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WO2014027995A1 - Outils de biologie moléculaire pour ingénierie algale - Google Patents

Outils de biologie moléculaire pour ingénierie algale Download PDF

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WO2014027995A1
WO2014027995A1 PCT/US2012/050628 US2012050628W WO2014027995A1 WO 2014027995 A1 WO2014027995 A1 WO 2014027995A1 US 2012050628 W US2012050628 W US 2012050628W WO 2014027995 A1 WO2014027995 A1 WO 2014027995A1
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buffer
algal cell
algal
composition
sugars
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PCT/US2012/050628
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English (en)
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Farzad Haerizadeh
Todd Peterson
Wen Chen
Ewa Lis
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Life Technologies Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated

Definitions

  • This invention relates generally to the field of algal cell biology. More specifically, it relates to compositions and methods of cloning and expressing nucleic acids molecules in Algae.
  • Algal biotechnology has strong potential to solve pressing challenges relating to the availability of food, renewable energy and climate change. Yet the tools for algal biotechnology do not currently allow for swift adoption of the organisms for synthetic biology applications. Main areas of research that need attention are: high level transgene expression, targeted integration, homologous recombination, elimination of silencing, ability to efficiently deliver large sections of heterologous DNA as well as robust tools for production organisms. Methods and compositions delineated below seek to address these challenges thru commercialization of advanced synthetic biology toolkits for algal hosts.
  • the present invention provides nucleic acids, vectors, plasmids, host cells, buffers and methods for cloning genes and expressing proteins in algal cells. These methods and compositions provide a collection of tools for manipulating the genome of an algal cell and cloning and selection of desired strains so that a high level expression of genes of interest may be obtained. Some embodiments provide for a composition for transformation of an algal cell with DNA the composition comprising one or more sugars, and a biological buffer.
  • the one or more sugars are selected from the group consisting of sucrose, fructose, maltose, trehalose, sorbitol, maltitol, erythrytol, mannitol, xylose, raffilose and lactose.
  • the one or more sugars are present at a total concentration of from 40mM to lOOmM.
  • the biological buffer is selected from the group consisting of Bis-Tris Propane, TRIS, AMPD, TABS, AMPSO, CHES and CAPSO and in other embodiments the concentration of the biological buffer is from 5mM to lOOmM and may have a pH from 8 to 10.
  • Some embodiments provide for an isolated nucleic acid which exhibits promoter activity in an algal cell.
  • Another embodiment may be an algal cell comprising an isolated nucleic acid sequence which exhibits promoter activity.
  • Further embodiments may be methods for performing homologous recombination in an algal cell comprising co-transforming the algal cell with a protein that enhances homologous recombination.
  • Other embodiments may provide for a cyanobacteria derived from cyanobacteria strain BC104 comprising one or more resistance markers and one or more promoters.
  • a further embodiment may be a plasmid comprising an origin of replication and plasmid maintenance regions derived from pANL.
  • Another embodiment may be an algal cell large capacity vector capable of replicating in Chlorella.
  • a further embodiment may be an algal cell comprising an isolated nucleic acid which encodes an RNA polymerase under the control of an inducible promoter the isolated nucleic acid further comprising a reporter gene under control by the same inducible promoter.
  • Figure 1 shows the yield of transformants when different sugars are used in the transformation buffer.
  • Figure 2 shows the transformation efficiency when using the optimized transformation buffer at different pH.
  • Figure 3 shows the transformation efficiency when using the optimized transformation buffer with the Bio-Rad Gene Pulser® II and Neon® electroporation devices.
  • Algae or algal cell refer to plants or cells belonging to the subphylum Algae of the phylum Thallophyta.
  • the algae are unicellular, photosynthetic, oxygenic algae and are non-parasitic plants without roots, stems or leaves; they contain chlorophyll and have a great variety in size, from microscopic to large seaweeds.
  • Green algae belonging to Eukaryota-Viridiplantae-Chlorophyta-Chlorophyceae, can be used. Blue-green, red, or brown algae may also be used.
  • Exemplary algae for which the methods and reagents described herein may be used include those of the genus Chlamydomonas and the genus Chlorella.
  • Cyanobacteria are photosynthetic bacteria which require light, inorganic elements, nitrogen sources, water and a carbon source, generally C0 2 , to metabolize and grow. Cyanobacteria are photosynthetic prokaryotes which carry out oxygenic photosynthesis. The main product of the metabolic pathway of Cyanobacteria during aerobic conditions is oxygen and carbohydrates. Exemplary cyanobacteria include those found in Donald Bryant, The Molecular Biology of Cyanobacteria, published by Kluwer Academic Publishers (1994).
  • Synechococcus such as Synechococcus lividus and Synechococcus elongatus
  • Synechocystis such as Synechocystis minervae, such as Synchocystis Sp PCC 6803.
  • nucleic acid As used herein, a nucleic acid is a sequence of contiguous nucleotides (riboNTPs, dNTPs or ddNTPs, or combinations thereof) of any length, which may encode a full-length polypeptide or a fragment of any length thereof, or which may be non-coding. As used herein, the terms “nucleic acid molecule” and “polynucleotide” may be used interchangeably.
  • Transformation is a process for introducing heterologous DNA into a plant cell, plant tissue, or plant.
  • Transformed plant cells, plant tissue, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • Transformed, transgenic, and recombinant refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.
  • Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • a promoter is an example of a transcriptional regulatory sequence, and is specifically a nucleic acid sequence generally described as the 5'-region of a gene located proximal to the start codon. The transcription of an adjacent nucleic acid segment is initiated at the promoter region. A repressible promoter's rate of transcription decreases in response to a repressing agent. An inducible promoter's rate of transcription increases in response to an inducing agent. A constitutive promoter's rate of transcription is not specifically regulated, though it can vary under the influence of general metabolic conditions.
  • a gene that is codon-optimized for expression in an organism is a gene whose nucleotide sequence has been altered with respect to the original nucleotide sequence, such that one or more codons of the nucleotide sequence has been changed to a different codon that encodes the same amino acid, in which the new codon is used more frequently in genes of the organism of interest than the original codon.
  • the degeneracy of the genetic code provides that all amino acids except form methionine and tryptophan are encoded by more than one codon.
  • arginine, leucine, and serine are encoded by different six different codons; glycine, alanine, valine, threonine, and proline are encoded by four different codons.
  • Many organisms use certain codons to encode a particular amino acid more frequently than others.
  • a gene may be codon-optimized to change one or more codons to new codons (preferred codons) that are among those used more frequently in the genes of the host organism (referred to as the codon preference of the organism).
  • a codon-optimized gene or nucleic acid molecule of the invention need not have every codon altered to conform to the codon preference of the intended host organism, nor is it required that altered codons of a codon-optimized gene or nucleic acid molecule be changed to the most prevalent codon used by the organism of interest.
  • a codon-optimized gene may have one or more codons changed to codons that are used more frequently that the original codon(s), whether or not they are used most frequently in the organism to encode a particular amino acid.
  • the transformation buffer may be comprised of one or more sugars and a buffer salt to regulate pH.
  • Suitable sugars include but are not limited to sucrose, fructose, maltose, trehalose, sorbitol, maltitol, erythrytol, mannitol, xylose, raffilose and lactose.
  • the total sugar concentration may be from 40mM to lOOmM, 50mM to lOOmM, 60mM to lOOmM, 70mM to lOOmM, 40mM to 90mM, 40mM to 80mM, 40mM to 70mM or 40mM to 60mM.
  • concentration of any one sugar may be from lOmM to lOOmM.
  • the pH of the transformation buffer may be from pH 7 to pH 10, pH 8 to pH 10, pH 9 to pH 10, pH 7 to pH 9 or pH 7 to pH 8.
  • a number of Biological Buffers suitable for this pH range are available from Sigma- Aldrich (St. Louis, MO) including, but not limited to, Bis-Tris Propane, TRIS, AMPD, TABS, AMPSO, CHES and CAPSO.
  • the concentration of the buffer may be from 5mM to lOOmM, 5mM to 90mM, 5mM to 80mM, 5mM to 70mM, 5mM to 60mM, 5mM to 50mM, lOmM to lOOmM, 20mM to lOOmM, 30mM to lOOmM, 40mM to lOOmM or 50mM to lOOmM.
  • viral genomes may be sheared or cleaved by a unique Chlorella virus encoded restriction endonuclease. Each fragement may then be cloned into a bidireactional reporter vector and screened. Ideally, reporters that are amenable to FACS sorting such as GFP will be used. Strong promoters will be isolated and validated with additional reporter genes. Candidate promoters will also be evaluated for maintenance of expression as a function of culture time to address silencing.
  • Chlorella is a widely used platform organism in algal biotechnology with well established mass culture. It is typically grown for consumption as a health supplement or animal feed due to its high protein content as well as high levels of polyunsaturated fatty acids. Chlorella is also now increasingly used for biofuels research due to its high lipid content as well as ability for many strains to grow under heterotrophic conditions. [0021 ] Initial reports show that genetic manipulation of Chlorella is feasible however the tools for this organism are in their infancy.
  • Functional chlorella viral promoter elements will be evaluated in a selected set of Chlorella strains such as: C. vulgaris, C. protothecoid.es, C. pyrenoidosa, C. ellipsoidea. Reporter gene expression from the promoters may be tested together with evaluation of common resistance markers such as hygromycin and their optimization for use in Chlorella. Development of additional resistance markers with mass culture potential such as glyphosate resistance may be of value as well. Lastly feasibility of performing targeted integration in Chlorella will be established.
  • Targeted integration can alleviate the need for screening large numbers of clones and can generally result in more uniform expression levels across different clones however it does not address the inability to disrupt genes in green algae in a targeted fashion. The need still exists for homologous recombination driven gene disruption to enable quick generation of e.g. deletion mutants or promoter
  • Strategies to increase the rate of homologous recombination may include: co- transformation with RecA or other proteins such as eukaryotic Rad51 homologs, evaluation of factors that affect recombination efficiency (e.g. DNA amount, length of homology, electroporation conditions, cell culture conditions), use of DNA single or double strand break agents or irradiation, as well as effects of cell cycle.
  • factors that affect recombination efficiency e.g. DNA amount, length of homology, electroporation conditions, cell culture conditions
  • use of DNA single or double strand break agents or irradiation as well as effects of cell cycle.
  • Chlamydomonas chloroplast Characterization of genes that facilitate this process in the chloroplast given its' small size appears feasible with consequent expression of them in the nucleus and re-evaluation of efficiency of nuclear homologous
  • Cyanobacteria have several traits that make them attractive production hosts. They have a wide range of metabolic capabilities while having little nutritional requirements. Some cyanobacteria not only fix carbon dioxide but also fix nitrogen reducing the need for fertilizer. They can tolerate high pH, high light intensity (including protection from UV light) and often high salt - traits that offer crop protection. Many cyanobacteria also produce mucilaginous envelope that protects them against predators and/or desiccation. Most importantly, cyanobacteria being prokaryotic are easy to manipulate genetically and offer advantages of cistronic expression as well as small genomes that can be more easily characterized - important traits for synthetic biology hosts.
  • BC104 (or BL0902) was isolated in Imperial Valley, CA and developed as a production strain due to presence of many favorable traits.
  • BC104 belongs to the Leptolyngbya sp., is filamentous, shows robust growth in 20-40 °C temperature range, can tolerate high pH (pH 11) and urea (used for predator control), grows in up to 0.5M salt (sea water concentration) and tolerates high solar irradiance.
  • the growth rate of BC104 exceeds that of Spirulina in laboratory culture and is on par with Spirulina outdoors with excellent culture stability.
  • BC104 can also be harvested using similar screening methods used for Spirulina.
  • BC104 has been shown to accumulate >25 fatty acids / dry cell weight following conversion to FAME. In addition to these desirable qualities, BC104 is amenable to transformation that is reliable, efficient, and stable. Transformation may be demonstrated by the
  • the organism' s genome will be sequenced and annotated.
  • Synechococcus elongatus and Synechocystis Conjugation is more widely used and has many advantages such as high efficiency of DNA transfer, low species selectivity and capacity to transfer very large DNA segments with limits typically imposed by the receipient organism rather than transfer capacity. Conjugation from E.coli has been sucessfully used to deliver DNA to many cyanobacterial species such as: Synechococcus elongatus PCC7942, Anabaena PCC7120, Nostoc punctijorme ATCC 29133, Cyanothece sp. ATCC 51142, Synechococcus sp. WH8102,
  • next step is establishment of a universal plasmid or small subset of plasmids with broad host range specificity.
  • a good candidate for origin of replication is based on RSF1010 plasmids (oriV, mob, rep) which has been shown to replicate, albeit with poor efficiency in some cases, in distant species of cyanobacteria: Synechococcus elongatus PCC7942, Synechocystis PCC6803, Synechocystis PCC6714, Anabaena PCC7120, Cyanothece sp. ATCC 51142, Leptolyngbya sp. BL0902 (BC104).
  • Improvement of host range specificity in addition to replication efficiency in cyanobacteria can be done by sequential mutagenesis and selection in a group of cyanobacterial strains of interest.
  • RSFIOIO appears to have poor stability in Synechococcus elongatus PCC7942.
  • Development of the large endogenous plasmid pANL for high capacity cloning may prove to be a more short term solution for this platform organism.
  • pANL is 46 kb in length, 53% GC content and encodes 58 orfs.
  • the replication origin and plasmid maintenance origins from pANL will first be minimized in size while maintaining functionality and then combined with yeast elements to allow high order assembly as well as elements to enable conjugation from E. coli if necessary.
  • the hybrid vector will be evaluated for stability as well as DNA carrying capacity.
  • T7 RNA polymerase is an RNA polymerase from T7 bacteriophage with extremely high specificity towards the T7 promoter, high processivity and low error rate.
  • T7 RNA polymerase is commonly used in E. coli expression platforms (e.g. BL21 DE3) and has been successfully applied to drive expression from the T7 promoter in several organisms including: S. cerevisiae mitochondria, E. coli, Bacillus megaterium and Pseudomonas.
  • E. cerevisiae mitochondria e.g. BL21 DE3
  • E. coli E. coli
  • Bacillus megaterium eudomonas.
  • Recently the Voigt lab at UCSF has developed variants of T7 RNA polymerases with altered processivity and specificity as well as a suite of T7 promoters of different strength.
  • T7 RNA polymerase/T7 promoter system to express genes in the chloroplast. Both wild-type T7 RNA polymerase as well as the low processivity (Voigt lab) T7 RNA polymerase (codon optimized for chloroplast expression) will be evaluated.
  • the polymerase may be placed under control of an inducible promoter to enable regulated levels of expression.
  • a reporter gene such as GFP may be placed under control of the T7 promoter and the expression of GFP following induction of T7 RNA polymerase will be evaluated.
  • Example 1 Transformation Yield with Transformation Buffers Having Different Sugar Compositions
  • electroporation buffer at a concentration of 2 x 10 cells/ml.
  • 250 ⁇ 1 cells were mixed with 2 ⁇ g of Vl-Gus-Scal linear DNA and incubated at 4°C for 5min.
  • the reaction mixture was transferred to a pre-chilled cuvette and then electroporation performed in a Bio-Rad Genepulser® II apparatus with settings of 500V, 50mF and 800W.
  • the reactions were set on the bench for 15min for resting and then transferred into 10ml of TAP media with 40mM sucrose to recover over night with light. Transformation efficiency was determined by plating 1/100 of each reaction. The results are shown in Figure 1.
  • Example 2 Transformation Yield with Transformation Buffers Having Different pH
  • Wild type Chlamydomonas reinhardtii cells were washed twice with 2.5ml of an electroporation buffer comprising 40mM sucrose, lOmM sorbitol and 10mm CHES adjusted to a pH of between 8.5 and 9.5. After washing, the cells were resuspended in the electroporation buffer at a concentration of 2 x 10 cells/ml. For each electroporation reaction, 250 ⁇ 1 cells were mixed with 2 ⁇ g of Vl-Gus-Scal linear DNA and incubated at 4°C for 5min.
  • reaction mixture was transferred to pre-chilled cuvettes and then electroporation performed in a Bio-Rad Genepulser® II apparatus with settings of 500V, 50mF and 800W.
  • the reactions were set on the bench for 15min for resting and then transferred into 10ml of TAP media with 40mM sucrose to recover over night with light. Transformation efficiency was determined by plating 1/100 of each reaction. The results are shown in Figure 2.
  • Wild type Chlamydomonas reinhardtii cells were washed twice with 2.5ml of an electroporation buffer comprising 40mM sucrose, lOmM sorbitol and 10mm CHES adjusted to a pH of 9.25. After washing, the cells were resuspended in the electroporation buffer at a concentration of 2 x 10 cells/ml. For each electroporation reaction, 250 ⁇ 1 cells were mixed with 2 ⁇ g of DNA and incubated at 4°C for 5min.
  • reaction mixture was transferred to pre-chilled cuvettes and then electroporation performed in a Bio-Rad Genepulser® II or Neon® apparatus with settings of 500V, 50mF and 800W.
  • the reactions were set on the bench for 15min for resting and then transferred into 10ml of TAP media with 40mM sucrose to recover over night with light. Transformation efficiency was determined by plating 1/100 of each reaction. The results are shown in Figure 3.

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Abstract

La présente invention concerne des compositions et des procédés de manipulation génétique de cellules algales. Les compositions et les procédés permettent un transfert amélioré d'un matériau génétique dans des cellules algales et le clonage et la sélection de cellules génétiquement modifiées. L'expression de protéines codées par le matériau génétique sera améliorée par les procédés et les compositions de l'invention.
PCT/US2012/050628 2012-08-12 2012-08-13 Outils de biologie moléculaire pour ingénierie algale WO2014027995A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10280428B2 (en) 2011-08-12 2019-05-07 Life Technologies Corporation Molecular biology tools for algal engineering

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