soluble protein concentrate was loaded on a 30 mL Sephacryl 100-HR gel filtration column driven by gravity flow (FIG. 23 A). Elution fractions El through E14 (FIG. 23B) were collected for further analysis.
[00242] The eluted samples where then separated on a 16% native PAGE gel. A photograph of the unstained gel (showing the reddish-colored heme fractions) is shown in FIG. 24A. A duplicate gel that has been stained with Coomassie Brilliant Blue stain to identify total protein is shown in FIG. 24B. The left lane of both gels is the control sample (water soluble extract; labeled "CE".
Example 18
Quantitation and Analysis of Cyanobacterially-produced Heme-containing Protein
[00243] Two methods can be used for determination of the concentration of heme- containing proteins in the bulk extraction. One method is the use of absorbance spectroscopy with known extinction coefficients. The ferric form of GlbN from Synechocystis strain PCC 6803 has a main absorbance peak at 410 nm. In this way, one can determine the relative induction of the heme-containing protein in the induced versus either non-induced or wild type strains.
[00244] As an example, FIG. 25 depicts the relative absorbance spectra of cell extracts from Cyanobacterium sp. ABICyanol expressing the cyanoglobin (GlbN) protein from
Synechocystis strain PCC 6803. AB4005 contains two PcpcB-glbN copies in tandem and an inducible sRNA to knockdown expression of the heme oxygenase ABICyanol . I_orf29000. AB4005 cells were grown in either the absence or presence of 10 μΜ copper-EDTA to induce the anti-HOl sRNA. The absorbance peak at 410 nm (arrow) shows the ferric form of GlbN from Synechocystis strain PCC 6803.
[00245] Another method of analysis involves heme staining following gel electrophoresis.
This method is used to determine accumulation levels in producers since the heme component of heme-containing proteins remains bound to the protein as it migrates through the gel. Molecular weights of all heme proteins present can also be determined using this method.
Example 19
Scale-up of Modified Cyanobacteria to Produce Heme-containing Protein
[00246] A culture of modified cyanobacteria is scaled-up to flasks, then 1 liter containers, then 5 liter containers, then to outdoor vertical photobioreactors. The host cells are then grown without induction of the inserted genes for most of the length of the culture period (such as, for example, about 20 days). Near the end of the culture period (approximately 2 to 4 days prior to culture harvest), the production of the heme-containing protein is induced. At this time, other modifications can also be induced, such as modifications to increase the heme that is available for binding. The culture can then be harvested, and the cells can be dried to produce a dried biomass material containing the heme-containing protein. Alternatively, the total protein can be isolated from the cell material. Further, the heme-containing protein can be substantially purified from the total protein fraction, if desired.
Example 20
Cell Extracts: Total Protein Assay
[00247] Protein concentration in the cellular extracts is determined using the Bio-Rad DC protein assay Kit (Bio-Rad; Hercules, CA; Cat# 500-0111) according to the manufacturer's instructions. Bovine serum albumin is used as the standard.
Example 21
Production of an Iron-rich Food Product from Cyanobacteria
[00248] A modified strain of Arthrospira platensis is modified as described in one of the above examples, to produce large amounts of an exogenous heme-containing protein. The culture is grown for 3 weeks in an outdoor photobioreactor. The culture is harvested, and the biomass is dried. The resulting biomass has a heme-bound iron content that is much higher than the iron content of wild type Arthrospira, and can thus be used as an iron-rich food supplement that is also rich in other nutrients. The food supplement can be formed into flakes, a powder, a tablet, or a capsule.
Example 22
Production of an Iron-rich Food Product and Phycocyanin from Cyanobacteria
[00249] A modified strain of Arthrospira platensis is modified as described in the above example, except that a substantial amount of the phycocyanin is isolated from the biomass before it is processed for its use as an iron-rich food supplement. In this way, two valuable products, phycocyanin and an iron-rich food supplement, can be obtained from the cyanobacterial culture. If desired, the product can be pasteurized or sterilized during the processing step. Example 23
Preparation of a Product Rich in Heme-containing Protein
[00250] Extraction and isolation of a protein product enriched with a heme-containing protein is achieved by removal of the lipid and hydrophobic fraction of the biomass with a nonpolar solvent. The remaining material is then precipitated using a pH shift. The protein-rich fraction is collected. The protein-rich material is desalted by filtration or buffer exchange. Optionally, the desalted protein-rich material is then dried to a fine powder.
Example 24
Production of a meat-like protein meal rich in a heme-containing protein
[00251] The protein-rich material of the above example is obtained and formulated into a vegetarian, meat-like food product. The gene encoding the heme-containing protein can be derived from a cyanobacterial source, an algal source, a plant source, or from an animal source.
Example 25
Heme Apoprotein Production
[00252] If desired, the heme-containing protein can be first produced in the cell as an apoprotein without bound heme, and can then be extracted from the cell biomass, purified, then further modified chemically to add in the heme-containing moiety.
[00253] Although the present invention has been described in considerable detail with reference to certain embodiments thereof, other embodiments are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the embodiments contained therein.

Claims

CLAIMS What is Claimed is:
1. A genetically modified cyanobacterium comprising a polynucleotide encoding a heterologous heme-containing protein.
2. The genetically modified cyanobacterium of claim 1, wherein said polynucleotide encodes a leghemoglobin or a cyanoglobin.
3. The genetically modified cyanobacterium of claim 2, wherein said polynucleotide has a nucleic acid sequence that is at least 85% identical to the polypeptide sequence of a plant-derived leghemoglobin or a cyanobacterial cyanoglobin.
4. The genetically modified cyanobacterium of any one of the above claims, wherein the cyanobacterium is from a genus selected from the group consisting of Arthrospim sp.,
Synechocystis, Synechococcus, Acaryochloris, Anabaena, Aphanothece, Thermosynechococcus, Chamae siphon, Chroococcus, Cyanobacterium, Cyanobium, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus, Prochloron, Chroococcidiopsis, Cyanocystis, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria, Xenococcus, Arthrospira, Borzia, Crinalium, Geitlerinema, Halospirulina, Leptolyngbya, Limnothrix, Lyngbya,
Microcoleus, Cyanodictyon, Aphanocapsa, Oscillatoria, Planktothrix, Prochlorothrix,
Pseudanabaena, Spirulina, Starria, Symploca, Trichodesmium, Tychonema, Anabaenopsis, Aphanizomenon, Calothrix, Cyanospira, Cylindrospermopsis, Cylindrospermum, Nodularia, Nostoc, Chlorogloeopsis, Fischerella, Geitleria, Nostochopsis, Iyengariella, Stigonema, Rivularia, Scytonema, Tolypothrix, Cyanothece, Phormidium, and Adrianema.
5. The genetically modified cyanobacterium of any one of the above claims, wherein the polynucleotide is codon optimized for optimal expression in the cyanobacterium.
6. The genetically modified cyanobacterium of any one of the above claims, wherein the polynucleotide is linked to a constitutive promoter.
7. The genetically modified cyanobacterium of any of claims 1-5, wherein the polynucleotide is linked to an inducible promoter.
8. The genetically modified cyanobacterium of claim 6 or 7, wherein the promoter is selected from the group consisting of PrbcL, PntcA, PnblA, PisiA, PpetJ, PpetE, PcorT, PsmtA, PziaA, PsigB, PlrtA, PhtpG, PhspA, PclpBl, PhliB, PggpS, PpsbA2, PpsaA, PnirA, PnarB, PnrtA, PisiB, PnrsB, PlrtA, PmrgA, PpstS, and PcrhC, PpetJ, PpsbD, PnblA, PrpoA, PisiB, PrnpA, PrpsL, PpsbD, PcpcB, PnirA*2, PnirA*3, PnirA*4, PmntC, PrpsL*4, Prbc*, PrpsL*4, PcpcB (SEQ JO NO: 55), PnirA (SEQ TO NO: 25), PlrtA, PmrgA, PnblA, PggpS, PpetJ (SEQ TO NO: 28), PppsA, PrnpA (SEQ ID NO: 49), PpstS, Porf0128 (SEQ ID NO: 34), Porfl486 (SEQ ID NO: 35), Porf3164 (SEQ ID NO: 39), Porf3293 (SEQ ID NO: 36), Porf3621 (SEQ ID NO: 37), Porf3635 (SEQ ID NO: 38), Porf3858, Porfl071 (SEQ ID NO: 30), Porfl072 (SEQ ID NO: 40), Porfl074 (SEQ ID NO: 41), Porfl075 (SEQ ID NO: 42), Porfl542 (SEQ ID NO: 43), Porfl823 (SEQ ID NO: 44), Porfl824, Porf3126 (PsmtA) (SEQ ID NO: 26), Porf3389, Porf0221 (SEQ ID NO: 31), Porf0222 (SEQ ID NO: 45), Porf0223 (SEQ ID NO: 32), Porf0316 (SEQ ID NO: 33), Porf3232 (SEQ ID NO: 46), Porf3461 (PpetJ) (SEQ ID NO: 28), Porf3749, and (SEQ ID NO: 47).
9. The genetically modified cyanobacterium of any one of the above claims, wherein the polynucleotide is located on an endogenous or heterologous extrachromosomal plasmid.
10. The genetically modified cyanobacterium of any of claims 1-8, wherein said polynucleotide is integrated into the chromosome.
11. The genetically modified cyanobacterium of any one of the above claims, further comprising a modification that increases the heme level in the cyanobacterium.
12. The genetically modified cyanobacterium of claim 11, wherein the genetic modification that increases the heme level in the cyanobacterium is a knockdown of endogenous heme oxygenase.
13. The genetically modified cyanobacterium of claim 12, wherein the knockdown of the heme oxygenase is due to the presence of a polynucleotide that generates a small RNA that is capable of reducing expression of the endogenous heme oxygenase.
14. The genetically modified cyanobacterium of claim 13, wherein the polynucleotide that encodes the heterologous heme-containing protein and the polynucleotide that generates the small RNA capable of reducing the expression of endogenous heme oxygenase are operably linked to an inducible promoter.
15. The genetically modified cyanobacterium of claim 13, wherein the polynucleotide that encodes the heterologous heme-containing protein and the polynucleotide that generates the small RNA capable of reducing the expression of endogenous heme oxygenase are linked to different inducible promoters, inducible by different chemical compounds.
16. The genetically modified cyanobacterium of any one of the above claims comprising a polynucleotide encoding a heterologous heme-containing protein and a small RNA able to reduce endogenous expression of heme oxygenase, wherein the polynucleotide for the heme- containing protein and the small RNA are linked to the same inducible promoter.
17. The genetically modified cyanobacterium of claim 11, wherein the modification increases the flux through ferrochelatase.
18. The genetically modified cyanobacterium of claim 17, wherein the modification that increases the flux through ferrochelatase is selected from the group consisting of: a knockdown of the gene encoding magnesium chelatase (MgCh), the overexpression of ferrochelatase (FeCh), the knockdown of gun4 (MgCh activator), a gun4(W192A) mutation, magnesium-starvation, iron supplementation, and nitrogen-starvation.
19. The genetically modified cyanobacterium of any one of the above claims, further comprising a modification that results in a lower amount of phycocyanin.
20. A method of producing heme-containing protein in a cyanobacterium, comprising:
a) growing the genetically modified cyanobacterium of any of claims 1-10 under conditions to produce said heme-containing protein; and
b) obtaining the heme-containing protein from the cyanobacterium.
21. A method of producing heme-containing protein in a cyanobacterium, comprising:
a) growing the genetically modified cyanobacterium of any of claims 11-19 under conditions to produce said heme-containing protein and under conditions to increase the heme level in the cyanobacterium; and
b) obtaining the heme-containing protein from the cyanobacterium.
22. A dried biomass containing the genetically modified cyanobacterium of any of claims 1-19.
23. A dried meal (or bulk protein) obtained from the genetically modified cyanobacterium of any of claims 1-19.
24. A partially purified heme-containing protein, obtained from the genetically modified cyanobacterium of any of claims 1-19.
25. An iron-rich nutritional supplement comprising the material of any one of claims 22-24.
26. A food product made from the heme-containing protein made from the genetically modified cyanobacterium of any of claims 1-19.
27. The food product of claim 26, wherein the food product is a meat substitute.
28. The food product of claim 27, wherein the food product has a red color.
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