WO2014016612A1 - Méthodes et compositions utilisées pour augmenter la productivité et la viabilité de cellules d'algue - Google Patents

Méthodes et compositions utilisées pour augmenter la productivité et la viabilité de cellules d'algue Download PDF

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WO2014016612A1
WO2014016612A1 PCT/GB2013/052008 GB2013052008W WO2014016612A1 WO 2014016612 A1 WO2014016612 A1 WO 2014016612A1 GB 2013052008 W GB2013052008 W GB 2013052008W WO 2014016612 A1 WO2014016612 A1 WO 2014016612A1
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metacaspase
cell
gene
algal
conditions
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PCT/GB2013/052008
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English (en)
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Jon PITTMAN
Patrick Gallois
Andrew Dean
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The University Of Manchester
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor

Definitions

  • This invention relates to a method for increasing the productivity of an algal cell. Also provided is a method for improving the resistance of an algal cell to Programmed Cell Death. Also provided is an algal cell modified to have the capacity for increased productivity, and/or improved resistance to Programmed Cell Death, and an algae population comprising such cells. Also provided is a genetic construct comprising a nucleic acid molecule the expression of which is capable of causing down regulation of a metacaspase in an algal cell, thus enabling an increase in the productivity of the cell, and/or improving resistance to Programmed Cell Death.
  • Algae are utilised for commercial applications, for example as a source of biofuel or in the production of chemical, pharmaceutical or nutritional products. They have the potential to provide high product yields, but this is dependent upon robust cultivation.
  • Typical environmental stresses include nutrient deficiencies, high light stress, virus infection, and salt stress, for example.
  • the stress can be lethal, which in low cost cultivation systems, such as open ponds, can result in complete collapse of the algal population and loss of the culture. This is far from desirable, as inoculation and culture start-up is expensive.
  • Lower levels of stress (referred to as sub-lethal or as non-lethal stress) may impact the productivity of the cell population but may not lead to cell death and collapse of the population.
  • Sub-lethal environmental stresses which can include mild salt accumulation or treatment and low nutrient conditions, do not trigger Programmed Cell Death and so do not cause significant reductions in cell number or lead to complete collapse of an algal population.
  • sub-lethal stresses will cause a reduction in biomass production through reduced growth rate, which will reduce product yields.
  • algal cells in low nutrient conditions significantly enhances the synthesis per cell of oils that are precursors for biofuel production.
  • growth in low nutrient conditions reduces the overall biomass of the algal culture. This reduction in biomass means that synthesis of oil induced by low nutrient conditions is not currently commercially viable. Collapse of an algal population may occur due to stress-induced Programmed Cell Death.
  • Programmed Cell Death is an intrinsic control mechanism used by animals, plants and algae, to minimise the impact of individual cells which may threaten the cell population. It therefore serves as a quality control mechanism, and has been shown to be involved in various aspects of growth and development.
  • the size of the cell population may take precedent over the quality of the cells. This is true of processes such as biomass production, for example. In such processes, it is beneficial to use cells which are better able to withstand environmental stresses, such that a larger cell population may be achieved, with less risk of collapse due to stress. Such populations have the potential to provide higher product yield as a result.
  • Metacaspases are an ancient class of protein that have been demonstrated to regulate Programmed Cell Death in higher plants and fungi. Metacaspases function as cysteine dependent protease enzymes, but are genetically distinct from distantly related Programmed Cell Death regulators such as the caspases in animals.
  • WO2004/081 168 describes a novel class of plant metacaspase genes, which the authors describe for use in modulating Programmed Cell Death in plants.
  • the metacaspase described in contrast to known caspases, has a recognition site with an R or a K at position P1 .
  • the authors show that down-regulation of expression of the novel metacaspase decreases Programmed Cell Death in plants.
  • the Arabidopsis metacaspase is over-expressed in tobacco.
  • Type 1 and Type 2 metacaspase genes Two types can be distinguished based upon sequence variation. These are referred to as Type 1 and Type 2. Fungi such as Saccharomyces cerevisiae only have Type 1 metacaspase, whereas higher plants have been shown to have a variety of both Type 1 and Type 2 metacaspases.
  • WO201 1/056886 describes a method of increasing resistance to Programmed Cell Death in algae. This is achieved by expressing in algal cells a mammalian, anti-apoptotic gene, such as a Bcl-2 family member.
  • Bidle & Bender (Euk. Cell (2008), 7(2): 223-236) look at gene expression of six metacaspases in marine phytoplankton, and use a broad spectrum caspase inhibitor, z- VAD-FMK to analyse the relationship with Programmed Cell Death.
  • z- VAD-FMK a broad spectrum caspase inhibitor
  • the authors rely on observations of gene expression and do not establish any direct link between an algal metacaspase and regulation of Programmed Cell Death.
  • the present invention aims to overcome or ameliorate this problem in the art.
  • a method of generating a modified algal cell which shows increased productivity compared to a non-modified algal cell when maintained under identical conditions comprising modifying the algal cell by down-regulating a metacaspase therein.
  • a method of generating a modified algal cell which shows improved resistance to Programmed Cell Death compared to a non-modified algal cell when maintained under identical conditions, wherein the method comprises modifying the algal cell by down-regulating a metacaspase therein.
  • productivity is meant the generation of biomass by the algal cell and/or the generation of produce (also referred to as output) of a cell (e.g. generation of lipids, fatty acids, pigments, vitamins and minerals, etc) over a defined window of time.
  • An increase in biomass i.e. the weight of a cell or population
  • An increase in the output of a cell may be due to increased biomass of the cell and/or other factors.
  • the cell may be maintained under normal (standard) conditions or under sub-lethal conditions, for example as defined herein. The latter is preferred.
  • lethal conditions the cells generated according to the present invention show increased resistance to Programmed Cell Death.
  • the present invention provides a method of generating a modified algal cell which shows increased productivity compared to a non-modified algal cell when maintained under identical conditions, wherein the method comprises i) modifying the algal cell by down-regulating a metacaspase therein; and ii) maintaining the cell under standard or sub- lethal conditions.
  • the present invention provides a method of generating a modified algal cell which shows improved resistance to Programmed Cell Death compared to a non-modified algal cell when maintained under identical conditions, wherein the method comprises i) modifying the algal cell by down-regulating a metacaspase therein; and ii) maintaining the cell under lethal conditions.
  • the metacaspase is an algal metacaspase.
  • it is a metacaspase which is native to the algal cell to be modified (i.e. expressed from a native algal cell gene), although it is conceivable within the scope of the invention that the metacaspase is not native to the cell, for example it has been introduced to the cell in a previous cell modification procedure.
  • the metacaspase may be expressed by a recombinant gene, for example on a vector such as plasmid, or a recombinant gene which has been introduced into the algal cell genome.
  • the metacaspase is metacaspase Type 1 (MC1 ) or metacaspase Type 2 (MC2).
  • the present invention provides a method of generating a modified algal cell which show increased productivity compared to a non-modified algal cell when maintained under identical conditions, wherein the method comprises modifying the cell by down-regulating Type 1 metacaspase or Type 2 metacaspase therein.
  • the method further comprises the step of maintaining the cell under standard or sub-lethal conditions.
  • the present invention also provides a method of generating a modified algal cell which shows improved resistance to Programmed Cell Death compared to a non-modified algal cell when maintained under identical conditions, wherein the method comprises modifying the cell by down-regulating Type 1 metacaspase or Type 2 metacaspase therein.
  • the method further comprises the step of maintaining the cell under lethal conditions.
  • Down regulation of a metacaspase may be conducted in any suitable way, acting at either the transcriptional or translational level, or on the transport or processing of RNA, or at the protein level. This may comprise using chemical means, for example to introduce a mutation which serves to down regulate expression of the metacaspase or expression of a gene encoding a gene product which regulates the expression or activity of the metacaspase, or to directly affect the activity of the metacaspase. Alternatively, the down regulation may be mediated by genetic means which may serve to down regulate expression of the metacaspase or affect the expression of a gene product which controls the expression or activity of the metacaspase.
  • the method of the invention comprises chemically treating an algal cell to increase the productivity of the algal cell, and/or improve the cells resistance to Programmed Cell Death, compared to a non-modified algal cell when maintained under identical conditions.
  • the chemical treatment induces mutations in the algal cell DNA.
  • mutations are induced either in a gene encoding the metacaspase, regulatory elements controlling expression of the metacaspase, or in a gene expressing a gene product which controls expression or activity of the metacaspase, or a regulatory element of said gene.
  • the method comprises a) chemically treating an algal cell to induce a mutation therein; b) maintaining the cell under standard or sub-lethal conditions; and c) identifying a mutated algal cell which shows increased productivity compared to a non-mutated cell maintained under identical conditions.
  • the invention may also provide a method which comprises a) chemically treating an algal cell to induce a mutation therein; b) maintaining the cell under lethal conditions; and c) identifying a mutated algal cell which shows improved resistance to Programmed Cell Death compared to a non-mutated cell maintained under identical conditions.
  • Mutagens are known to persons skilled in the art, and include for example physical methods such as UV, X-ray, gamma rays, and chemicals such as ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), nitrosoguanidine, biological methods such as zinc-finger nucleases and many others.
  • Such mutagens may cause DNA or RNA strand breaks, DNA or RNA point mutation induction, DNA or RNA insertion, DNA or RNA deletion, DNA amplification, DNA translocation or inversion.
  • a method of generating a modified algal cell which shows increased productivity compared to a non-modified algal cell when maintained under identical conditions comprises: i) introducing into the cell a heterologous nucleic acid molecule which is capable of (either directly or through an expression product thereof) down-regulating a metacaspase in an algal cell; and ii) optionally maintaining the cell under standard or sub-lethal conditions.
  • a of generating a modified algal cell which show improved resistance to Programmed Cell Death compared to a non-modified algal cell when maintained under identical conditions wherein the method comprises: i) introducing into the cell a heterologous nucleic acid molecule which is capable of (either directly or through an expression product thereof) down-regulating a metacaspase in an algal cell; and ii) optionally maintaining the cell under lethal conditions.
  • the metacaspase is Type 1 metacaspase or Type 2 metacaspase.
  • the heterologous nucleic acid sequence may encode a gene product which down-regulates expression of the gene encoding the metacaspase, or may encode a gene product which affects the activity of the metacaspase.
  • the heterologous nucleic acid molecule may encode a gene product which affects the expression of the metacaspase by interfering with transcription or translation of the protease gene, or processing or transport of transcription products, or by placing the metacaspase gene under altered control.
  • the heterologous nucleic acid molecule may bind to the gene or its transcript (for example as an antisense sequence or miRNA) to block transcription or translation, or may cause degradation of the transcript (i.e. encodes an siRNA or RNase dependent antisense) or may alter the gene, for example by insertional mutagenesis, or may encode a different regulatory sequence to place the gene under reduced expression, or may encode a gene product which interferes with the regulatory elements or factors which bind the regulatory element complex.
  • the heterologous nucleic acid molecule may encode a gene product which affects the activity of the metacaspase.
  • the heterologous nucleic acid molecule may encode an inhibitor of the metacaspase, or may encode a gene product which controls the expression or activity of a member of the cascade, and thus has an indirect affect on the expression or activity of the metacaspase.
  • a cell modified in this way may be referred to as a "knockdown" or “knockout” cell.
  • the knockout or knockdown may be permanent or transient (e.g. a temporary change in metacaspase gene expression).
  • the modification to the algal cell to knockout or knockdown the metacaspase is a heritable modification, such that descendants of the algal cell comprise the same modification and also show a capacity for increased productivity and/or increased resistance to Programmed Cell Death.
  • a modified algal cell or algae population which has increased productivity compared to a non-modified cell or population when maintained under identical conditions, wherein the algal cell or algae population has been modified to have down-regulated metacaspase expression and/or activity.
  • the metacaspase may be Type 1 metacaspase or Type 2 metacaspase.
  • the conditions are standard conditions or sub-lethal conditions.
  • a modified algal cell which has improved resistance to Programmed Cell Death compared to a non-modified cell when maintained under identical conditions, where the algal cell has been modified to have down-regulated metacaspase expression and/or activity.
  • the metacaspase may be Type 1 metacaspase or Type 2 metacaspase.
  • the conditions are lethal conditions.
  • the cell may be genetically modified (i.e. transgenic).
  • the cell comprises a heterologous nucleic acid molecule which provides a capacity for increased productivity under sub-lethal stress conditions and/or improves the cell's resistance to Programmed Cell Death.
  • the heterologous nucleic acid molecule down regulates a metacaspase in an algal cell.
  • the metacaspase may be Type 1 metacaspase or Type 2 metacaspase.
  • the heterologous nucleic acid molecule is capable of mediating down regulation of a metacaspase, as described above in relation to genetic modification of the algal cell.
  • the heterologous nucleic acid molecule may encode an antisense nucleic acid molecule, an RNA interference molecule such as an siRNA, a microRNA, a modified version of a gene encoding a metacaspase or a gene which regulates expression or activity of the metacaspase, or a modified version of a gene regulatory element of said genes, or a nucleic acid molecule which encodes a negative regulator of expression or activity of the metacaspase (e.g. a transcription factor or a competitive or non-competitive inhibitor).
  • the heterologous nucleic acid molecule may be provided in the algal cell as a genetic construct, for example a vector, or a construct adapted for homologous or non-homologous recombination.
  • the cell may be chemically modified, and comprise a mutation which provides a capacity for increased productivity and/or improves the cells resistance to Programmed Cell Death.
  • the chemically induced mutation is a genetic mutation, for example as described herein.
  • an algal cell of the present invention may comprise a mutation in a gene encoding the metacaspase (for example metacaspase 1 or metacaspase 2), in a gene encoding a gene product which expression of the metacaspase as defined herein, or a regulatory element of a gene, and exhibit increased productivity as a cell or population and/or improved resistance to Programmed Cell Death.
  • the mutation may be a point mutation, strand break, insertion, inversion, deletion, amplification or translocation of a gene or gene product encoding a metacaspase; or a gene encoding a gene product which regulates expression or activity of the metacaspase, or in a regulatory element of such a gene.
  • a genetic construct comprising a nucleic acid molecule which when introduced into an algal cell and expressed, has the effect of improving the productivity of the cell or a population of such cells and/or improving the cells resistance to Programmed Cell Death.
  • the nucleic acid molecule down regulates a metacaspase in an algal cell, preferably a Type 1 or Type 2 metacaspase in an algal cell.
  • the heterologous nucleic acid molecule is capable of mediating down regulation of a metacaspase, as described above in relation to genetic modification of the algal cell.
  • the genetic construct comprises a nucleic acid molecule, encoding a heterologous nucleic acid molecule as described herein.
  • the genetic construct may comprise one or more regulatory elements for expression of the nucleic acid molecule preferably operably linked to the nucleic acid molecule.
  • the genetic construct may be adapted to allow integration of the nucleic acid molecule into the genome of the algal cell, for example by homologous recombination.
  • an algal cell or algae population as described herein in the production of one or more of a plastic, detergent, non-toxic biodegradable polymer, animal feed, fertiliser, nutraceutical, pharmaceutical, biofuel and cosmetic.
  • Figure 1 shows: Nucleic acid sequences of C. reinhardtii MC1 (A) and MC2 (B) cDNA with the target regions underlined in bold which were used for artificial microRNA knockdown design.
  • Figure 2 shows: Growth of pChlamiRNA3-MC1 a and pChlamiRNA3-MC2a knockdown C. reinhardtii 24 hours (A) and 4 hours (B) after treatment with 12.5 mM H 2 0 2 at day 7 of the growth phase (stationary phase cells). Absorbance at 680 nm was used to determined cell density and is shown as the % absorbance relative to the initial value before H 2 0 2 treatment.
  • C denotes the control strain transformed with empty pChlamiRNA3 vector
  • 1 a and 2a denotes the MC1 a and MC2a knockdown lines.
  • Figure 3 shows: (A) Growth of pChlamiRNA2-MC1 b line 4 (MC1 b4) C. reinhardtii cells in comparison with wild type (CW15) cells under non-stressed conditions over time in liquid TAP medium, as determined by absorbance (680 nm) measurement. (B) Growth of MC1 b4 and wild type (CW15) cells following the addition of 7.5 mM or 12.5 mM H 2 0 2 at day 4 of the growth phase (exponential phase cells), as indicated by the arrow. Wild type cells exhibited complete cell death immediately following treatment with either concentration of H 2 0 2 .
  • Figure 4 shows (A) Growth of pChlamiRNA2-MC1 b line 4 (MC1 b4) C.
  • Figure 5 shows: (A) Stationary phase dry weight biomass of pChlamiRNA2-MC1 b line 4 (MC1 b4) and wild type (CW15) under sub-lethal salt stress (0.08M NaCI) conditions. (B) Exponential phase dry weight biomass productivity (mg day "1 ) of pChlamiRNA2-MC1 b line 4 (MC1 b4) and wild type (CW15) under the same conditions (0.08 M NaCI). Error bars are +/- S.E.
  • Figure 6 shows: (A) Stationary phase dry weight biomass of pChlamiRNA2-MC1 b line 4 (MC1 b4) and wild type (CW15) under non-stressed conditions (Standard TAP media) and under sub-lethal stress, namely Low-P (0.01 mM P-P0 4 ) and Low-N (0.7mM N- NH 4 ) TAP media. (B) Exponential phase dry weight biomass productivity of pChlamiRNA2- MC1 b line 4 (MC1 b4) and wild type (CW15) under the same conditions as in A. (C) Relative lipid yield per ml of culture as determined by Nile red fluorometry during stationary phase.
  • (D) Mean cell diameter of pChlamiRNA2-MC1 b line 4 (MC1 b4) and wild type (CW15) during stationary phase under the same conditions as in A. Under both non-stress, and sub-lethal stress, the MC1 b4 knockdown line gives higher biomass, and faster growth rate. Under sub lethal stress conditions (Low N and Low P) MC1 b4 lines have a higher lipid yield per unit culture volume when compared to wild type (CW15). Error bars are minimum and maximum values recorded.
  • the present invention is based upon the surprising observation that down regulation of a metacaspase, particularly Type 1 or Type 2 metacaspase, in an algal cell or population of algae increases the productivity of the cell (i.e. biomass and/or cellular output), even when maintained under sub-lethal stress conditions.
  • a metacaspase particularly Type 1 or Type 2 metacaspase
  • the productivity of the cell i.e. biomass and/or cellular output
  • sub-lethal stresses cause a reduction in biomass due to reduced growth rate. This in turn typically leads to reduced product yields.
  • the oil synthesis per cell may be increased but the overall biomass of the algae population is reduced due to low growth rate, and therefore the overall yield is reduced.
  • the present inventors have found that down regulating a metacaspase in an algal cell or population of algal cells has the effect of increasing the biomass and the produce per cell (e.g.
  • the present invention is further based upon the finding that down regulation of a metacaspase, particularly Type 1 or Type 2 metacaspase, can increase a cell's resistance to Programmed Cell Death and therefore increases its viability, particularly under conditions of lethal stress which might usually result in Programmed Cell Death of the cell.
  • a method of increasing productivity may include modifying an algal population i.e. modifying a plurality of algal cells in a population, or modifying a cell and generating a population of modified cells therefrom.
  • the metacaspase may be a Type 1 or Type 2 metacaspase.
  • a Type 1 metacaspase is preferably a protein encoded by the nucleic acid sequence of Figure 1A (SEQ ID. NO 1 ) or having the amino acid sequence encoded by Figure 1A.
  • a Type 1 metacaspase may be a protein encoded by a nucleic acid sequence which has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence of Figure 1A or a fragment thereof, or which is substantially homologous to the amino acid sequence encoded by Figure 1 A.
  • a Type 2 metacaspase is preferably a protein encoded by the nucleic acid sequence of Figure 1 B (SEQ ID.
  • a Type 2 metacaspase may be a protein encoded by a nucleic acid sequence which has at least 70% %, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence of Figure 1 B 1A or a fragment thereof, or which is substantially homologous to the amino acid sequence encoded by the sequence of Figure 1 B.
  • proteins which are substantially identical or homologous to a protein encoded by the sequence of Figure 1A or 1 B will share substantially the same functional characteristics as a Type 1 or Type 2 metacaspase, respectively, encoded by the sequence of Figure 1 A or 1 B.
  • a fragment of a sequence of Figure 1 may comprise a contiguous sequence of at least 10, 20, 30, 40, 50, 100, 150, 200, 300, 400 or 500, 1000 or 1500 nucleotides.
  • sequence identity of a variant or functionally equivalent sequence to the sequences of Figure 1 is determined by comparing the two aligned sequences, or fragments thereof, over a pre-determined comparison window, and determining the number of positions at which identical residues occur.
  • the comparison window may comprise all or part of the sequence, for example a window comprising 10, 20, 30, 40, 50, 100, 200, 500 or 1000 nucleotides or more.
  • sequence identity is expressed as a percentage.
  • the measurement of sequence identity of a nucleotide sequence is a method well known to those skilled in the art, using computer implemented mathematical algorithms such as ALIGN (Version 2.0), GAP, BESTFIT, BLAST (Altschul ei a/ J. Mol. Biol.
  • the present invention relates to down regulating a native algal protein.
  • the metacaspase is preferably a native algal metacaspase.
  • native is meant that the gene is present in a naturally occurring algal cell, and has not been introduced into the cell by the hand of man.
  • down regulate means that either the expression of the metacaspase, or the activity of the metacaspase, or both, is reduced compared to the corresponding levels in an unmodified cell of the same type, under identical conditions.
  • Down-regulation may be permanent or transient.
  • a reduction in gene expression levels may be determined by any suitable technique, many of which are known and available to persons skilled in the art. Examples of such techniques include Northern Blotting, PCR (for example RT-qPCR), Western Blotting, "tag based” technologies like Serial analysis of gene expression (SAGE) and RNA-Seq or Next-generation sequencing (NGS).
  • Down regulation may include knockdown of the gene, whereby the expression and/or activity is reduced compared to wild-type, or knockout, where the expression or activity of the gene is completely abolished compared to wild type.
  • the expression of a gene is the process of interpreting the information provided by the DNA sequence to provide a gene product, such as an RNA or peptide or protein.
  • Reference to gene expression may include one or more of the steps of gene transcription to produce RNA, RNA processing or modification, transport of RNA, translation of an RNA sequence to an amino acid sequence, protein folding and transport. Interference with any one or more of these steps may result in down regulation of the gene product (e.g. the protease), resulting in down regulated expression.
  • the activity of a metacaspase for the purposes of the present invention mean its ability to cleave a protein, and to have a functional role in mediating growth, productivity and Programmed Cell Death of an algal cell under conditions of stress (e.g. sub-lethal or lethal).
  • Down regulating the activity of the protease means that it has a reduced or abolished ability to cleave proteins, and a reduced or abolished ability to mediate its role in the cascades relating to growth, productivity and leading to Programmed Cell Death.
  • the down regulation may be indicated by reduced expression product levels as described above, or reduced activity which may be measured by assaying for reaction products using known techniques.
  • the increased productivity of a cell or cell population is observed for modified cells as described herein under standard conditions and under conditions of sub-lethal stress.
  • Increased survivability and/or viability of a modified cell as described herein is preferably observed under (or after) conditions of lethal stress (i.e. which would otherwise result in Programmed Cell Death).
  • Standard growth conditions are known to persons skilled in the art, and typically include media optimised for maximal growth, which includes non-limiting macro- and micro-nutrient, 0 2 and C0 2 concentrations, optimised light, and temperature, although a skilled person will appreciate that for any particular algae strain or culture system there will be variations in the standard conditions to obtain optimal growth.
  • Micronutrients include various trace metals, vitamin thiamin (Bi), cyanocobalamin (B 12 ), and biotin.
  • Macro nutrients include iron, nitrogen and phosphate.
  • Typical standard growth conditions are 16-27 ⁇ ; salinity of 12-40g/l (for a marine alga); light intensity of 5000- 20000 lux; a photoperiod range of 16:8 to 24:0 of light:dark hours, and a pH of between 7 and 9.
  • standard conditions include sufficient aeration.
  • Stress conditions include stress challenge by a biological agents, such as insects, fungi, bacteria, viruses, nematodes, viroids, mycoplasmas, etc.; or challenge by environmental factors including evaporation and dilution, nutrient deficiency, radiation levels, air pollution (ozone, acid rain, sulphur dioxide), temperature (hot and cold extremes), herbicide damage, pesticide damage, or other agricultural practices (e.g. over-fertilization, chemical sprays, etc.), salt stress, or the use of hydrogen peroxide as a proxy for environmental stress.
  • Sub-lethal stress conditions are well known to those in the art, typically include below optimal nutrients (e.g.
  • Sub-lethal stress conditions typically do not induce cell death, and the cell or population will undergo growth and production, albeit at lower levels than when maintained under standard conditions. A cell will usually be viable under sub-lethal conditions.
  • sub-lethal stress conditions include batch cultivation in Tris-acetate-phosphate (TAP) medium with 80 mM NaCI (for a freshwater alga), or TAP medium with initial nitrogen concentration reduced to 0.7 mM, or TAP medium with initial phosphorus concentration reduced to 0.01 mM, or cultivation at 37 °C, although a skilled person will appreciate that for any particular algae strain or culture system there will be variations in the conditions to obtain cells displaying symptoms of sublethal stress.
  • markers for sub-lethal stress conditions include low cellular chlorophyll levels, and/or decreased specific growth rate, and/or increased storage lipid (triacylglycerol) synthesis, and/or increased starch synthesis, which are statistically significant when measured relative to standard growth conditions, as defined earlier.
  • a statistically significant increase may be between 2 to in excess of 100%.
  • a population will preferably show at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or in excess of 100% increase of the sub-lethal stress marker than in standard growth conditions.
  • a statistically significant decrease may be between 2 to 100%.
  • a population will preferably show at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% decrease of the sub-lethal stress marker than in standard growth conditions.
  • Lethal stress may be defined as the conditions under which a cell becomes stressed and shows signs of aging or death, as described below.
  • Lethal stress conditions include, for example addition to exponentially growing cells of: 7.5 mM hydrogen peroxide, 200 mM NaCI (for a freshwater alga), 50 ⁇ cadmium, cultivation at 44 9 C, although a skilled person will appreciate that for any particular algae strain or culture system there will be variations in the conditions to obtain cells displaying symptoms of lethal stress.
  • Indicators of lethal stress conditions include markers of Programmed Cell Death: 1 ) loss of plasma membrane integrity, which can be measured for example using dyes such as Evans blue, trypan blue, SYTOX® green, SYTOX® orange or propidium iodide; 2) DNA fragmentation, the extent of which can be measured for example using TUNEL reaction kits or electrophoresis using agarose gel); 3) enzymatic activity, measured for example using in vivo enzymatic assays using preferably cell permeable caspase-substrates or fluorescein diacetate (FDA) vital dye viability test or in vitro methods using algal protein extracts and preferably caspase substrates; 4) a drop in the cytosolic pH, measured using for example an in vivo pH probe.
  • FDA fluorescein diacetate
  • a cell is described herein as being maintained under sub-lethal or lethal conditions, this may be all or part of a culture period. Where it is a part of a culture period, the cell may be maintained under one or more other conditions (standard, sub-lethal or lethal) for the remainder of the period. Thus, it may be maintained under the same conditions for a defined culture period, or under any combination of two or more of standard, sub lethal or lethal conditions for a defined culture period. For example, a cell may be maintained under standard conditions prior to a period of lethal conditions, and then standard conditions again. Alternatively, a culture may be maintained under standard conditions followed by sub-lethal conditions. In particular where lethal conditions are concerned, preferably these will be part of a culture period.
  • a cell is described as being capable of increased productivity, or showing increased productivity, or similar, this means that the cell has increased productivity compared to a non-modified cell when maintained under identical conditions, and when placed under conditions suitable for cell viability, for example standard or sub-lethal conditions as defined herein.
  • Biomass can be measured at the cell or population level in a number of ways, including those known to persons skilled in the art. Simple methods include measuring the mass of algae (wet or dried) in a defined volume, or counting the number of cells. Other methods include measuring the growth rate, measuring cell size (e.g. diameter), and measuring carbon mass.
  • the output of a cell can be measured in terms of measuring the yield of a particular cellular product, such as lipid from a predefined volume of culture. An increase in output is observed where there is a higher yield per unit of culture than from the same unit of a non-modified culture.
  • the culture may be batch culture, continuous or semi-continuous culture.
  • Reference herein to maintaining a cell under specified conditions preferably refers to a defined window of time.
  • a defined culture period may be 1 , 2, 3, 4, 5 10, 15, 20 days or more. Maintaining the cell under specified conditions may refer to the whole of this culture period, or any part of the culture period which may be specified in 1 , 2, 3, 4, 5, 6 etc up to 24 or more hours, or in days and parts thereof.
  • a defined time period for culture begins after any modification of the cell, and when the cell is placed under the specified conditions.
  • a method of the invention includes the step of removing a sample from a cell culture, and assaying for one or more markers of productivity, for example as described herein. Further, the method may comprise assaying a sample of the culture for markers of productivity at 1 , 2, 3 or more time intervals during a defined culture period.
  • Increased survivability and/or viability under conditions which would typically lead to Programmed Cell Death can be measured in any number of ways. Typically, this is assessed for an algal population, but may be assessed for a single algal cell by measuring the lifespan of the cell under conditions of stress. For an algal population, increased survivability and/or viability, which indicates increased resistance to Programmed Cell Death may be assessed by the presence or absence of cell death markers, or by the number of cells present in the population.
  • Cell death markers include 1 ) loss of plasma membrane integrity, which can be measured for example using dyes such as Evans blue, trypan blue, SYTOX® green, SYTOX® orange or propidium iodide; 2) DNA fragmentation, the extent of which can be measured for example using TUNEL reaction kits or electrophoresis using agarose gel); 3) enzymatic activity, measured for example using in vivo enzymatic assays using preferably cell permeable caspase-substrates or fluorescein diacetate (FDA) vital dye viability test or in vitro methods using algal protein extracts and preferably caspase substrates; 4) a drop in the cytosolic pH, measured using for example an in vivo pH probe.
  • dyes such as Evans blue, trypan blue, SYTOX® green, SYTOX® orange or propidium iodide
  • DNA fragmentation the extent of which can be measured for example using TUNEL reaction kits or electrophoresis using agarose gel
  • Cell counting methods include microscopy, optical density and flow cytometry.
  • a preferred method of assessing a cell for viability comprises 1 ) incubate algae with SYTOX® green (Invitrogen) at 250 nM until a maximum staining is achieved, preferably 15 min, and score dead cells using fluorescence microscopy with excitation around 504 nm and collection at 510-560 nm; or 2) incubate algae with fluorescein diacetate (Sigma- Aldrich) at 5 g/mL, preferably for 20 to 30 minutes and score dead cells using fluorescence microscopy with excitation around 494 nm and collection at 505-525 nm; or 3) incubate algae with propidium iodide (Sigma-Aldrich) at 10 ⁇ g/mL, staining preferably for 5 minutes and score dead cells using fluorescence microscopy with excitation around 536 nm and collection at 610-650 nm.
  • a method of the invention includes the step of removing a sample from a cell culture, and assaying for one or more markers of Programmed Cell Death, for example as described herein.
  • protease expression and/or activity levels can be determined, using methods available in the art.
  • a wild type cell will show a 6x to 8x increase in protease activity upon activation of Programmed cell Death.
  • a modified cell will show less than 5x, 4x, 3x or 2x increase in protease activity under conditions of stress.
  • An increased resistance to Programmed Cell Death may be observed by measuring survivability after a specified time from induction of lethal stress in a modified algal population, and comparing to the degree of survivability for a non-modified algal population.
  • An algal cell population is said to show increased resistance to Programmed Cell Death if the population shows a statistically significant increase in survival rate under conditions of stress, when compared to the survival rate of unmodified algal cell population.
  • the survival rate is the percentage of cells which do not exhibit a death marker under conditions of stress.
  • a statistically significant survival rate may be between 2 to 100%.
  • a population of the present invention will preferably show at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% greater numbers of cells surviving the stress conditions than in the wild type population.
  • An increased resistance to Programmed Cell Death may also be measured in terms of the time taken for a specified percentage of cells in a population to exhibit death markers, under conditions of stress. A cell population may be said to be resistance if this time is greater by a statistically significant amount compared to an unmodified algal cell population.
  • a method of the invention may further comprise assaying a sample of the culture for markers of Programmed Cell Death at 1 , 2, 3 or more time intervals during a defined culture period.
  • Increased resistance is measured against the resistance, as defined above, of a cell or population of the same type under identical conditions, which has not been modified as defined herein.
  • the present invention also relates to maintaining an algae culture by i) modifying a cell to down regulate a metacaspase therein; ii) maintaining a population of cells generated from i) in culture; iii) transferring survivors of any lethal conditions to standard culture conditions or altering any lethal culture conditions to standard or sub-lethal conditions.
  • algae include both unicellular and multicellular organisms. These include marine algae, freshwater algae and symbiotic algae such as in reefs, sea sponges and lichens. These include algae in the Groups: Chlorophyceae, Charophyceae, Ulvophyceae, and algae of the Stramenopile supergroup, which include the diatoms.
  • Preferred algal cells or algae for use in the present invention will be those which are suitable for use as a source of any one or more of plastics, detergents, nontoxic biodegradable polymers, animal feeds, fertiliser, nutraceuticals, pharmaceuticals, biofuels and cosmetics.
  • algal cells or algae are Ankistrodesmus, Botryococcus braunii, Chlorella spp, Dunaliella spp, Cyclotella Dl- 35, Hantzschia DI-160, Nannochloris spp, Nannochloropsis spp, Nitzschia, Phaeodactylum tricornutum, Scenedesmus spp, Stichococcus, Tetraselmis suecica, Thalassiosira pseudonana, Crpthecodinium cohnii, Neochloris spp, Haematococcus pluvialis, Porphyridium cruentum, Coccomyca C-169, Phaeodactylum tricornutum, Schizochytrium spp., Crypthecodinium spp., Nitzschia spp., Isochrysis spp., Tetraselmis spp., Spirulina spp.
  • An algae population may be maintained in an open or closed system, or in a bioreactor or any other suitable system known to persons skilled in the art.
  • the method comprises increasing the productivity of an algal cell and/or improving an algal cell's resistance to Programmed Cell Death by introducing into the algal cell a heterologous nucleic acid molecule which is capable of down regulating (either directly or via an expression product thereof) a metacaspase in the algal cell.
  • the metacaspase may be Type 1 metacaspase or Type 2 metacaspase.
  • the metacaspase may have a role in regulating growth, productivity, and activating or regulating Programmed Cell Death of an algal cell.
  • Any suitable method may be used to introduce a nucleic acid molecule into a cell, many of which will be known and available to persons skilled in the art. The methods for introduction will depend upon the type of cell.
  • Suitable methods include, for example, direct gene transfer methods such as microinjection, microprojectile mediated transformation (preferably using a particle gun) and electroporation, or other methods such as vortexing in the presence of DNA coated microfibers or liposome mediated transformation, or the glass bead method, or Agrobacterium tumefaciens and equivalent DNA transfer methods in bacterium.
  • the nucleic acid molecule may act within the cell to down regulate a metacaspase, as described herein.
  • the nucleic acid molecule may either directly or through expression of a gene product, down regulate expression of the metacaspase.
  • the nucleic acid molecule comprises an Open Reading Frame i.e. a series of nucleotide triplets (codons) coding for amino acids without any termination codons, and preferably translatable into a peptide.
  • the nucleic acid molecule encodes an RNA, which binds to, and down regulates expression of the metacaspase from the native algal gene encoding the same.
  • the RNA molecule may be a short (or small) interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA, riboswitch RNA or antisense RNA.
  • siRNA short interfering RNA
  • shRNA short hairpin RNA
  • microRNA microRNA
  • riboswitch RNA riboswitch RNA
  • antisense RNA A microRNA is preferred.
  • the RNA may be artificial or naturally occurring.
  • the nucleic acid molecule encodes an RNA which is antisense to all or a portion of a metacaspase as defined herein.
  • the nucleic acid molecule encodes an RNA which is antisense to at least a portion of a sequence of the Type 1 or Type 2 metacaspase gene of Figure 1.
  • exemplary portions of the Type 1 and Type 2 metacaspase genes to which an RNA may be substantially complementary to are 5'- GGGGCCTCTCTTATATGTTAG-3' (SEQ ID NO. 3) and 5'- TTGGTTCTTAGGTCAATGTGA-3' of Type 1 SEQ ID NO. 4) and (5'- ACGTTTCCGGTGATTTGATAC-3' SEQ ID NO. 5) of Type 2.
  • RNA substantially complementary of one of these sequences is able to inhibit expression of the metacaspase, and consequently increase the productivity of an algal cell or algae population under sub-lethal stress conditions and/or inhibit Programmed Cell Death of the genetically modified algal cell. It may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% complementary to the defined sequence.
  • an antisense is any nucleic acid molecule which is specifically hybridisable or specifically complementary to either RNA or DNA, preferably the plus strand.
  • the antisense nucleic acid molecule will hybridise to the corresponding mRNA, forming a double-stranded molecule.
  • a short (or small) interfering RNA (si RNA), short hairpin RNA (shRNA), microRNA will typically be antisense to the mRNA or plus strand of the DNA encoding the protease or a gene regulatory element thereof.
  • An antisense sequence binds or stably binds to a target nucleic acid molecule if a sufficient amount of the sequence forms base pairs with the sequence of the target.
  • Binding can be detected by physical or functional properties of the double stranded complex, for example whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription and translation.
  • Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures.
  • nucleic acid molecule or protein or peptide means that it has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra chromosomal DNA and RNA, and proteins.
  • Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids, proteins and peptides.
  • a nucleic acid molecule is a deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form. It includes within its scope known analogues of natural nucleotides, wherein the analogues hybridise to nucleic acids in a manner similar to the naturally occurring nucleotides.
  • a nucleic acid molecule may be any length, preferably a length suited to any particular embodiment of the invention.
  • a nucleic acid molecule of the present invention will have a length between 20 and 2500 nucleotides, the size depending on the method being used. For example, antisense could be up to the size of the target gene mRNA size, e.g. 2064 nucleotides for CrMC1.
  • a method of generating a modified algal cell which shows increased productivity compared to a non-modified algal cell when maintained under identical conditions comprises: i) introducing into the cell a heterologous nucleic acid molecule which is capable of (either directly or through an expression product thereof) down-regulating a metacaspase in an algal cell; and ii) optionally maintaining the cell under standard or sub-lethal conditions.
  • a method of generating a modified algal cell which show improved resistance to Programmed Cell Death compared to a non- modified algal cell when maintained under identical conditions comprises: i) introducing into the cell a heterologous nucleic acid molecule which is capable of (either directly or through an expression product thereof) down-regulating a metacaspase in an algal cell; and ii) optionally maintaining the cell under lethal conditions.
  • the metacaspase may be Type 1 metacaspase or Type 2 metacaspase.
  • the nucleic acid molecule is heterologous (i.e. non-native) to the algal cell. It is envisaged, however, that in some embodiments the nucleic acid molecule may be a native algal molecule, which under appropriate expression conditions is able to effect down regulation of the metacaspase. Such expression conditions include, for example, being operably linked to a sequence to which it is not normally linked, or it is under control by different regulatory elements than normal. In some embodiments the nucleic acid molecule or part thereof may be identical to the target sequence.
  • nucleic acid molecule is not native to an algal cell, it may be desirable to use codon optimisation in order to maximising efficient translation of the nucleic acid molecule. This may be appropriate where the expression product of the nucleic acid molecule which causes down regulation of the metacaspase in the algal cell is a protein.
  • the nucleic acid molecule is provided in a genetic construct, which is introduced into an algal cell.
  • the nucleic acid molecule is operably linked to a regulatory element, which is any nucleic acid sequence which regulates expression (i.e. transcription or translation) of a coding sequence to which it is operably linked.
  • regulatory sequences include promoters, enhancers, transcription terminators, initiation codons, splicing signals including acceptor and donor splice sites, stop codons, amber or ochre codons, transcription factor binding sites, ribosome binding sites, IRES, and targeting sequences such as cell compartmentalisation signals (e.g.
  • a genetic construct of the invention may comprise any one or more (two, three, four, five, six etc) regulatory sequences.
  • One or more regulatory elements may be provided in a 5' UTR. Additionally, a 3' UTR may also be provided.
  • a promoter with reference to the present invention may be defined as a control sequence that directs transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter may optionally include distal enhancer or repressor elements that can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included for use in the present invention.
  • any constitutive or inducible promoter can be used. They may be transcriptionally regulated promoters, chemically inducible promoters, and light activated promoters. In contrast to inducible promoters, constitutive promoters function under most environmental conditions. Many different constitutive promoters can be utilized with respect to the methods of this disclosure.
  • Preferred promoters for use in the present invention include the HSP70A-RBCS2 tandem promoter and the PSAD promoter (Fischer, N. & Rochaix, J. D. 2001 . The flanking regions of psaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii. Mol. Genet. Genom.
  • a nucleic acid molecule of the invention may be operably linked to a selectable marker.
  • the construct may comprise a selectable marker.
  • the selectable marker may be operably linked to the nucleic acid molecule.
  • the selectable marker may be controlled independently to the nucleic acid molecule, for example under the control of a separate promoter and/or other regulatory elements.
  • Suitable selection markers will be well known to those skilled in the art, and may include any nucleic acid sequence which, upon expression, provides a detectable phenotype.
  • the expressed polypeptide is detectable within the cell, preferably without adversely affecting the cell.
  • the selection marker may be green fluorescent protein or an enzyme, such as luciferase which generates a signal when contacted with a suitable agent.
  • Other selective markers include those which confer upon the cell a selective ability to grow in certain conditions, for example in the absence of specified nutrients, or in the presence of an agent which would otherwise be adverse to the cell. Examples of selectable markers include those that confer antimetabolite resistance (e.g.
  • dihydrofolate reductase dihydrofolate reductase
  • neomycin phosphotransferase kanamycin and paromycin
  • hygromycin hygromycin
  • trpB hisD
  • mannose-6-phosphate isomerise orthinine decarboxylase
  • demainase from Aspergillus terreus.
  • Additional selectable markers include those that confer herbicide resistance, for example, phosphinothricin acetyltransferase gene, a mutant EPSP-synthase, a mutant acetolactate synthase, a mutant psbA, or a mutant protoporphyrinogen oxidase, or other markers conferring resistance to a herbicide such as glufosinate.
  • Selectable markers include polynucleotides that confer dihydrofolate reductase (DHFR) or neomycin resistance for eukaryotic cells and tetracycline; ampicillin resistance for prokaryotes such as E. coli; and bleomycin, gentamycin, glyphosphate, hygromicin, kanamycin, methotrexate, phleomycin, phosphinotricin, spectinomycin, streptomycin, sulphonamide and sulfonylurea resistance in plants.
  • Preferred selection markeds include resistance to cryptopleurine and emetine, S-23142, sulfonylurea herbicides, zeomycin or paromomycin selection due to the presence of the aphVIII gene.
  • a preferred selection marker for use in the present invention is one which provides a nutrient auxotrophy selection, preferably an arginine auxotrophy selection due to the presence of the ARG7 gene (alternative markers including NIA1 (NIT1 ) (can grow on nitrate), NIC7 (rescue mutants to nicotianamide prototrophy).
  • NIA1 NIT1
  • NIC7 rescue mutants to nicotianamide prototrophy
  • Suitable genetic constructs for use in the present invention include vectors including for example viral vectors, plasmids (circular or linear), phagemids, linear DNA.
  • Preferred genetic constructs for use in the present invention include pChlamiRNA2 and pChlamiRNA3 (as described by Molnar et al. 2009. Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant Journal 58: 165-174).
  • a vector may include one or more selectable markers, as defined herein, one or more cloning sites, for example to allow homologous recombination with native algal nucleic acid, one or more regulatory elements as defined herein, and one or more origin or replication.
  • nucleic acid molecules as defined herein may be provided in a single vector, where desired. These may be under separate regulatory control, or be operably linked to the same regulatory elements. All or part of a nucleic acid molecule or genetic construct may become integrated into the algal genome. Preferred mechanisms for integration of a nucleic acid molecule into a genome include homologous recombination and non-homologous end joining (illegitimate recombination). Alternatively, all or part of the nucleic acid molecule or genetic construct may remain in the cytoplasm, or in the mitochondria or chloroplast.
  • a method of generating a modified algal cell which shows increased productivity compared to a non- modified cell maintained under identical conditions by a) introducing into the algal cell a heterologous nucleic acid molecule which is capable of (either directly or through an expression product thereof) down-regulating a metacaspase in an algal cell; b) allowing integration of the nucleic acid molecule into the genome of the algal cell; and c) expressing the nucleic acid molecule in the algal cell.
  • the method may further comprise maintaining the cell under standard or sub-lethal conditions.
  • a method of generating a modified algal cell which shows increased resistance to Programmed Cell Death compared to a non-modified cell maintained under identical conditions by a) introducing into the cell a heterologous nucleic acid molecule which is capable of (either directly or through an expression product thereof) down-regulating a metacaspase in an algal cell; b) allowing integration of the nucleic acid molecule into the genome of the algal cell; and c) expressing the nucleic acid molecule in the algal cell.
  • the method may further comprise maintaining the cell under lethal conditions.
  • the metacaspase may be Type 1 metacaspase or Type 2 metacaspase.
  • the nucleic acid molecule may be as defined herein.
  • operably linked means that two or more nucleic acid molecules are positioned such that one affects the function of the other, i.e. they are placed in a functional relationship with each other.
  • function includes expression, for example the pattern of expression.
  • the two or more nucleic acid molecules which are operably linked may function as a single unit.
  • Reference herein to protein may be used interchangeably with the term "polypeptide”.
  • nucleic acid molecules provided herein may be recombinant.
  • recombinant is meant that the nucleic acid molecule has been manipulated, and is no longer identical to that found in nature (for example in terms of its location, sequence, or linkage to other nucleic acid molecules).
  • recombinant can mean that the nucleic acid molecule has been removed from where it is naturally found, and may be provided, for example, in a manipulated form for example in a vector or isolated form.
  • the methods of the invention further comprise the step of culturing the modified algal cell to form an algae population.
  • the present invention also provides a method of increasing the productivity of an algal cell as described herein and further comprising harvesting a cellular product from the cell.
  • the product may be a lipid, vitamin, trace metal, fatty acid, or other.
  • the method may include any suitable harvesting method, for example enzymatic digestion, chemical disruption, mechanical disruption and/or centrifugation.
  • the present invention also provides an algal cell, which has been modified as defined herein.
  • the algal cell comprises a genetic modification (e.g. a mutation or transfection with exogenous nucleic acid) which causes down regulation of a metacaspase.
  • the algal cell exhibits increased productivity under standard and sub-lethal conditions, and/or increased resistance to Programmed Cell Death, preferably exhibiting an increased lifespan under lethal conditions e.g. as defined herein.
  • descendants of an algal cell of the invention which inherit a modification as defined herein and as such exhibits increased productivity under standard and sub-lethal conditions and/or increased resistance to Programmed Cell Death under lethal conditions, preferably exhibiting an increased lifespan under lethal conditions.
  • a cell of the invention may comprise all or part of a genetic construct as defined herein, optionally integrated into the genome, and/or all or part of a nucleic acid molecule as defined herein, and/or an antisense molecule as defined herein.
  • the present invention preferably also provide algae, wherein preferably at least 40%, 50%, 60%, 70%, 80% or at least 90% of the algae have been modified to be capable of increased productivity and/or to have an improved resistance to Programmed Cell Death, as defined herein.
  • An algal cell population is a group of cells, substantially consisting of algal cells of the same type. The population may be of any size, for example ranging from 0.5x10 6 to 20 x 10 6 cells/ml.
  • each reference to "a" or "one or more” independently includes one, two, three, four, five, or six or more.
  • DNA constructs were generated which were used to produce transgenic algae cells for knocking down either the MC1 or MC2 gene.
  • the complementary DNA (cDNA) sequence of the two C. reinhardtii MC genes (MC1 and MC2 shown in figure 1)) were identified and cDNA sequence corresponding to specific 21 nucleotide (nt) regions of the MC1 and MC2 cDNA were selected in order to generate an artificial microRNA (amiRNA) which will specifically target and knock-down expression of the MC1 or MC2 mRNA.
  • cDNA complementary DNA sequence of the two C. reinhardtii MC genes (MC1 and MC2 shown in figure 1)
  • nt specific 21 nucleotide regions of the MC1 and MC2 cDNA
  • MC1a and MC1b Two 21 nt regions of MC1 (5'-GGGGCCTCTCTTATATGTTAG-3' (SEQ ID NO 6) and 5'- TTGGTTCTTAGGTCAATGTGA-3' (SEQ ID NO 7)) referred to as MC1a and MC1b, respectively, and one 21 nt region of MC2 (5'-ACGTTTCCGGTGATTTGATAC-3' (SEQ ID NO 8)) referred to as MC2a, were selected that were efficient at generating the desired MC-knock-down phenotype.
  • MC target sequences were used to design two complementary 90 nt oligonucleotides (which were synthesised by MWG Eurofins, Germany) which correspond to a Spel restriction enzyme site followed by the target sequence, followed by a 42 nt spacer sequence (5'- tctcgctgatcggcaccatgggggtggtggtgatcagcgcta-3' (SEQ ID NO 9)), followed by the reverse complement of the target sequence, then a Spel restriction enzyme site.
  • the two oligonucleotides were annealed together by boiling for 5 min then gradually cooling to generate a double stranded DNA (dsDNA) encoding the amiRNA precursor.
  • the dsDNA oligonucleotide was purified using a Qiagen PCR clean-up kit (Qiagen). This strategy to generate an amiRNA precursor DNA and the design of the spacer sequence was as described by Molnar et al. (2009 Plant Journal 58: 165-174) except that the design of the MC1 and MC2 target region sequence was unique to this present invention.
  • the amiRNA precursor dsDNA oligonucleotides for MC1a, MC1b, or MC2a were ligated into either the pChlamiRNA2 or pChlamiRNA3 expression plasmids (as described by Molnar et al. 2009 Plant Journal 58: 165-174).
  • the pChlamiRNA2 plasmid provides arginine auxotrophy selection due to the presence of the ARG7 gene, and gene expression is driven by the HSP70A-RBCS2 tandem promoter.
  • the pChlamiRNA3 plasmid provides paromomycin selection due to the presence of the aphVIII gene, and gene expression is driven by the PSAD promoter.
  • the plasmids were purchased from the Chlamydomonas Resource Center at the University of Minnesota, USA.
  • All plasmids were isolated and purified from host Escherichia coli DH5a bacteria using a QIAprep mini plasmid isolation kit (Qiagen) and digested with Spel restriction enzyme (Roche) then treated with calf intestinal alkaline phosphatase (Roche) to dephosphorylate the cut ends of the plasmid DNA and make them compatible to enable cloning of the dsDNA MC amiRNA precursor oligonucleotides.
  • the linear plasmid DNA was purified using a Qiagen PCR clean-up kit (Qiagen).
  • the dsDNA oligonucleotide was treated with T4 polynucleotide kinase (Roche) to phosphorylate the DNA ends.
  • the linearised pChlamiRNA2 or pChlamiRNA3 plasmids and MC1a, MC1b or MC2a oligonucleotides were ligated using T4 DNA ligase (Roche) to fuse the cut DNA ends and yield circular plasmid DNA of pChlamiRNA2 or pChlamiRNA3 containing either MC1a, MC1b or MC2a amiRNA precursor dsDNA sequence.
  • the plasmids were transformed into competent DH5a E. coli using the heat-shock method then the bacteria were selected on ampicillin growth media. Colonies that grew following selection were isolated and plasmid DNA was isolated using a QIAprep mini plasmid isolation kit (Qiagen) and the plasmid sequence was confirmed by DNA sequencing.
  • C. reinhardtii strain CCAP 1 1/32CW15+ arg- purchased from the Culture Collection of Algae and Protozoa at the Scottish Association of Marine Sciences, UK.
  • C. reinhardtii was maintained in Tris-acetate- phosphate (TAP) medium, as described by Harris ⁇ The Chlamydomonas Sourcebook, Academic Press, San Diego, 1989), and grown at 22°C with a 16h light:8 h dark light regime and illuminated with cool white fluorescent tubes at approximately 100 ⁇ photon m "2 s "1 .
  • TAP Tris-acetate- phosphate
  • reinhardtii transformation was performed using the glass bead method. Plasmid DNA was linearised with Kpnl restriction enzyme prior to transformation. C. reinhardtii cells were collected by centrifugation and resuspended in TAP medium to give a concentration of approximately 1 x 10 8 cells ml "1 . Polyethylene glycol (M r 6000) to a final concentration of 5% (w/v), 50 ⁇ g of boiled salmon sperm DNA, 300 mg of acid-washed glass beads (0.5 mm diameter) and 10 ⁇ g of linearised plasmid DNA was added to the cells.
  • M r 6000 Polyethylene glycol
  • the cells were vortexed for 15 sec then spread onto solid TAP medium containing 1 .5% (w/v) agar, for the pChlamiRNA2 transformed cells, and with 10 ⁇ g ml "1 paromomycin and 50 g ml "1 arginine for selective growth of the pChlamiRNA3 transformed cells. Colonies that grew on the plates were transferred to fresh selective plates then inoculated in liquid TAP medium to generate cell cultures for verification of plasmid transformation and MC mRNA expression level.
  • Genomic DNA was isolated from 2ml of dense C. reinhardtii culture following centrifugation of the cells and resuspension in 500 ⁇ of CTAB buffer (2% (w/v) hexadecyl trimethyl ammonium bromide, 100 mM Tris-HCI (pH 8.0), 20 mM EDTA (pH 8.0), 1 .4 M NaCI, 1 % PVP-40).
  • CTAB buffer 2% (w/v) hexadecyl trimethyl ammonium bromide, 100 mM Tris-HCI (pH 8.0), 20 mM EDTA (pH 8.0), 1 .4 M NaCI, 1 % PVP-40.
  • the cell-CTAB solution was incubated at 65 °C for 1 h then mixed with an equal volume of phenol/chloroform/isoamylalcohol (25:24:1 ).
  • aqueous phase was extracted and gDNA was precipitated from this by addition of an equal volume of isopropanol, then collected by centrifugation and washed with 70% ethanol.
  • PCR was performed using Fast-Taq DNA polymerase (Roche), standard buffer and dNTPs, and oligonucleotide primers that were complementary to the pChlamiRNA2 and 3 plasmid sequence to amplify transgene DNA from the C. reinhardtii gDNA.
  • a PCR cycling condition of 95°C melting temperature, 60°C annealing temperature and 72°C elongation temperature, for 35 cycles as used and amplified DNA was visualised by agarose gel electrophoresis, to confirm transgene integration.
  • RNA was isolated from C. reinhardtii by mixing 1 ml of TRIzol reagent (Invitrogen) in an isolated cell pellet containing approximately 5-10 x 10 6 cells. Following centrifugation, the supernatant was incubated at room temperature for 5 mins then extracted with 0.2 ml of chloroform. Following further centrifugation, the aqueous phase was mix with 0.5 ml isopropanol to precipitate RNA, which was collected by centrifugation and washed with 70% ethanol. First strand cDNA was synthesised from DNase-treated RNA using an oligo dT 18 primer and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's protocol.
  • TRIzol reagent Invitrogen
  • MC gene expression was quantified from the cDNA by quantitative real-time PCR using 12.5 ⁇ of a SYBR Green core qPCR kit master mix solution (Eurogentec), 2.5 ⁇ of cDNA, 8.5 ⁇ of sterile deionised water and 0.75 ⁇ of both a forward and reverse oligonucleotide primer set, designed against MC1 or MC2 cDNA sequence (MC1 forward: 5'- GATGACGAGCTGAACCGCAT-3 (SEQ ID NO 10)'; MC1 reverse: 5'- CAATTACCGCGTGAAGCGT-3' (SEQ ID NO 1 1 ); MC2 forward: 5'- ACCAAGCCCGGTGTGAAGT-3' (SEQ ID NO 12); MC2 reverse: 5'- TGGTCCAAAAGGGTCCCAG-3' (SEQ ID NO 13)), and performed on an ABI Prism 7000 machine (Applied Biosystems) according to the manufacturer's instructions, and using the standard SYBR Green detection program and normalised to 18S and
  • FIG. 1 shows an example with the MC1 b cell line compared to control (wild type) cells. Control cells have complete cell death while the MC1 b cells show no effect in response to 7.5 mM H 2 0 2 and 12.5 mM H 2 0 2 led to an initial drop in cell number followed by renewed growth.
  • MC1 b knockdown cells and wild type (control) cells were also grown for an extended period of time up to 10 weeks in low nutrient liquid growth medium, Jaworski's medium (JM) and grown at 22 °C with a 16h light:8 h dark light regime and illuminated with cool white fluorescent tubes at approximately 100 ⁇ photon m "2 s "1 .
  • JM Jaworski's medium
  • lipid (triacylglycerol) production was determined in the cultures by measurement of lipids by staining with the fluorescent lipid stain Nile Red (9-diethylamino-5H- benzo[a]phenoxazine-5-one) (as described by Dean et al 2010 Bioresource Technology 101 :4499-4507).
  • MC1 b4 lines Under sub-lethal stress conditions that induce lipid synthesis (Low N and Low P) MC1 b4 lines have a higher lipid yield per unit culture volume when compared to wild type. Therefore in sub-lethal stressed cells, MC knockdown lines have more biomass and more volumetric oil yield compared to wild type cells. Under lethal stress conditions, the higher survival of the MC1 b4 line also leads to increased biomass and oil yield.

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Abstract

Cette invention concerne une méthode permettant d'augmenter la productivité d'une cellule d'algue ou d'une population d'algues dans des conditions de stress classiques ou sublétales. L'invention concerne également l'amélioration de la résistance de cellules d'algue à la mort cellulaire programmée dans des conditions létales. Les méthodes consistent à réguler à la baisse l'expression d'une métacaspase dans la cellule de l'algue native. L'invention concerne également des cellules d'algue pouvant accroître leur productivité dans des conditions de stress sublétales, et des cellules d'algue plus résistantes à la mort cellulaire programmée.
PCT/GB2013/052008 2012-07-26 2013-07-26 Méthodes et compositions utilisées pour augmenter la productivité et la viabilité de cellules d'algue WO2014016612A1 (fr)

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US9113607B1 (en) 2015-03-25 2015-08-25 Heliae Development, Llc Methods for treating a culture of Haematococcus pluvialis for contamination using hydrogen peroxide
ITUB20160845A1 (it) * 2016-02-18 2017-08-18 Micoperi Blue Growth S R L Metodo e terreno di coltivazione di una biomassa microalgale
WO2017214077A1 (fr) * 2016-06-08 2017-12-14 Radhakrishna Sureshkumar Culture et récolte écoénergétique de micro-algues à l'aide d'une transition sol-gel thermoréversible
WO2018064037A1 (fr) 2016-09-30 2018-04-05 Heliae Development Llc Procédés d'application de toxicité d'acétate et d'induction d'absorption d'acétate dans des cultures de microalgues
EP3283090A4 (fr) * 2015-04-15 2018-10-24 Synthetic Genomics, Inc. Mutants srp54 chloroplastiques d'algues

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9113607B1 (en) 2015-03-25 2015-08-25 Heliae Development, Llc Methods for treating a culture of Haematococcus pluvialis for contamination using hydrogen peroxide
US9347034B1 (en) 2015-03-25 2016-05-24 Heliae Development Llc Methods for preventing lysis in a culture of haematococcus pluvialis using hydrogen peroxide
US9347035B1 (en) 2015-03-25 2016-05-24 Heliae Development Llc Haematococcus pluvialis culture compositions
US9392793B1 (en) 2015-03-25 2016-07-19 Heliae Development Llc Methods for treating a culture of haematococcus pluvialis for contamination using salt
US9447375B1 (en) 2015-03-25 2016-09-20 Heliae Development Llc Methods for treating a culture of Haematococcus pluvialis for lysis using hydrogen peroxide
US9447373B1 (en) 2015-03-25 2016-09-20 Heliae Development Llc Methods for treating a culture of haematococcus pluvialis for contamination using salt and hydrogen peroxide
US9447374B1 (en) 2015-03-25 2016-09-20 Heliae Development Llc Methods for preventing a chytrid infection in a culture of Haematococcus pluvialis using hydrogen peroxide
EP3283090A4 (fr) * 2015-04-15 2018-10-24 Synthetic Genomics, Inc. Mutants srp54 chloroplastiques d'algues
US10544424B2 (en) 2015-04-15 2020-01-28 Synthetic Genomics, Inc. Algal chloroplastic SRP54 mutants
ITUB20160845A1 (it) * 2016-02-18 2017-08-18 Micoperi Blue Growth S R L Metodo e terreno di coltivazione di una biomassa microalgale
WO2017214077A1 (fr) * 2016-06-08 2017-12-14 Radhakrishna Sureshkumar Culture et récolte écoénergétique de micro-algues à l'aide d'une transition sol-gel thermoréversible
WO2018064037A1 (fr) 2016-09-30 2018-04-05 Heliae Development Llc Procédés d'application de toxicité d'acétate et d'induction d'absorption d'acétate dans des cultures de microalgues

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